elisa plates  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Thermo Fisher elisa plates
    1:7:7 stoichiometry of Baz, DE-cad, and Arm at SAJs. (A–D) Mapping protein distributions. (A, top) X-y planes are shown with single-cell sides boxed. (middle) Montages of single-cell sides shown from apical surface to 12 µm basal (0.3-µm z steps). SAJ is shown in a bracket. Dots indicate five SAJs and are shown at 3-µm intervals. (bottom) SAJ parameters are shown (50 sides each). (B) SAJ RFI profiles are shown of DE-cad∷GFP ( n = 288), Arm∷GFP ( n = 229), and Baz∷GFP ( n = 188). (C) IM RFI profiles ( n = 50 each) are shown. (D) Maps of SAJ and IM RFIs are shown over cell cortex. (E–G) Embryo protein counts are shown. (E) Purified <t>GST-GFP</t> is shown (12% SDS-PAGE; coomassie). (F) Example <t>ELISA</t> assay is shown with GST-GFP standard curve, and Baz∷GFP late cellularization embryos are shown. (G) Graph of the example standard curve is shown (done in duplicate), and the example Baz sample is indicated in red. (H) Levels of GFP-tagged proteins versus endogenous proteins in WT embryos (3–7-h embryo lysates; 6% SDS-PAGE). Blots were probed with DE-cad, Arm, Baz, and β-tubulin antibodies (non–GFP-tagged portion of DE-cad∷GFP detected; Oda and Tsukita, 1999 ). (I) Protein counts are shown. (J) Single-plane images are shown with the same coverslips and settings. Late cellularization is shown. RFIs were measured at the 10 brightest SAJs and equal IM areas. Normalized means ± SD are shown for five embryos each. WB, Western blot.
    Elisa Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/elisa plates/product/Thermo Fisher
    Average 99 stars, based on 430 article reviews
    Price from $9.99 to $1999.99
    elisa plates - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions"

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200812146

    1:7:7 stoichiometry of Baz, DE-cad, and Arm at SAJs. (A–D) Mapping protein distributions. (A, top) X-y planes are shown with single-cell sides boxed. (middle) Montages of single-cell sides shown from apical surface to 12 µm basal (0.3-µm z steps). SAJ is shown in a bracket. Dots indicate five SAJs and are shown at 3-µm intervals. (bottom) SAJ parameters are shown (50 sides each). (B) SAJ RFI profiles are shown of DE-cad∷GFP ( n = 288), Arm∷GFP ( n = 229), and Baz∷GFP ( n = 188). (C) IM RFI profiles ( n = 50 each) are shown. (D) Maps of SAJ and IM RFIs are shown over cell cortex. (E–G) Embryo protein counts are shown. (E) Purified GST-GFP is shown (12% SDS-PAGE; coomassie). (F) Example ELISA assay is shown with GST-GFP standard curve, and Baz∷GFP late cellularization embryos are shown. (G) Graph of the example standard curve is shown (done in duplicate), and the example Baz sample is indicated in red. (H) Levels of GFP-tagged proteins versus endogenous proteins in WT embryos (3–7-h embryo lysates; 6% SDS-PAGE). Blots were probed with DE-cad, Arm, Baz, and β-tubulin antibodies (non–GFP-tagged portion of DE-cad∷GFP detected; Oda and Tsukita, 1999 ). (I) Protein counts are shown. (J) Single-plane images are shown with the same coverslips and settings. Late cellularization is shown. RFIs were measured at the 10 brightest SAJs and equal IM areas. Normalized means ± SD are shown for five embryos each. WB, Western blot.
    Figure Legend Snippet: 1:7:7 stoichiometry of Baz, DE-cad, and Arm at SAJs. (A–D) Mapping protein distributions. (A, top) X-y planes are shown with single-cell sides boxed. (middle) Montages of single-cell sides shown from apical surface to 12 µm basal (0.3-µm z steps). SAJ is shown in a bracket. Dots indicate five SAJs and are shown at 3-µm intervals. (bottom) SAJ parameters are shown (50 sides each). (B) SAJ RFI profiles are shown of DE-cad∷GFP ( n = 288), Arm∷GFP ( n = 229), and Baz∷GFP ( n = 188). (C) IM RFI profiles ( n = 50 each) are shown. (D) Maps of SAJ and IM RFIs are shown over cell cortex. (E–G) Embryo protein counts are shown. (E) Purified GST-GFP is shown (12% SDS-PAGE; coomassie). (F) Example ELISA assay is shown with GST-GFP standard curve, and Baz∷GFP late cellularization embryos are shown. (G) Graph of the example standard curve is shown (done in duplicate), and the example Baz sample is indicated in red. (H) Levels of GFP-tagged proteins versus endogenous proteins in WT embryos (3–7-h embryo lysates; 6% SDS-PAGE). Blots were probed with DE-cad, Arm, Baz, and β-tubulin antibodies (non–GFP-tagged portion of DE-cad∷GFP detected; Oda and Tsukita, 1999 ). (I) Protein counts are shown. (J) Single-plane images are shown with the same coverslips and settings. Late cellularization is shown. RFIs were measured at the 10 brightest SAJs and equal IM areas. Normalized means ± SD are shown for five embryos each. WB, Western blot.

    Techniques Used: Purification, SDS Page, Enzyme-linked Immunosorbent Assay, Western Blot

    2) Product Images from "Macrophage migration inhibitory factor mediates peripheral nerve injury-induced hypersensitivity by curbing dopaminergic descending inhibition"

    Article Title: Macrophage migration inhibitory factor mediates peripheral nerve injury-induced hypersensitivity by curbing dopaminergic descending inhibition

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2017.271

    Effects of ISO-1 on migration inhibitory factor (MIF) and monoamine content in L-SC and VTA following chronic constriction injury (CCI). The MIF inhibitor ISO-1 was administered i.t. or i.c.v. for 2 weeks from day 7 post-nerve injury. In L-SC and VTA, the MIF and monoamine content was detected with ELISA and HPLC, respectively, at post-injury day 21. The turnover effect of i.t. ISO-1 for MIF and dopamine in L-SC is shown in a and b (* P
    Figure Legend Snippet: Effects of ISO-1 on migration inhibitory factor (MIF) and monoamine content in L-SC and VTA following chronic constriction injury (CCI). The MIF inhibitor ISO-1 was administered i.t. or i.c.v. for 2 weeks from day 7 post-nerve injury. In L-SC and VTA, the MIF and monoamine content was detected with ELISA and HPLC, respectively, at post-injury day 21. The turnover effect of i.t. ISO-1 for MIF and dopamine in L-SC is shown in a and b (* P

    Techniques Used: Migration, Enzyme-linked Immunosorbent Assay, High Performance Liquid Chromatography

    Migration inhibitory factor (MIF) and dopamine content in L-SC and VTA in CCI mice over the 70-day observation period. MIF and dopamine content in microdialysate obtained from L-SC and VTA were detected with ELISA and HPLC, respectively, on days 0, 1, 2, 4, 7, 14, 21, 35, 49 and 70 post CCI. Time-dependent MIF content upregulation in L-SC and VTA, as well as inversed dopamine content downregulation, are shown in a and b (* P
    Figure Legend Snippet: Migration inhibitory factor (MIF) and dopamine content in L-SC and VTA in CCI mice over the 70-day observation period. MIF and dopamine content in microdialysate obtained from L-SC and VTA were detected with ELISA and HPLC, respectively, on days 0, 1, 2, 4, 7, 14, 21, 35, 49 and 70 post CCI. Time-dependent MIF content upregulation in L-SC and VTA, as well as inversed dopamine content downregulation, are shown in a and b (* P

    Techniques Used: Migration, Mouse Assay, Enzyme-linked Immunosorbent Assay, High Performance Liquid Chromatography

    3) Product Images from "Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL"

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073375

    Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.
    Figure Legend Snippet: Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.

    Techniques Used: Derivative Assay, Construct, Cotransfection, FLAG-tag, Transfection, Plasmid Preparation, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Incubation, MTT Assay, Viability Assay

    Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).
    Figure Legend Snippet: Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Produced, Plasmid Preparation, Immunoprecipitation, SDS Page, Transfection, Incubation, Recombinant

    Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.
    Figure Legend Snippet: Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.

    Techniques Used: Construct, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Produced, SDS Page

    Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.
    Figure Legend Snippet: Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.

    Techniques Used: Expressing, Staining, Transfection, Plasmid Preparation, MTT Assay, Viability Assay, Incubation, Produced, Enzyme-linked Immunosorbent Assay, Blocking Assay

    Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.
    Figure Legend Snippet: Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.

    Techniques Used: Activity Assay, Derivative Assay, Cotransfection, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Construct, MTT Assay, Transfection

    4) Product Images from "In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA"

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211035

    Least squares linear regression analysis between OD values obtained in indirect ELISA testing sera from non-infected cattle in response to rUS9-FhLAP (rUS9-FhLAP response; X-axis) and LPS from E . coli serotype O55:B5 (LPS response; Y-axis). Each point represents the paired OD values obtained for each serum. Blue lines represent the 95% upper and lower confidence levels.
    Figure Legend Snippet: Least squares linear regression analysis between OD values obtained in indirect ELISA testing sera from non-infected cattle in response to rUS9-FhLAP (rUS9-FhLAP response; X-axis) and LPS from E . coli serotype O55:B5 (LPS response; Y-axis). Each point represents the paired OD values obtained for each serum. Blue lines represent the 95% upper and lower confidence levels.

    Techniques Used: Indirect ELISA, Infection

    Analysis of soluble E . coli proteins binding to Nickel Affinity Gel columns. (A) SDS-PAGE analysis of M15 E . coli soluble proteins present in the starting sample (lane 1), in the non-retained fraction (lane 2), and in the eluted fraction (lane 3, concentrated). The relative mobility of MW standards is shown on the left. (B) Least squares linear regression analysis comparing OD values obtained in indirect ELISA for sera from non-infected cattle (n = 35) using as target IMAC-purified proteins from non-transformed (M15 response; X-axis) or transformed (rUS9-FhLAP response; Y-axis) M15 E . coli cells. Each point represents the paired OD values obtained for each serum in the indirect ELISAs. Blue lines represent the 95% upper and lower confidence levels.
    Figure Legend Snippet: Analysis of soluble E . coli proteins binding to Nickel Affinity Gel columns. (A) SDS-PAGE analysis of M15 E . coli soluble proteins present in the starting sample (lane 1), in the non-retained fraction (lane 2), and in the eluted fraction (lane 3, concentrated). The relative mobility of MW standards is shown on the left. (B) Least squares linear regression analysis comparing OD values obtained in indirect ELISA for sera from non-infected cattle (n = 35) using as target IMAC-purified proteins from non-transformed (M15 response; X-axis) or transformed (rUS9-FhLAP response; Y-axis) M15 E . coli cells. Each point represents the paired OD values obtained for each serum in the indirect ELISAs. Blue lines represent the 95% upper and lower confidence levels.

    Techniques Used: Binding Assay, SDS Page, Indirect ELISA, Infection, Purification, Transformation Assay

    ELISA OD values obtained testing sera from Fasciola –free (open circles) and Fasciola –infected (closed circles) cattle. Each point in the figure represents the mean OD value (two replicates) obtained for one individual serum sample. IND = sera tested in indirect ELISA with rUS9-FhLAP antigen as target. US9 = sera tested in capture ELISA with mAb US9 coupled to ELISA plates and rUS9-FhLAP as target antigen. MM3 = sera tested in capture ELISA with mAb MM3 (MM3-ELISA; reference test) and Fasciola ESAs as target antigen. Horizontal lines represent the cut-off values (red dashed line: cut-off 1; blue line: cut-off 2). Cut-off 1 and cut-off 2 values for each ELISA were as follows: OD = 1.174 and 0.963 (IND); OD = 0.111 and 0.084 (US9); OD = 0.075 and 0.059 (MM3).
    Figure Legend Snippet: ELISA OD values obtained testing sera from Fasciola –free (open circles) and Fasciola –infected (closed circles) cattle. Each point in the figure represents the mean OD value (two replicates) obtained for one individual serum sample. IND = sera tested in indirect ELISA with rUS9-FhLAP antigen as target. US9 = sera tested in capture ELISA with mAb US9 coupled to ELISA plates and rUS9-FhLAP as target antigen. MM3 = sera tested in capture ELISA with mAb MM3 (MM3-ELISA; reference test) and Fasciola ESAs as target antigen. Horizontal lines represent the cut-off values (red dashed line: cut-off 1; blue line: cut-off 2). Cut-off 1 and cut-off 2 values for each ELISA were as follows: OD = 1.174 and 0.963 (IND); OD = 0.111 and 0.084 (US9); OD = 0.075 and 0.059 (MM3).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection, Indirect ELISA

    Least squares linear regression analysis comparing non-inhibited and inhibited (MTFSVPIS peptide) sera from non-infected cattle (A) or sera from F . hepatica naturally-infected cattle (B). Each point represents the paired OD values obtained for each serum in indirect ELISA using rUS9-FhLAP as target antigen. Blue lines represent the 95% upper and lower confidence levels.
    Figure Legend Snippet: Least squares linear regression analysis comparing non-inhibited and inhibited (MTFSVPIS peptide) sera from non-infected cattle (A) or sera from F . hepatica naturally-infected cattle (B). Each point represents the paired OD values obtained for each serum in indirect ELISA using rUS9-FhLAP as target antigen. Blue lines represent the 95% upper and lower confidence levels.

    Techniques Used: Infection, Indirect ELISA

    ELISA OD values obtained testing sera from Fasciola –free (open circles) and Fasciola –infected (closed circles) sheep. Each point in the figure represents the mean OD value (two replicates) obtained for one individual serum sample. IND = sera tested in indirect ELISA with rUS9-FhLAP antigen as target. US9 = sera tested in capture ELISA with mAb US9 coupled to ELISA plates and rUS9-FhLAP as target antigen. MM3 = sera tested in capture ELISA with mAb MM3 (MM3-ELISA; reference test). Horizontal lines represent the cut-off values (red dashed line: cut-off 1; blue line: cut-off 2). Cut-off 1 and cut-off 2 values for each ELISA were as follows: OD = 0.205 and 0.16 (IND); OD = 0.114 and 0.089 (US9); OD = 0.009 and 0.007 (MM3).
    Figure Legend Snippet: ELISA OD values obtained testing sera from Fasciola –free (open circles) and Fasciola –infected (closed circles) sheep. Each point in the figure represents the mean OD value (two replicates) obtained for one individual serum sample. IND = sera tested in indirect ELISA with rUS9-FhLAP antigen as target. US9 = sera tested in capture ELISA with mAb US9 coupled to ELISA plates and rUS9-FhLAP as target antigen. MM3 = sera tested in capture ELISA with mAb MM3 (MM3-ELISA; reference test). Horizontal lines represent the cut-off values (red dashed line: cut-off 1; blue line: cut-off 2). Cut-off 1 and cut-off 2 values for each ELISA were as follows: OD = 0.205 and 0.16 (IND); OD = 0.114 and 0.089 (US9); OD = 0.009 and 0.007 (MM3).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection, Indirect ELISA

    Least squares linear regression analysis obtained comparing non-infected (A) and Fasciola -infected (B) cattle sera OD values obtained in indirect ELISA and US9-ELISA. Each point represents the paired OD values obtained for each serum by both methods. Blue lines represent the 95% upper and lower confidence levels.
    Figure Legend Snippet: Least squares linear regression analysis obtained comparing non-infected (A) and Fasciola -infected (B) cattle sera OD values obtained in indirect ELISA and US9-ELISA. Each point represents the paired OD values obtained for each serum by both methods. Blue lines represent the 95% upper and lower confidence levels.

    Techniques Used: Infection, Indirect ELISA, Enzyme-linked Immunosorbent Assay

    Analysis of the expression of the chimeric recombinant protein rUS9-FhLAP in E . coli and optimization of its use in ELISA. (A) SDS-PAGE and WB analysis of rUS9-FhLAP expression. Lane 1: MW markers. Lane 2: Coomasie blue staining of the soluble protein fraction obtained after induction with 0.5 mM IPTG. Lane 3: Coomasie blue staining of rUS9-FhLAP after purification by IMAC. Lane 4: WB analysis of rUS9-FhLAP revealed with mAb US9 as primary antibody. Lane 5: WB analysis of rUS9-FhLAP revealed with HRP-conjugated anti-polyhistidine. (B) ELISA OD values obtained testing different concentrations of rUS9-FhLAP (range: 0.7–22 μg/mL) as target antigen in capture ELISA with mAb US9. (C) ELISA OD values obtained testing different concentrations of rUS9-FhLAP (range: 0.3–11 μg/mL) as target antigen in indirect ELISA. The analysis was carried out with a serum from a Fasciola -infected sheep as primary antibody.
    Figure Legend Snippet: Analysis of the expression of the chimeric recombinant protein rUS9-FhLAP in E . coli and optimization of its use in ELISA. (A) SDS-PAGE and WB analysis of rUS9-FhLAP expression. Lane 1: MW markers. Lane 2: Coomasie blue staining of the soluble protein fraction obtained after induction with 0.5 mM IPTG. Lane 3: Coomasie blue staining of rUS9-FhLAP after purification by IMAC. Lane 4: WB analysis of rUS9-FhLAP revealed with mAb US9 as primary antibody. Lane 5: WB analysis of rUS9-FhLAP revealed with HRP-conjugated anti-polyhistidine. (B) ELISA OD values obtained testing different concentrations of rUS9-FhLAP (range: 0.7–22 μg/mL) as target antigen in capture ELISA with mAb US9. (C) ELISA OD values obtained testing different concentrations of rUS9-FhLAP (range: 0.3–11 μg/mL) as target antigen in indirect ELISA. The analysis was carried out with a serum from a Fasciola -infected sheep as primary antibody.

    Techniques Used: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Staining, Purification, Indirect ELISA, Infection

    5) Product Images from "Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity"

    Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp201

    The Pol β polymorphism R137Q is defective in polymerase activity. ( A ) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP- 32 P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. ( B ) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. ( C ) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32 P as shown in figure. ( D ) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.
    Figure Legend Snippet: The Pol β polymorphism R137Q is defective in polymerase activity. ( A ) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP- 32 P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. ( B ) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. ( C ) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32 P as shown in figure. ( D ) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.

    Techniques Used: Activity Assay, Labeling, Incubation, Purification, Nucleic Acid Electrophoresis, Avidin-Biotin Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Adsorption

    6) Product Images from "Significant role for IRF3 in both T cell and APC effector functions during T cell responses"

    Article Title: Significant role for IRF3 in both T cell and APC effector functions during T cell responses

    Journal: Cellular immunology

    doi: 10.1016/j.cellimm.2016.08.015

    IRF3 deficiency reduces T cell cytokine and Granzyme B expression during T cell responses with APCs. Primary splenic mononuclear cells (2.5 × 10 6 ) from C57Bl/6 and IRF3KO mice were stimulated with 1 µg/ml anti-CD3 with or without 1 µg/ml anti-CD28 with or without 25 µg/ml poly I:C for 24 or 48 h. Forty eight h supernatant levels of IFN-γ ( A ) and IL-17 ( B ) were assayed by ELISA, mRNA levels for GrB ( C ) and Foxp3 (D) were determined by qRT-PCR. Data are means ± SE, * indicates p
    Figure Legend Snippet: IRF3 deficiency reduces T cell cytokine and Granzyme B expression during T cell responses with APCs. Primary splenic mononuclear cells (2.5 × 10 6 ) from C57Bl/6 and IRF3KO mice were stimulated with 1 µg/ml anti-CD3 with or without 1 µg/ml anti-CD28 with or without 25 µg/ml poly I:C for 24 or 48 h. Forty eight h supernatant levels of IFN-γ ( A ) and IL-17 ( B ) were assayed by ELISA, mRNA levels for GrB ( C ) and Foxp3 (D) were determined by qRT-PCR. Data are means ± SE, * indicates p

    Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    7) Product Images from "IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity"

    Article Title: IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151722

    IFN-γR signaling is required for Spt-GC formation and IgG production. (A and B) The percentages of B220 + Fas hi PNA hi GC B cells (A) and CD4 + CXCR5 hi PD-1 hi Tfh cells (B) were obtained from flow cytometric analysis of spleen cells of 3- and 6-mo-old B6 and B6.IFN-γR 1 −/− mice. Each symbol represents a mouse ( n = 11–15). (C) Representative histological images of spleen sections from 6-mo-old mice ( n = 5 per group) stained with the GC B cell marker PNA and anti-IgD. Bars, 150 µm. (D) Flow cytometric analysis of CD86 expression (MFI) on total B220 + B cells at 3 and 6 mo of age ( n = 5 mice per group). Error bars are mean ± SD. (E and F) Numbers of IgG + (E) and IgM + (F) splenic AFCs in 6-mo-old mice of indicated strains ( n = 5–9). (G–K) Analysis of serum titers of IgG, IgG 1 , IgG 2b, IgG 2c , and IgM Abs in 6-mo-old mice by ELISA. Each symbol represents a mouse ( n = 6–8). The data shown are the cumulative results of two or three independent experiments. Statistical values were determined using an unpaired, nonparametric, Mann–Whitney Student’s t test. Horizontal lines indicate mean values. *, P
    Figure Legend Snippet: IFN-γR signaling is required for Spt-GC formation and IgG production. (A and B) The percentages of B220 + Fas hi PNA hi GC B cells (A) and CD4 + CXCR5 hi PD-1 hi Tfh cells (B) were obtained from flow cytometric analysis of spleen cells of 3- and 6-mo-old B6 and B6.IFN-γR 1 −/− mice. Each symbol represents a mouse ( n = 11–15). (C) Representative histological images of spleen sections from 6-mo-old mice ( n = 5 per group) stained with the GC B cell marker PNA and anti-IgD. Bars, 150 µm. (D) Flow cytometric analysis of CD86 expression (MFI) on total B220 + B cells at 3 and 6 mo of age ( n = 5 mice per group). Error bars are mean ± SD. (E and F) Numbers of IgG + (E) and IgM + (F) splenic AFCs in 6-mo-old mice of indicated strains ( n = 5–9). (G–K) Analysis of serum titers of IgG, IgG 1 , IgG 2b, IgG 2c , and IgM Abs in 6-mo-old mice by ELISA. Each symbol represents a mouse ( n = 6–8). The data shown are the cumulative results of two or three independent experiments. Statistical values were determined using an unpaired, nonparametric, Mann–Whitney Student’s t test. Horizontal lines indicate mean values. *, P

    Techniques Used: Single-particle Tracking, Flow Cytometry, Mouse Assay, Staining, Marker, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Critical role of T-bet in Spt-GC development and Th1 Ab responses. (A and B) Percentages of B220 + Fas hi PNA hi GC B cells (A) and CD4 + CXCR5 hi PD-1 hi Tfh cells (B) obtained from flow cytometric analysis of spleen cells of 3-mo-old B6 and B6. T-bet −/− mice. Each symbol represents a mouse ( n = 3–4). (C) Representative images of spleen sections from 3-mo-old B6 and B6. T-bet −/− mice stained with GL7, anti-CD4, and anti-IgD. GC area measurement is shown in the right panel ( n = 3–4 mice per group). Bars, 150 µm. (D–F) Total serum Ab titers of IgG (D), IgG 2b (E), and IgG 2c (F) measured by ELISA ( n = 3–4 per group). (G) Maintenance of ex vivo–generated GC B cell phenotype of B6 and B6. T-bet −/− mice in media alone or with IFN-γ. (H) Quantitative RT-PCR analysis of IFN-γ transcripts in ex vivo–generated GC B cells (as described in G) from B6 and B6. T-bet −/− mice ( n = 3–4 mice per group). The data shown in G and H are representative of three independent experiments. In D–F, statistical analysis was performed by one-way ANOVA with a follow-up Tukey multiple-comparison test. In A–C, G, and H, statistical values were determined using an unpaired, nonparametric, Mann–Whitney Student’s t test. Error bars are mean ± SD. *, P
    Figure Legend Snippet: Critical role of T-bet in Spt-GC development and Th1 Ab responses. (A and B) Percentages of B220 + Fas hi PNA hi GC B cells (A) and CD4 + CXCR5 hi PD-1 hi Tfh cells (B) obtained from flow cytometric analysis of spleen cells of 3-mo-old B6 and B6. T-bet −/− mice. Each symbol represents a mouse ( n = 3–4). (C) Representative images of spleen sections from 3-mo-old B6 and B6. T-bet −/− mice stained with GL7, anti-CD4, and anti-IgD. GC area measurement is shown in the right panel ( n = 3–4 mice per group). Bars, 150 µm. (D–F) Total serum Ab titers of IgG (D), IgG 2b (E), and IgG 2c (F) measured by ELISA ( n = 3–4 per group). (G) Maintenance of ex vivo–generated GC B cell phenotype of B6 and B6. T-bet −/− mice in media alone or with IFN-γ. (H) Quantitative RT-PCR analysis of IFN-γ transcripts in ex vivo–generated GC B cells (as described in G) from B6 and B6. T-bet −/− mice ( n = 3–4 mice per group). The data shown in G and H are representative of three independent experiments. In D–F, statistical analysis was performed by one-way ANOVA with a follow-up Tukey multiple-comparison test. In A–C, G, and H, statistical values were determined using an unpaired, nonparametric, Mann–Whitney Student’s t test. Error bars are mean ± SD. *, P

    Techniques Used: Single-particle Tracking, Flow Cytometry, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Ex Vivo, Generated, Quantitative RT-PCR, MANN-WHITNEY

    8) Product Images from "Takinib, a selective TAK1 inhibitor, broadens the therapeutic efficacy of TNFα inhibition for cancer and autoimmune disease"

    Article Title: Takinib, a selective TAK1 inhibitor, broadens the therapeutic efficacy of TNFα inhibition for cancer and autoimmune disease

    Journal: Cell chemical biology

    doi: 10.1016/j.chembiol.2017.07.011

    Takinib induces apoptosis in RA FLS and reduces IL-6 secretion. A RA FLS cells in the presence and absence of TNF were treated with titrations of Takinib and 5ZO for 48h. Caspase 3/7 activity was measured using the fluorogenic substrate (DEVD) 2 -R110. Data are expressed as RFU and normalized to yield % caspase induction (n=2, mean ± SEM). B Caspase induction after treatment with TAKINIB, 219, 220, 5ZO or tofacitinib for 48h in the presence of TNF (n=6, mean ± SEM). C RA FLS were treated with indicated compounds and IL-6 secretion was measured using human IL-6 ELISA (n=5, mean ± SEM). D Proposed mechanism of Takinib induction of apoptosis through inhibition of complex 1 signaling.
    Figure Legend Snippet: Takinib induces apoptosis in RA FLS and reduces IL-6 secretion. A RA FLS cells in the presence and absence of TNF were treated with titrations of Takinib and 5ZO for 48h. Caspase 3/7 activity was measured using the fluorogenic substrate (DEVD) 2 -R110. Data are expressed as RFU and normalized to yield % caspase induction (n=2, mean ± SEM). B Caspase induction after treatment with TAKINIB, 219, 220, 5ZO or tofacitinib for 48h in the presence of TNF (n=6, mean ± SEM). C RA FLS were treated with indicated compounds and IL-6 secretion was measured using human IL-6 ELISA (n=5, mean ± SEM). D Proposed mechanism of Takinib induction of apoptosis through inhibition of complex 1 signaling.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Inhibition

    9) Product Images from "Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice against Cryptococcus neoformans Infection ▿"

    Article Title: Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice against Cryptococcus neoformans Infection ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01161-06

    Relative affinities of recombinant human IgG subclasses for target antigen. OD, optical density. (A) Direct antigen binding by recombinant antibodies. Serially diluted recombinant IgGs were allowed to bind to GXM immobilized on ELISA plates. The EC 50 (ng/well) of each isotype is indicated. (B) Competitive binding of recombinant IgG to GXM in the presence of a high-affinity peptide mimetic of GXM (P206). A fixed concentration (0.5 μg/ml) of each IgG was incubated for 1 h with various concentrations of P206 and then added to ELISA plates coated with GXM. The concentration of peptide (μg/ml) that inhibited 50% of binding (IC 50 ) is indicated.
    Figure Legend Snippet: Relative affinities of recombinant human IgG subclasses for target antigen. OD, optical density. (A) Direct antigen binding by recombinant antibodies. Serially diluted recombinant IgGs were allowed to bind to GXM immobilized on ELISA plates. The EC 50 (ng/well) of each isotype is indicated. (B) Competitive binding of recombinant IgG to GXM in the presence of a high-affinity peptide mimetic of GXM (P206). A fixed concentration (0.5 μg/ml) of each IgG was incubated for 1 h with various concentrations of P206 and then added to ELISA plates coated with GXM. The concentration of peptide (μg/ml) that inhibited 50% of binding (IC 50 ) is indicated.

    Techniques Used: Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation

    10) Product Images from "In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120"

    Article Title: In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

    Journal: Virology

    doi: 10.1016/j.virol.2007.10.044

    Reactivity of mAbs to V3, C1, and C5 with gp120/mAb complexes. (A) gp120 or gp120/mAb complexes were captured on the ELISA plates by polyclonal anti-C5 Abs and reacted with biotinylated mAbs specific for V3 (694/98D and 447/52D) or C1 (EH21). Alternatively, gp120 or the complexes were directly coated onto ELISA plates and reacted with biotinylated mAbs specific for C5 (1331A). The gp120/mAb complexes were prepared with 1 μg/ml of recombinant gp120 LAI and 2 μg/ml of mAbs to the CD4bs (654D) or C2 (1006-30D) and serially diluted by 2 fold. Gp120 mixed with a control mAb (1418) was also included as a control. The x-axis shows the gp120 concentrations in each of the gp120/mAb mixtures. Relative binding of the biotinylated mAbs was determined using alkaline phosphatase-conjugated streptavidin. Means and standard deviations were calculated from duplicate wells. Data from one of five repeated experiments are shown. *, p
    Figure Legend Snippet: Reactivity of mAbs to V3, C1, and C5 with gp120/mAb complexes. (A) gp120 or gp120/mAb complexes were captured on the ELISA plates by polyclonal anti-C5 Abs and reacted with biotinylated mAbs specific for V3 (694/98D and 447/52D) or C1 (EH21). Alternatively, gp120 or the complexes were directly coated onto ELISA plates and reacted with biotinylated mAbs specific for C5 (1331A). The gp120/mAb complexes were prepared with 1 μg/ml of recombinant gp120 LAI and 2 μg/ml of mAbs to the CD4bs (654D) or C2 (1006-30D) and serially diluted by 2 fold. Gp120 mixed with a control mAb (1418) was also included as a control. The x-axis shows the gp120 concentrations in each of the gp120/mAb mixtures. Relative binding of the biotinylated mAbs was determined using alkaline phosphatase-conjugated streptavidin. Means and standard deviations were calculated from duplicate wells. Data from one of five repeated experiments are shown. *, p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay

    Reactivity of serum Abs from mice immunized with rgp120 or gp120-mAb complexes with V3, C1, C5, and gp120 core. Sera collected from the final bleed were tested by ELISA using peptides representing the V3, C1 and C5 regions of gp120 and against core gp120 protein lacking V1/V2, V3, and the N and C termini. Peptides or core gp120 were coated on the ELISA plates and reacted with serially diluted sera. Mouse Ab binding was assessed by alkaline-phosphatase-conjugated secondary antibody to mouse IgG. Note the differences in the scales of y-axis for the right and left graphs.
    Figure Legend Snippet: Reactivity of serum Abs from mice immunized with rgp120 or gp120-mAb complexes with V3, C1, C5, and gp120 core. Sera collected from the final bleed were tested by ELISA using peptides representing the V3, C1 and C5 regions of gp120 and against core gp120 protein lacking V1/V2, V3, and the N and C termini. Peptides or core gp120 were coated on the ELISA plates and reacted with serially diluted sera. Mouse Ab binding was assessed by alkaline-phosphatase-conjugated secondary antibody to mouse IgG. Note the differences in the scales of y-axis for the right and left graphs.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

    Ig classes induced in mice immunized with gp120 in the presence or absence of mAbs. Mice were immunized with gp120 alone, gp120 complexed with different anti-gp120 mAbs, or PBS. For comparison, mice were also immunized with gp120 combined with anti-parvovirus mAbs (1418 or 860-55D). The results obtained with sera from two different immunization experiments are shown in A and B. (A) Sera were collected a week after the final boost, serially diluted, and tested for titers of gp120-specific IgG and IgA. (B) For detection of gp120-specific IgG1, IgG2a, and IgG2b, sera collected one week after each immunization were used and tested at a dilution of 1:640. Gp120 LAI was captured on ELISA plates coated with sheep anti-C5 antibody and reacted with diluted mouse sera. Rabbit antibodies against different mouse Ig subtypes and subclasses and alkaline-phosphatase-conjugated anti-rabbit IgG were used for detection along with alkaline phosphatase substrates p-nitrophenyl phosphate (OD 405 ) or PhosphaGlow chemiluminescent. Means and standard deviations were derived from duplicate wells.
    Figure Legend Snippet: Ig classes induced in mice immunized with gp120 in the presence or absence of mAbs. Mice were immunized with gp120 alone, gp120 complexed with different anti-gp120 mAbs, or PBS. For comparison, mice were also immunized with gp120 combined with anti-parvovirus mAbs (1418 or 860-55D). The results obtained with sera from two different immunization experiments are shown in A and B. (A) Sera were collected a week after the final boost, serially diluted, and tested for titers of gp120-specific IgG and IgA. (B) For detection of gp120-specific IgG1, IgG2a, and IgG2b, sera collected one week after each immunization were used and tested at a dilution of 1:640. Gp120 LAI was captured on ELISA plates coated with sheep anti-C5 antibody and reacted with diluted mouse sera. Rabbit antibodies against different mouse Ig subtypes and subclasses and alkaline-phosphatase-conjugated anti-rabbit IgG were used for detection along with alkaline phosphatase substrates p-nitrophenyl phosphate (OD 405 ) or PhosphaGlow chemiluminescent. Means and standard deviations were derived from duplicate wells.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay

    Detection of Abs to the CD4-binding site of gp120 in sera of mice immunized with the gp120/Ab complexes. Serum Abs to the CD4bs was measured by ELISA by comparing serum reactivity to gp120 in the presence or absence of sCD4. Sera were collected after the last immunization from mice immunized with gp120 combined with mAbs to CD4bs (654-D and 559/64D), mAb to V3 (694/98D), or control mAb (1418), or no antigen (PBS). These sera were serially diluted and reacted with gp120 captured on the wells in the presence or absence of sCD4 (1 μg/ml). Means and standard deviations from duplicate wells are shown. There is no statistical significant difference between Ab reactivities in the presence versus absence of sCD4.
    Figure Legend Snippet: Detection of Abs to the CD4-binding site of gp120 in sera of mice immunized with the gp120/Ab complexes. Serum Abs to the CD4bs was measured by ELISA by comparing serum reactivity to gp120 in the presence or absence of sCD4. Sera were collected after the last immunization from mice immunized with gp120 combined with mAbs to CD4bs (654-D and 559/64D), mAb to V3 (694/98D), or control mAb (1418), or no antigen (PBS). These sera were serially diluted and reacted with gp120 captured on the wells in the presence or absence of sCD4 (1 μg/ml). Means and standard deviations from duplicate wells are shown. There is no statistical significant difference between Ab reactivities in the presence versus absence of sCD4.

    Techniques Used: Binding Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Genetic and pharmacological validation of TAK1 inhibition in macrophages as a therapeutic strategy to effectively inhibit TNF secretion"

    Article Title: Genetic and pharmacological validation of TAK1 inhibition in macrophages as a therapeutic strategy to effectively inhibit TNF secretion

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-35189-7

    Takinib reduces TNF in a murine LPS challenge. FVB mice treated i.p. with varying concentrations of Takinib, once (qw) or twice weekly (qbw), caused no significant adverse events or weight change over 8 weeks of chronic treatment ( a ). Bone marrow derived macrophages (BMDM’s) were plated at (2 × 10 6 /well) in a 24-well plate and treated with M-CSF (20 ng/mL) for 72 hours followed by a 24 hour rest period prior to activation with LPS (10 ng/mL) and IFNγ (50 ng/mL) and treatment with Takinib, at the indicated concentrations, or vehicle for 24 hours. Cytokine production was sampled from cell supernatant and examined by ELISA ( b ) (One-way ANOVA with Dunnett’s, n = 3± SEM). C57/bl6 male and female mice were sacrificed prior to LPS challenge and baseline TNF and IL-6 serum concentrations were examined by ELISA ( c,d ) (Student t-test, n = 3± SEM, 3 males, 3 females). C57/bl6 male and female mice were injected intraperitoneally (i.p.) with LPS (100 μg/kg in PBS) followed immediately by vehicle or Takinib (50 mg/kg in DMSO). 1 hour later, mice were sacrificed and blood samples clotted in centrifuge for 10 minutes at 3,000 rpm. Concentration of serum cytokines TNF and IL-6 determined by ELISA ( c,d ) (Student t-test, n = 5± SEM).
    Figure Legend Snippet: Takinib reduces TNF in a murine LPS challenge. FVB mice treated i.p. with varying concentrations of Takinib, once (qw) or twice weekly (qbw), caused no significant adverse events or weight change over 8 weeks of chronic treatment ( a ). Bone marrow derived macrophages (BMDM’s) were plated at (2 × 10 6 /well) in a 24-well plate and treated with M-CSF (20 ng/mL) for 72 hours followed by a 24 hour rest period prior to activation with LPS (10 ng/mL) and IFNγ (50 ng/mL) and treatment with Takinib, at the indicated concentrations, or vehicle for 24 hours. Cytokine production was sampled from cell supernatant and examined by ELISA ( b ) (One-way ANOVA with Dunnett’s, n = 3± SEM). C57/bl6 male and female mice were sacrificed prior to LPS challenge and baseline TNF and IL-6 serum concentrations were examined by ELISA ( c,d ) (Student t-test, n = 3± SEM, 3 males, 3 females). C57/bl6 male and female mice were injected intraperitoneally (i.p.) with LPS (100 μg/kg in PBS) followed immediately by vehicle or Takinib (50 mg/kg in DMSO). 1 hour later, mice were sacrificed and blood samples clotted in centrifuge for 10 minutes at 3,000 rpm. Concentration of serum cytokines TNF and IL-6 determined by ELISA ( c,d ) (Student t-test, n = 5± SEM).

    Techniques Used: Mouse Assay, Derivative Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Injection, Concentration Assay

    12) Product Images from "Assessment of ELISA as Endpoint in Neuronal Cell-based Assay for BoNT Detection using hiPSC derived neurons"

    Article Title: Assessment of ELISA as Endpoint in Neuronal Cell-based Assay for BoNT Detection using hiPSC derived neurons

    Journal: Journal of pharmacological and toxicological methods

    doi: 10.1016/j.vascn.2017.04.013

    Plate selection for ELISA. HIP Neurons were exposed to serial dilutions of BoNT/A1, and cell lysates were analyzed for cleaved SNAP-25 by ELISA using either a Nunc Polystyrene Maxisorp plate (Nunc) or a Thermo Scientific Immulon Immunoassay plate (Immulon). The signal was detected with ultra TMB. The average and standard deviations of 4 replicates is shown. Dose response curves were fitted using Prims 6 software and a nonlinear regression (variable slope, four parameter).
    Figure Legend Snippet: Plate selection for ELISA. HIP Neurons were exposed to serial dilutions of BoNT/A1, and cell lysates were analyzed for cleaved SNAP-25 by ELISA using either a Nunc Polystyrene Maxisorp plate (Nunc) or a Thermo Scientific Immulon Immunoassay plate (Immulon). The signal was detected with ultra TMB. The average and standard deviations of 4 replicates is shown. Dose response curves were fitted using Prims 6 software and a nonlinear regression (variable slope, four parameter).

    Techniques Used: Selection, Enzyme-linked Immunosorbent Assay, Software

    13) Product Images from "Anti‐β2‐glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression"

    Article Title: Anti‐β2‐glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.13229

    Dual‐reactivity of WB‐6 to cardiolipin‐β 2 ‐glycoprotein I (CL‐β 2 GPI) and DNA. (a) Direct‐binding enzyme‐linked immunosorbent assay (ELISA) to test the binding activity of WB‐6 to β 2 GPI, CL and CL‐β 2 GPI. (b) Direct‐binding ELISA to show the binding activity of WB‐6 and 2C10 to calf‐thymus (CT) native DNA. Mouse immunoglobulin (Ig)G2b, kappa monoclonal MPC‐11 is an isotype‐matched control without DNA‐binding activity. (c) Inhibition ELISA in which binding activity of WB‐6 to calf thymus (CT)‐DNA was inhibited by preincubation with poly(dT), poly(dA‐dT) or CT‐DNA.
    Figure Legend Snippet: Dual‐reactivity of WB‐6 to cardiolipin‐β 2 ‐glycoprotein I (CL‐β 2 GPI) and DNA. (a) Direct‐binding enzyme‐linked immunosorbent assay (ELISA) to test the binding activity of WB‐6 to β 2 GPI, CL and CL‐β 2 GPI. (b) Direct‐binding ELISA to show the binding activity of WB‐6 and 2C10 to calf‐thymus (CT) native DNA. Mouse immunoglobulin (Ig)G2b, kappa monoclonal MPC‐11 is an isotype‐matched control without DNA‐binding activity. (c) Inhibition ELISA in which binding activity of WB‐6 to calf thymus (CT)‐DNA was inhibited by preincubation with poly(dT), poly(dA‐dT) or CT‐DNA.

    Techniques Used: Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Inhibition

    14) Product Images from "B-Cell-Intrinsic Type 1 Interferon Signaling Is Crucial for Loss of Tolerance and the Development of Autoreactive B Cells"

    Article Title: B-Cell-Intrinsic Type 1 Interferon Signaling Is Crucial for Loss of Tolerance and the Development of Autoreactive B Cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.06.046

    IFNαR 1 Is Required for Dysregulated Spt-AFC and Spt-GC Formation in B6. Sle1b Mice (A) Flow cytometric analysis of surface expression of IFNαR 1 on B220 + Fas low GL-7 low non-GC B cells (red), B220 + Fas h GL-7 hi GC B cells (blue), CD4 + PD-1 low CXCR5 low non-Tfh (orange), and CD4 + PD-1 hi CXCR5 hi Tfh (green) in female B6 and B6. Sle1b mice. Error bars represent mean ± SEM. (B and C) The percentages of GC B cells (B) and Tfh cells (C) in splenocytes from 3- and 6-month-old female mice. (D) Spleen sections from 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice (5 mice per group) were stained with anti-CD4 Ab (red) GL-7 (green) and anti-IgD Ab (blue), and GC areas were measured for 10 GCs per spleen section (right panels). The scale bars represent 50 μm. (E and F) Each symbol represents a measured GC. IgM + (E) and IgG + (F) AFCs in the spleens of 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice are shown. (G-J) Analysis of serum titers of IgM (G), IgG (H), IgG1 (I), and IgG2c (J) antibodies in 6-month-old mice by ELISA. Each symbol represents a mouse, and these data represent at least two-three independent experiments. Statistical significance was determined by one-way ANOVA with a follow-up Tukey multiple-comparison test (NS, not significant, *p
    Figure Legend Snippet: IFNαR 1 Is Required for Dysregulated Spt-AFC and Spt-GC Formation in B6. Sle1b Mice (A) Flow cytometric analysis of surface expression of IFNαR 1 on B220 + Fas low GL-7 low non-GC B cells (red), B220 + Fas h GL-7 hi GC B cells (blue), CD4 + PD-1 low CXCR5 low non-Tfh (orange), and CD4 + PD-1 hi CXCR5 hi Tfh (green) in female B6 and B6. Sle1b mice. Error bars represent mean ± SEM. (B and C) The percentages of GC B cells (B) and Tfh cells (C) in splenocytes from 3- and 6-month-old female mice. (D) Spleen sections from 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice (5 mice per group) were stained with anti-CD4 Ab (red) GL-7 (green) and anti-IgD Ab (blue), and GC areas were measured for 10 GCs per spleen section (right panels). The scale bars represent 50 μm. (E and F) Each symbol represents a measured GC. IgM + (E) and IgG + (F) AFCs in the spleens of 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice are shown. (G-J) Analysis of serum titers of IgM (G), IgG (H), IgG1 (I), and IgG2c (J) antibodies in 6-month-old mice by ELISA. Each symbol represents a mouse, and these data represent at least two-three independent experiments. Statistical significance was determined by one-way ANOVA with a follow-up Tukey multiple-comparison test (NS, not significant, *p

    Techniques Used: Single-particle Tracking, Mouse Assay, Flow Cytometry, Expressing, Staining, Enzyme-linked Immunosorbent Assay

    15) Product Images from "Growth Associated Protein 43 (GAP-43) as a Novel Target for the Diagnosis, Treatment and Prevention of Epileptogenesis"

    Article Title: Growth Associated Protein 43 (GAP-43) as a Novel Target for the Diagnosis, Treatment and Prevention of Epileptogenesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17377-z

    Quantified GAP-43 protein expression in the brain. ( A ) Near-infrared (NIF) western blots normalized to GAPDH show GAP-43 protein in CD and non-CD brains at each time point after PTZ induced seizures. Western blot image was cropped to remove protein ladder. ( B ) GAP-43 protein in the brain as measured by ELISA in CD rats compared to non-CD rats at each time point. ( C ) ELISA results show a higher level of GAP-43 protein in CD rats as compared to non-CD rats 120 days after PTZ induced acute seizures. Error bars represent standard error of the mean. Sample sizes: Non-CD + Saline n = 5, + PTZ Day 2 n = 4, + PTZ Day 15 n = 4, + PTZ Day 30 n = 4. CD + Saline n = 4, + PTZ Day 2 n = 4, + PTZ Day 15 n = 4, + PTZ Day 30 n = 4.
    Figure Legend Snippet: Quantified GAP-43 protein expression in the brain. ( A ) Near-infrared (NIF) western blots normalized to GAPDH show GAP-43 protein in CD and non-CD brains at each time point after PTZ induced seizures. Western blot image was cropped to remove protein ladder. ( B ) GAP-43 protein in the brain as measured by ELISA in CD rats compared to non-CD rats at each time point. ( C ) ELISA results show a higher level of GAP-43 protein in CD rats as compared to non-CD rats 120 days after PTZ induced acute seizures. Error bars represent standard error of the mean. Sample sizes: Non-CD + Saline n = 5, + PTZ Day 2 n = 4, + PTZ Day 15 n = 4, + PTZ Day 30 n = 4. CD + Saline n = 4, + PTZ Day 2 n = 4, + PTZ Day 15 n = 4, + PTZ Day 30 n = 4.

    Techniques Used: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    16) Product Images from "Characterization of Neospora caninum Surface Protein NcSRS2 Based on Baculovirus Expression System and Its Application for Serodiagnosis of Neospora Infection"

    Article Title: Characterization of Neospora caninum Surface Protein NcSRS2 Based on Baculovirus Expression System and Its Application for Serodiagnosis of Neospora Infection

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.39.11.3987-3991.2001

    Specificity of recombinant NcSRS2 against anti- N. caninum serum. ELISA using NLA or the recombinant NcSRS2 expressed by Ba/SRS2p44 or Ba/SRS2p41 was carried out using anti- N. caninum (A) or anti- T. gondii (B) mouse serum (1:250). The antigens (Ag) from the culture supernatants (sup) and the cell lysates (cell) of recombinant baculovirus-infected Sf9 cells were prepared as described in Materials and Methods.
    Figure Legend Snippet: Specificity of recombinant NcSRS2 against anti- N. caninum serum. ELISA using NLA or the recombinant NcSRS2 expressed by Ba/SRS2p44 or Ba/SRS2p41 was carried out using anti- N. caninum (A) or anti- T. gondii (B) mouse serum (1:250). The antigens (Ag) from the culture supernatants (sup) and the cell lysates (cell) of recombinant baculovirus-infected Sf9 cells were prepared as described in Materials and Methods.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Infection

    Examination of bovine sera. Minimum and maximum values (Min-Max), 25 and 75% percentiles (25%–75%), and median values of ELISA using the NLA or the recombinant NcSRS2 prepared from Ba/SRS2p44-infected cells (SRS2) are shown. Bovine sera that were N. caninum IFAT positive (P) ( n = 40) and negative (N) ( n = 40), which were randomly selected, were examined by ELISA. The cutoff values were determined by the mean value plus two standard deviations from bovine sera that were IFAT negative (recombinant NcSRS2 ELISA, optical density = 0.189; NLA ELISA, optical density = 0.371).
    Figure Legend Snippet: Examination of bovine sera. Minimum and maximum values (Min-Max), 25 and 75% percentiles (25%–75%), and median values of ELISA using the NLA or the recombinant NcSRS2 prepared from Ba/SRS2p44-infected cells (SRS2) are shown. Bovine sera that were N. caninum IFAT positive (P) ( n = 40) and negative (N) ( n = 40), which were randomly selected, were examined by ELISA. The cutoff values were determined by the mean value plus two standard deviations from bovine sera that were IFAT negative (recombinant NcSRS2 ELISA, optical density = 0.189; NLA ELISA, optical density = 0.371).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant, Infection

    17) Product Images from "The Identification and Characterization of Two Novel Epitopes on the Nucleocapsid Protein of the Porcine Epidemic Diarrhea Virus"

    Article Title: The Identification and Characterization of Two Novel Epitopes on the Nucleocapsid Protein of the Porcine Epidemic Diarrhea Virus

    Journal: Scientific Reports

    doi: 10.1038/srep39010

    Mapping of PEDV N protein epitopes. ( a ) Schematic diagram of the epitope mapping. The segments that could be recognized by both PN-D4 and PN-D6 are highlighted in red; the segments that could only be recognized by PN-D4 are highlighted in blue; the segments that could only be recognized by PN-D6 are highlighted in green; and the segments in gray are those that could not be recognized by either PN-D4 or PN-D6. According to ( a ), the N gene was divided into mutually overlapping N1 and N2 fragments and was directionally cloned into the pGEX-6p-1 vector. ( b ) Six hours after the addition of 1 mM IPTG, the induced products of GST-N, GST-N1 and GST-N2 were processed and analyzed using SDS-PAGE (12% separating gel and 5% stacking gel). ( c ) Next, the GST fusion proteins GST-N, GST-N1 and GST-N2 were coated onto ELISA plates (0.5 μg/well) and were probed with MAbs PN-D4 and PN-D6. After the first round of identification, N1 and N2 were further divided and expressed in two series of fusion proteins, GST-N1-1-GST-N1-11 and GST-N2-1-GST-N2-11 ( d , h ), which were analyzed by Western blotting and ELISA using PN-D4 ( e , g ), PN-D6 ( i , k ), and anti-GST Tag antibody ( f , j ), respectively.
    Figure Legend Snippet: Mapping of PEDV N protein epitopes. ( a ) Schematic diagram of the epitope mapping. The segments that could be recognized by both PN-D4 and PN-D6 are highlighted in red; the segments that could only be recognized by PN-D4 are highlighted in blue; the segments that could only be recognized by PN-D6 are highlighted in green; and the segments in gray are those that could not be recognized by either PN-D4 or PN-D6. According to ( a ), the N gene was divided into mutually overlapping N1 and N2 fragments and was directionally cloned into the pGEX-6p-1 vector. ( b ) Six hours after the addition of 1 mM IPTG, the induced products of GST-N, GST-N1 and GST-N2 were processed and analyzed using SDS-PAGE (12% separating gel and 5% stacking gel). ( c ) Next, the GST fusion proteins GST-N, GST-N1 and GST-N2 were coated onto ELISA plates (0.5 μg/well) and were probed with MAbs PN-D4 and PN-D6. After the first round of identification, N1 and N2 were further divided and expressed in two series of fusion proteins, GST-N1-1-GST-N1-11 and GST-N2-1-GST-N2-11 ( d , h ), which were analyzed by Western blotting and ELISA using PN-D4 ( e , g ), PN-D6 ( i , k ), and anti-GST Tag antibody ( f , j ), respectively.

    Techniques Used: Clone Assay, Plasmid Preparation, SDS Page, Enzyme-linked Immunosorbent Assay, Western Blot

    Selection of MAbs against PEDV N protein. ( a ) Six MAbs were proven to be positive by IFA using PEDV inoculated cells. ( b ) The six positive clones were then tested by ELISA against GST-fusion N protein and GST-tag expressed in Escherichia coli .
    Figure Legend Snippet: Selection of MAbs against PEDV N protein. ( a ) Six MAbs were proven to be positive by IFA using PEDV inoculated cells. ( b ) The six positive clones were then tested by ELISA against GST-fusion N protein and GST-tag expressed in Escherichia coli .

    Techniques Used: Selection, Immunofluorescence, Clone Assay, Enzyme-linked Immunosorbent Assay

    18) Product Images from "The K167I variant of DNA polymerase β that is found in Esophageal Carcinoma patients impairs polymerase activity and BER"

    Article Title: The K167I variant of DNA polymerase β that is found in Esophageal Carcinoma patients impairs polymerase activity and BER

    Journal: Scientific Reports

    doi: 10.1038/srep15986

    The pol β variant K167I is defective in polymerase activity. ( A ) Polymerase activity assays were performed with biotin labeled 1-nt gapped DNA substrate (pol-GAP). Separated DNA polymerization products were pulled down using Sepharose-avidin beads. After washing, the amount of radio labeled nucleotides incorporated into the products was determined by liquid scintillation counting. We found that the K167I variant (0.25 ng: 5.2 ± 0.57, 0.5 ng: 11.3 ± 1.25, 1 ng: 18.5 ± 2.31, 2 ng: 26.2 ± 2.94, 4 ng: 32.1 ± 3.51, 6 ng: 35.7 ± 3.97, 8 ng: 37.8 ± 4.84) had about 30% primer extension activity compared to the WT enzyme (0.25 ng: 11.2 ± 1.52, 0.5 ng: 25.4 ± 3.45, 1 ng: 52.6 ± 5.79, 2 ng: 71.8 ± 7.04, 4 ng: 84.5 ± 7.95, 6 ng: 91.3 ± 8.89, 8 ng: 99.1 ± 8.97). ( B ) ELISA based isotherm adsorption assays of the DNA bindi ng affinity of K167I (0.0125 μg: 0.08 ± 0.005, 0.025 μg: 0.15 ± 0.011, 0.05 μg: 0.21 ± 0.015, 0.1 μg: 0.27 ± 0.021, 0.2 μg: 0.33 ± 0.024, 0.4 μg: 0.36 ± 0.023, 0.8 μg: 0.38 ± 0.028) and WT pol β (0.0125 μg: 0.09 ± 0.005, 0.025 μg: 0.17 ± 0.010, 0.05 μg: 0.24 ± 0.015, 0.1 μg: 0.31 ± 0.021, 0.2 μg: 0.36 ± 0.020, 0.4 μg: 0.38 ± 0.023, 0.8 μg: 0.41 ± 0.028). The DNA substrate was biotin labeled pol-GAP. ( C ) Quantification of the 5′dRP lyase activity in K167I (1 ng: 22.4 ± 2.79, 2 ng: 31.7 ± 3.04, 4 ng: 70.6 ± 7.15, 6 ng: 82.1 ± 7.92, 8 ng: 89.7 ± 8.25, 10 ng: 95.4 ± 8.74) and WT pol β (1 ng: 21.9 ± 2.31, 2 ng: 35.2 ± 2.94, 4 ng: 76.2 ± 6.89, 6 ng: 84.3 ± 7.64, 8 ng: 93.1 ± 8.73, 10 ng: 98.7 ± 8.81) were shown.
    Figure Legend Snippet: The pol β variant K167I is defective in polymerase activity. ( A ) Polymerase activity assays were performed with biotin labeled 1-nt gapped DNA substrate (pol-GAP). Separated DNA polymerization products were pulled down using Sepharose-avidin beads. After washing, the amount of radio labeled nucleotides incorporated into the products was determined by liquid scintillation counting. We found that the K167I variant (0.25 ng: 5.2 ± 0.57, 0.5 ng: 11.3 ± 1.25, 1 ng: 18.5 ± 2.31, 2 ng: 26.2 ± 2.94, 4 ng: 32.1 ± 3.51, 6 ng: 35.7 ± 3.97, 8 ng: 37.8 ± 4.84) had about 30% primer extension activity compared to the WT enzyme (0.25 ng: 11.2 ± 1.52, 0.5 ng: 25.4 ± 3.45, 1 ng: 52.6 ± 5.79, 2 ng: 71.8 ± 7.04, 4 ng: 84.5 ± 7.95, 6 ng: 91.3 ± 8.89, 8 ng: 99.1 ± 8.97). ( B ) ELISA based isotherm adsorption assays of the DNA bindi ng affinity of K167I (0.0125 μg: 0.08 ± 0.005, 0.025 μg: 0.15 ± 0.011, 0.05 μg: 0.21 ± 0.015, 0.1 μg: 0.27 ± 0.021, 0.2 μg: 0.33 ± 0.024, 0.4 μg: 0.36 ± 0.023, 0.8 μg: 0.38 ± 0.028) and WT pol β (0.0125 μg: 0.09 ± 0.005, 0.025 μg: 0.17 ± 0.010, 0.05 μg: 0.24 ± 0.015, 0.1 μg: 0.31 ± 0.021, 0.2 μg: 0.36 ± 0.020, 0.4 μg: 0.38 ± 0.023, 0.8 μg: 0.41 ± 0.028). The DNA substrate was biotin labeled pol-GAP. ( C ) Quantification of the 5′dRP lyase activity in K167I (1 ng: 22.4 ± 2.79, 2 ng: 31.7 ± 3.04, 4 ng: 70.6 ± 7.15, 6 ng: 82.1 ± 7.92, 8 ng: 89.7 ± 8.25, 10 ng: 95.4 ± 8.74) and WT pol β (1 ng: 21.9 ± 2.31, 2 ng: 35.2 ± 2.94, 4 ng: 76.2 ± 6.89, 6 ng: 84.3 ± 7.64, 8 ng: 93.1 ± 8.73, 10 ng: 98.7 ± 8.81) were shown.

    Techniques Used: Variant Assay, Activity Assay, Labeling, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Adsorption

    19) Product Images from "Glycosylation Patterns of HIV-1 gp120 Depend on the Type of Expressing Cells and Affect Antibody Recognition *"

    Article Title: Glycosylation Patterns of HIV-1 gp120 Depend on the Type of Expressing Cells and Affect Antibody Recognition *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.085472

    Binding of IgG from HIV-1-positive patients ( 1–13 ) and HIV-1 negative control subjects ( C1–C3 ) to purified native recombinant gp120 glycoproteins produced in 293T, HepG2, RD, CHO, and Jurkat cells was tested by ELISA. IgG in serum diluted 1:10,000 was allowed to bind to 96-well plates coated with an equal amount of our recombinant gp120 glycoproteins (0.5 μg/ml) and bound IgG antibodies were detected with anti-human IgG HRP-conjugated antibody. Mean values of absorbance and standard deviations are shown ( A ). Statistical analysis of differences in binding of serum IgG from HIV-1-positive subjects to individual gp120 preparations was performed by the Friedmann test, with results expressed as a box graph ( p
    Figure Legend Snippet: Binding of IgG from HIV-1-positive patients ( 1–13 ) and HIV-1 negative control subjects ( C1–C3 ) to purified native recombinant gp120 glycoproteins produced in 293T, HepG2, RD, CHO, and Jurkat cells was tested by ELISA. IgG in serum diluted 1:10,000 was allowed to bind to 96-well plates coated with an equal amount of our recombinant gp120 glycoproteins (0.5 μg/ml) and bound IgG antibodies were detected with anti-human IgG HRP-conjugated antibody. Mean values of absorbance and standard deviations are shown ( A ). Statistical analysis of differences in binding of serum IgG from HIV-1-positive subjects to individual gp120 preparations was performed by the Friedmann test, with results expressed as a box graph ( p

    Techniques Used: Binding Assay, Negative Control, Purification, Recombinant, Produced, Enzyme-linked Immunosorbent Assay

    20) Product Images from "A novel C3d-containing oligomeric vaccine provides insight into the viability of testing human C3d-based vaccines in mice"

    Article Title: A novel C3d-containing oligomeric vaccine provides insight into the viability of testing human C3d-based vaccines in mice

    Journal: Immunobiology

    doi: 10.1016/j.imbio.2017.10.002

    Direct in vivo comparison of the adjuvant effects of hC3d with the conventional adjuvant alum. Three groups (n = 4) of mice were immunized intra-peritoneally with hC3d-TTCF-hC4BP/saline (circles); TTCF/Alum (squares) or TTCF/saline (triangles) containing TTCF molar equivalents of 5 μg/animal, and boosted at day 28 (arrow) following initial immunization. Serum was collected on the days indicated and analysed for the presence of anti-TTCF IgG by ELISA. Average values (relative units (RU)) ± SD are shown. One-way ANOVA Tukey’s HSD test was used to define significance levels at 95% confidence (* p
    Figure Legend Snippet: Direct in vivo comparison of the adjuvant effects of hC3d with the conventional adjuvant alum. Three groups (n = 4) of mice were immunized intra-peritoneally with hC3d-TTCF-hC4BP/saline (circles); TTCF/Alum (squares) or TTCF/saline (triangles) containing TTCF molar equivalents of 5 μg/animal, and boosted at day 28 (arrow) following initial immunization. Serum was collected on the days indicated and analysed for the presence of anti-TTCF IgG by ELISA. Average values (relative units (RU)) ± SD are shown. One-way ANOVA Tukey’s HSD test was used to define significance levels at 95% confidence (* p

    Techniques Used: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Recombinant constructs and protein production. a) Tissue culture supernatant collected from CHO cells expressing constructs, diagrammatically indicated to left of blot, was prepared in reducing sample buffer and loaded on a 10% SDS-PAGE gel before blotting to nitrocellulose. Predicted monomer constructs: hC3d-TTCF-hC4BP: 124.2 kDa, TTCF-hC4BP: 89.5 kDa, hC3d-hC4BP: 72.1 kDa and hC4BP: 38.1 kDa were detected. (b) hC3d-TTCF-hC4BP supernatant in non-reducing (NR) and reducing (R) sample buffer was separated on 5% SDS-PAGE, transferred using a modified glycine-methanol transfer buffer containing 0.1% SDS at 35 V overnight followed by 100 V for 1hr. In both panels, proteins were detected using sheep anti-hC4BP antibody followed by donkey anti-sheep IgG-HRPO. The migration of protein size markers are indicated on the right of each blot. Predicted conformation is illustrated diagrammatically either side of blots. ELISA plate wells were coated with undiluted tissue culture supernatants from CHO cell transfectants (as indicated on x axis). Plate-bound constructs were detected with rabbit anti-hC3d followed by goat anti-rabbit HRPO (c), anti-TTCF (mAb 10G5) followed by goat anti-mouse HRPO (d), sheep anti-hC4BP followed by donkey anti-sheep HRPO (e). Supernatants collected from untransfected CHO cells were used to calculate relative units (RU). Panel (f) shows coomassie blue staining following SDS-PAGE analysis of approximately 1–2ug of affinity purified recombinant proteins. All results shown are representative of several similar experiments.
    Figure Legend Snippet: Recombinant constructs and protein production. a) Tissue culture supernatant collected from CHO cells expressing constructs, diagrammatically indicated to left of blot, was prepared in reducing sample buffer and loaded on a 10% SDS-PAGE gel before blotting to nitrocellulose. Predicted monomer constructs: hC3d-TTCF-hC4BP: 124.2 kDa, TTCF-hC4BP: 89.5 kDa, hC3d-hC4BP: 72.1 kDa and hC4BP: 38.1 kDa were detected. (b) hC3d-TTCF-hC4BP supernatant in non-reducing (NR) and reducing (R) sample buffer was separated on 5% SDS-PAGE, transferred using a modified glycine-methanol transfer buffer containing 0.1% SDS at 35 V overnight followed by 100 V for 1hr. In both panels, proteins were detected using sheep anti-hC4BP antibody followed by donkey anti-sheep IgG-HRPO. The migration of protein size markers are indicated on the right of each blot. Predicted conformation is illustrated diagrammatically either side of blots. ELISA plate wells were coated with undiluted tissue culture supernatants from CHO cell transfectants (as indicated on x axis). Plate-bound constructs were detected with rabbit anti-hC3d followed by goat anti-rabbit HRPO (c), anti-TTCF (mAb 10G5) followed by goat anti-mouse HRPO (d), sheep anti-hC4BP followed by donkey anti-sheep HRPO (e). Supernatants collected from untransfected CHO cells were used to calculate relative units (RU). Panel (f) shows coomassie blue staining following SDS-PAGE analysis of approximately 1–2ug of affinity purified recombinant proteins. All results shown are representative of several similar experiments.

    Techniques Used: Recombinant, Construct, Expressing, SDS Page, Modification, Migration, Enzyme-linked Immunosorbent Assay, Staining, Affinity Purification

    21) Product Images from "Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs"

    Article Title: Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs

    Journal: Journal of Virology

    doi: 10.1128/JVI.00369-18

    Vaccination regimens and characterization of binding antibody responses. (A) Vaccination regimens for guinea pigs immunized with a longitudinal prime/boost vaccination schedule. Animals were vaccinated at weeks 0, 4, 8, 12, 62, 66, 70, 74, 104, 108, 112, and 116 utilizing the listed vaccination regimens and bled 4 weeks after each vaccination, as well as at weeks 138 and 200. C97, C97ZA012 gp140; 92UG, 92UG037 gp140; Mos, mosaic gp140. Error bars represent the standard deviations. (B) Binding antibody titers for HIV-1 Env gp140s of different clades as measured by endpoint ELISAs. (C) Binding antibody titers for HIV-1 C97ZA012 gp140, showing specific isotype and subclass responses, as measured utilizing endpoint ELISAs. Error bars represent the standard deviations. (D) Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) × 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and > 50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.
    Figure Legend Snippet: Vaccination regimens and characterization of binding antibody responses. (A) Vaccination regimens for guinea pigs immunized with a longitudinal prime/boost vaccination schedule. Animals were vaccinated at weeks 0, 4, 8, 12, 62, 66, 70, 74, 104, 108, 112, and 116 utilizing the listed vaccination regimens and bled 4 weeks after each vaccination, as well as at weeks 138 and 200. C97, C97ZA012 gp140; 92UG, 92UG037 gp140; Mos, mosaic gp140. Error bars represent the standard deviations. (B) Binding antibody titers for HIV-1 Env gp140s of different clades as measured by endpoint ELISAs. (C) Binding antibody titers for HIV-1 C97ZA012 gp140, showing specific isotype and subclass responses, as measured utilizing endpoint ELISAs. Error bars represent the standard deviations. (D) Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) × 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and > 50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    22) Product Images from "Assessment of ELISA as Endpoint in Neuronal Cell-based Assay for BoNT Detection using hiPSC derived neurons"

    Article Title: Assessment of ELISA as Endpoint in Neuronal Cell-based Assay for BoNT Detection using hiPSC derived neurons

    Journal: Journal of pharmacological and toxicological methods

    doi: 10.1016/j.vascn.2017.04.013

    Plate selection for ELISA. HIP Neurons were exposed to serial dilutions of BoNT/A1, and cell lysates were analyzed for cleaved SNAP-25 by ELISA using either a Nunc Polystyrene Maxisorp plate (Nunc) or a Thermo Scientific Immulon Immunoassay plate (Immulon). The signal was detected with ultra TMB. The average and standard deviations of 4 replicates is shown. Dose response curves were fitted using Prims 6 software and a nonlinear regression (variable slope, four parameter).
    Figure Legend Snippet: Plate selection for ELISA. HIP Neurons were exposed to serial dilutions of BoNT/A1, and cell lysates were analyzed for cleaved SNAP-25 by ELISA using either a Nunc Polystyrene Maxisorp plate (Nunc) or a Thermo Scientific Immulon Immunoassay plate (Immulon). The signal was detected with ultra TMB. The average and standard deviations of 4 replicates is shown. Dose response curves were fitted using Prims 6 software and a nonlinear regression (variable slope, four parameter).

    Techniques Used: Selection, Enzyme-linked Immunosorbent Assay, Software

    23) Product Images from "Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs"

    Article Title: Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs

    Journal: Journal of Virology

    doi: 10.1128/JVI.00369-18

    Vaccination regimens and characterization of binding antibody responses. (A) Vaccination regimens for guinea pigs immunized with a longitudinal prime/boost vaccination schedule. Animals were vaccinated at weeks 0, 4, 8, 12, 62, 66, 70, 74, 104, 108, 112, and 116 utilizing the listed vaccination regimens and bled 4 weeks after each vaccination, as well as at weeks 138 and 200. C97, C97ZA012 gp140; 92UG, 92UG037 gp140; Mos, mosaic gp140. Error bars represent the standard deviations. (B) Binding antibody titers for HIV-1 Env gp140s of different clades as measured by endpoint ELISAs. (C) Binding antibody titers for HIV-1 C97ZA012 gp140, showing specific isotype and subclass responses, as measured utilizing endpoint ELISAs. Error bars represent the standard deviations. (D) Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) × 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and > 50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.
    Figure Legend Snippet: Vaccination regimens and characterization of binding antibody responses. (A) Vaccination regimens for guinea pigs immunized with a longitudinal prime/boost vaccination schedule. Animals were vaccinated at weeks 0, 4, 8, 12, 62, 66, 70, 74, 104, 108, 112, and 116 utilizing the listed vaccination regimens and bled 4 weeks after each vaccination, as well as at weeks 138 and 200. C97, C97ZA012 gp140; 92UG, 92UG037 gp140; Mos, mosaic gp140. Error bars represent the standard deviations. (B) Binding antibody titers for HIV-1 Env gp140s of different clades as measured by endpoint ELISAs. (C) Binding antibody titers for HIV-1 C97ZA012 gp140, showing specific isotype and subclass responses, as measured utilizing endpoint ELISAs. Error bars represent the standard deviations. (D) Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) × 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and > 50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    24) Product Images from "The Minimal Domain of Adipose Triglyceride Lipase (ATGL) Ranges until Leucine 254 and Can Be Activated and Inhibited by CGI-58 and G0S2, Respectively"

    Article Title: The Minimal Domain of Adipose Triglyceride Lipase (ATGL) Ranges until Leucine 254 and Can Be Activated and Inhibited by CGI-58 and G0S2, Respectively

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026349

    ELISA experiment demonstrating that ATGL254 interacts with CGI-58 and G0S2. A. ELISA plates were coated with purified ATGL254-MBP and then incubated with purified His-tagged CGI-58. Purified smt (the fusion tag for CGI-58) was used as a negative control. Detection was performed by anti-his-antibody, HRP-labeled secondary antibody and tetramethyl-benzidine as substrate. B. ELISA plates were coated with purified mG0S2 and then incubated with purified ATGL254-MBP. Purified MBP was used as negative control. Detection was performed using an anti-MBP (HRP conjugated) antibody and otherwise as in A. Data are presented as mean+SD and representative for 2 independent experiments (performed in triplicates). C. SDS-PAGE confirming the expression and purification of respective fusion proteins.
    Figure Legend Snippet: ELISA experiment demonstrating that ATGL254 interacts with CGI-58 and G0S2. A. ELISA plates were coated with purified ATGL254-MBP and then incubated with purified His-tagged CGI-58. Purified smt (the fusion tag for CGI-58) was used as a negative control. Detection was performed by anti-his-antibody, HRP-labeled secondary antibody and tetramethyl-benzidine as substrate. B. ELISA plates were coated with purified mG0S2 and then incubated with purified ATGL254-MBP. Purified MBP was used as negative control. Detection was performed using an anti-MBP (HRP conjugated) antibody and otherwise as in A. Data are presented as mean+SD and representative for 2 independent experiments (performed in triplicates). C. SDS-PAGE confirming the expression and purification of respective fusion proteins.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Purification, Incubation, Negative Control, Labeling, SDS Page, Expressing

    25) Product Images from "Innate Immunity Drives the Initiation of a Murine Model of Primary Biliary Cirrhosis"

    Article Title: Innate Immunity Drives the Initiation of a Murine Model of Primary Biliary Cirrhosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121320

    Increased serum AMAs in mice injected with 2-OA-BSA/OCH. Wild type mice were immunized with 2-OA-BSA and α-GalCer (group name: 2-OA/a-GC), OCH (group name: 2-OA/OCH) or PBS (group name: 2-OA/PBS) at weeks 0, 2, 4, 6 and 8. At week 12, serum levels of autoantibodies to mPDC-E2 were measured by ELISA. n = 9–10 mice per group. *, p
    Figure Legend Snippet: Increased serum AMAs in mice injected with 2-OA-BSA/OCH. Wild type mice were immunized with 2-OA-BSA and α-GalCer (group name: 2-OA/a-GC), OCH (group name: 2-OA/OCH) or PBS (group name: 2-OA/PBS) at weeks 0, 2, 4, 6 and 8. At week 12, serum levels of autoantibodies to mPDC-E2 were measured by ELISA. n = 9–10 mice per group. *, p

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    Lower serum levels of IFN-γ after OCH injection. C57BL/6 mice were intravenously injected with α-GalCer, OCH, or PBS. Serum samples were collected at 2 and 18 hours after α-GalCer, OCH, or PBS injection. IFN- γ (A) and IL-4 (B) were measured by ELISA. n = 10 mice per group. ***, p
    Figure Legend Snippet: Lower serum levels of IFN-γ after OCH injection. C57BL/6 mice were intravenously injected with α-GalCer, OCH, or PBS. Serum samples were collected at 2 and 18 hours after α-GalCer, OCH, or PBS injection. IFN- γ (A) and IL-4 (B) were measured by ELISA. n = 10 mice per group. ***, p

    Techniques Used: Injection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    26) Product Images from "The diagnostic targeting of a carbohydrate virulence factor from M.Tuberculosis"

    Article Title: The diagnostic targeting of a carbohydrate virulence factor from M.Tuberculosis

    Journal: Scientific Reports

    doi: 10.1038/srep10281

    Improvement of My2F12 assay sensitivity by serum denaturation. (A) Anti-LAM antibody titres from BCG vaccinated and non-vaccinated individuals. (B) Sandwich ELISA signal-to-noise ratio showing inverse correlation between levels of endogenous anti-LAM antibodies and limit of detection. A limit of detection (dotted line) is set at a ratio of 2 and noise is defined as the signal obtained with no LAM added (C) Significant reduction of levels of endogenous anti-LAM antibodies in serum (at 1:512 dilution) by Proteinase K and heat treatment. (D) Improvement in sensitivity of sandwich ELISA as after denaturation of endogenous anti-LAM antibodies in serum samples with proteinase K and heat treatment (+PK) as compared to untreated spiked serum (-PK) and PBS standards. All data shown are the average of three independent experiments and error bars show standard error of mean. Concentrations of ManLAM are that in whole blood spiked before coagulation. (* p
    Figure Legend Snippet: Improvement of My2F12 assay sensitivity by serum denaturation. (A) Anti-LAM antibody titres from BCG vaccinated and non-vaccinated individuals. (B) Sandwich ELISA signal-to-noise ratio showing inverse correlation between levels of endogenous anti-LAM antibodies and limit of detection. A limit of detection (dotted line) is set at a ratio of 2 and noise is defined as the signal obtained with no LAM added (C) Significant reduction of levels of endogenous anti-LAM antibodies in serum (at 1:512 dilution) by Proteinase K and heat treatment. (D) Improvement in sensitivity of sandwich ELISA as after denaturation of endogenous anti-LAM antibodies in serum samples with proteinase K and heat treatment (+PK) as compared to untreated spiked serum (-PK) and PBS standards. All data shown are the average of three independent experiments and error bars show standard error of mean. Concentrations of ManLAM are that in whole blood spiked before coagulation. (* p

    Techniques Used: Laser Capture Microdissection, Sandwich ELISA, Coagulation

    27) Product Images from "TLR2/MyD88 pathway-dependent regulation of dendritic cells by dengue virus promotes antibody-dependent enhancement via Th2-biased immunity"

    Article Title: TLR2/MyD88 pathway-dependent regulation of dendritic cells by dengue virus promotes antibody-dependent enhancement via Th2-biased immunity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22525

    Bystander activation of uninfected DCs by DenV-infected DCs in a TLR2-dependent manner (A) Phenotypic levels of infected and uninfected BMDC (DN3 + and DN3 − ). BMDC derived from wild-type BL/6 and TLR2 KO mice were infected with DenV2 (5.0 moi) and co-stained for intracellular DenV NS1 protein and surface CD11c molecule along with phenotypic markers at 24 h (black line) and 48 h (red line) pi. Mock-infected BMDC is shown in grey. (B) Fold-change of phenotypic levels in infected and uninfected BMDC. Expression of each surface molecule on DN3 + infected and DN3 − uninfected DCs was quantified by MFI. Values shown in graphs represent mean fold-change in MFI vs. mock infection in three independent experiments. (C) TNF-α and IL-6 expression in infected and uninfected BMDCs. TNF-α and IL-6 expression by infected and uninfected BL/6 and TLR2 KO BMDC was determined by intracellular cytokine staining of DN3 + (infected) and DN3 − (uninfected) cells at 24 h pi. (D) Dependence of IL-6 and TNF-α production in DenV-infected DCs on p38 and JNK MAPK. BMDC derived from wild-type BL/6 were infected with DenV2 (5.0 moi) in the presence or absence of p38 inhibitor (SB203580), JNK inhibitor (SP600125), and MEK inhibitor (PD98059). Levels of TNF-α and IL-6 cytokines in culture supernatants were determined by ELISA at 48 h pi. Data represent the average ± SE of wells evaluated in quadruplicates. (E) Phosphorylation levels of ERK and STAT3 signal molecules in infected and uninfected BMDC. Phosphorylation level of ERK and STAT3 signal molecules was determined by intracellular staining of DN3 + (infected) and DN3 − (uninfected) cells using mAbs against phosphorylated signal molecules at 24 h pi. Values in representative histograms denote average MFI levels derived from three independent experiments. * , p
    Figure Legend Snippet: Bystander activation of uninfected DCs by DenV-infected DCs in a TLR2-dependent manner (A) Phenotypic levels of infected and uninfected BMDC (DN3 + and DN3 − ). BMDC derived from wild-type BL/6 and TLR2 KO mice were infected with DenV2 (5.0 moi) and co-stained for intracellular DenV NS1 protein and surface CD11c molecule along with phenotypic markers at 24 h (black line) and 48 h (red line) pi. Mock-infected BMDC is shown in grey. (B) Fold-change of phenotypic levels in infected and uninfected BMDC. Expression of each surface molecule on DN3 + infected and DN3 − uninfected DCs was quantified by MFI. Values shown in graphs represent mean fold-change in MFI vs. mock infection in three independent experiments. (C) TNF-α and IL-6 expression in infected and uninfected BMDCs. TNF-α and IL-6 expression by infected and uninfected BL/6 and TLR2 KO BMDC was determined by intracellular cytokine staining of DN3 + (infected) and DN3 − (uninfected) cells at 24 h pi. (D) Dependence of IL-6 and TNF-α production in DenV-infected DCs on p38 and JNK MAPK. BMDC derived from wild-type BL/6 were infected with DenV2 (5.0 moi) in the presence or absence of p38 inhibitor (SB203580), JNK inhibitor (SP600125), and MEK inhibitor (PD98059). Levels of TNF-α and IL-6 cytokines in culture supernatants were determined by ELISA at 48 h pi. Data represent the average ± SE of wells evaluated in quadruplicates. (E) Phosphorylation levels of ERK and STAT3 signal molecules in infected and uninfected BMDC. Phosphorylation level of ERK and STAT3 signal molecules was determined by intracellular staining of DN3 + (infected) and DN3 − (uninfected) cells using mAbs against phosphorylated signal molecules at 24 h pi. Values in representative histograms denote average MFI levels derived from three independent experiments. * , p

    Techniques Used: Activation Assay, Infection, Derivative Assay, Mouse Assay, Staining, Expressing, Enzyme-linked Immunosorbent Assay

    DenV induces Ag-specific Th2 immune responses in TLR2/MyD88-dependent manner (A) Enhanced number of anti-OVA-IgG producing cells by re-stimulation of Ag in mice infected with DenV2. Splenocytes of wild-type BL/6, TLR2 KO, and MyD88 KO mice immunized i.p. with PBS (vehicle), OVA (50 μg), or OVA (50 μg) plus DenV2 infection (1×10 6 ffu/mouse) were prepared at 14 days post-immunization and used to enumerate anti-OVA-IgG producing cells using ELISPOT assay. Bars denote the average ± SE of the number of anti-OVA-IgG producing cells in million splenocytes obtained from four mice per group. Pictures are representative of ELISPOT in each group. (B) Levels and ratio of OVA-specific IgG isotypes (IgG 1 and IgG 2a ) in sera of DenV2-infected mice. Levels of IgG isotypes in sera were determined by ELISA at 14 days after mice were immunized with PBS, OVA, or OVA plus DenV2 infection. (C) Prominent production of Th2-type cytokines in splenocyte of DenV2-infected mice after re-stimulation with OVA protein. Splenocytes of BL/6, TLR2 KO, and MyD88 KO mice immunized with PBS, OVA, or OVA plus DenV2 infection were prepared at 14 dpi and re-stimulated with OVA protein (100 μg/ml) for 72 h. Th1/Th2-cytokine levels in culture supernatants of re-stimulated splenocytes were determined by sandwich ELISA. (D) DenV2 E-specific IgG levels and its isotype ratio in DenV-infected mice. BL/6, TLR2 KO, and MyD88 KO mice were infected i.p. with DenV2 (1×10 6 ffu/mouse) and boosted 7 days later. Levels of DenV2 E protein-specific IgG and its isotypes (IgG 1 and IgG 2a ) were determined by ELISA at 14 days after boosting immunization. (E, F) Th2-biased CD4 + T cell responses specific for DenV Ag in TLR2-dependent manner. Splenocytes of BL/6 and TLR2 KO mice infected with DenV2 were prepared at 14 dpi and stimulated with two CD4 + T-cell epitope peptides (NS3 198-212 and NS3 237-251 ) for 12 h. The frequency (E) and total absolute number (F) of CD4 + T cells producing Th1 (IFN-γ and TNF-α) and Th2 (IL-4 and IL-5) were evaluated by co-staining with intracellular cytokines and surface CD4 molecules. Dot plots are representatives of at least three independent experiments. Bars denote the average ± SE of levels derived from at least three independent experiments ( n = 3-4). * , p
    Figure Legend Snippet: DenV induces Ag-specific Th2 immune responses in TLR2/MyD88-dependent manner (A) Enhanced number of anti-OVA-IgG producing cells by re-stimulation of Ag in mice infected with DenV2. Splenocytes of wild-type BL/6, TLR2 KO, and MyD88 KO mice immunized i.p. with PBS (vehicle), OVA (50 μg), or OVA (50 μg) plus DenV2 infection (1×10 6 ffu/mouse) were prepared at 14 days post-immunization and used to enumerate anti-OVA-IgG producing cells using ELISPOT assay. Bars denote the average ± SE of the number of anti-OVA-IgG producing cells in million splenocytes obtained from four mice per group. Pictures are representative of ELISPOT in each group. (B) Levels and ratio of OVA-specific IgG isotypes (IgG 1 and IgG 2a ) in sera of DenV2-infected mice. Levels of IgG isotypes in sera were determined by ELISA at 14 days after mice were immunized with PBS, OVA, or OVA plus DenV2 infection. (C) Prominent production of Th2-type cytokines in splenocyte of DenV2-infected mice after re-stimulation with OVA protein. Splenocytes of BL/6, TLR2 KO, and MyD88 KO mice immunized with PBS, OVA, or OVA plus DenV2 infection were prepared at 14 dpi and re-stimulated with OVA protein (100 μg/ml) for 72 h. Th1/Th2-cytokine levels in culture supernatants of re-stimulated splenocytes were determined by sandwich ELISA. (D) DenV2 E-specific IgG levels and its isotype ratio in DenV-infected mice. BL/6, TLR2 KO, and MyD88 KO mice were infected i.p. with DenV2 (1×10 6 ffu/mouse) and boosted 7 days later. Levels of DenV2 E protein-specific IgG and its isotypes (IgG 1 and IgG 2a ) were determined by ELISA at 14 days after boosting immunization. (E, F) Th2-biased CD4 + T cell responses specific for DenV Ag in TLR2-dependent manner. Splenocytes of BL/6 and TLR2 KO mice infected with DenV2 were prepared at 14 dpi and stimulated with two CD4 + T-cell epitope peptides (NS3 198-212 and NS3 237-251 ) for 12 h. The frequency (E) and total absolute number (F) of CD4 + T cells producing Th1 (IFN-γ and TNF-α) and Th2 (IL-4 and IL-5) were evaluated by co-staining with intracellular cytokines and surface CD4 molecules. Dot plots are representatives of at least three independent experiments. Bars denote the average ± SE of levels derived from at least three independent experiments ( n = 3-4). * , p

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Staining, Derivative Assay

    TLR2/MyD88 pathway is required for recognition of dengue virus infection (A) Dependence of IL-6 and TNF-α production on TLR2/MyD88 pathway in DCs infected with DenV. BMDC from wild-type BL/6, TLR2, 3, 4, 9, or MyD88 KO mice were infected with DenV2 (5.0 moi), after which cytokine levels in culture supernatants were determined by ELISA at 48 h pi. (B) Ex vivo secretion of IL-6 and TFN-α from splenocytes of BL/6 and TLR2 KO mice. Splenocytes from BL/6 and TLR2 KO mice i.p. infected with DenV2 (1×10 6 ffu/mouse) were prepared at 48 h pi. Cytokine levels in culture supernatants were determined at indicated time after starting culture of splenocytes. (C) Levels of IL-6 and TNF-α in sera of BL/6 and TLR2 KO mice following DenV infection. Cytokine levels in sera collected at indicated times were determined by ELISA. (D) Dependence of phenotypic changes of Ag presentation-related markers on TLR2/MyD88 pathway in DenV2-infected DCs. BMDC derived from BL/6, TLR2 KO, and MyD88 KO mice were infected with DenV2 (5.0 moi) and used to stain activation markers at 24 h pi. (E) MFI ratio of phenotype markers to mock-infected DCs. Data denote the average ± SE of cytokines or MFI levels obtained from three independent experiments. * , p
    Figure Legend Snippet: TLR2/MyD88 pathway is required for recognition of dengue virus infection (A) Dependence of IL-6 and TNF-α production on TLR2/MyD88 pathway in DCs infected with DenV. BMDC from wild-type BL/6, TLR2, 3, 4, 9, or MyD88 KO mice were infected with DenV2 (5.0 moi), after which cytokine levels in culture supernatants were determined by ELISA at 48 h pi. (B) Ex vivo secretion of IL-6 and TFN-α from splenocytes of BL/6 and TLR2 KO mice. Splenocytes from BL/6 and TLR2 KO mice i.p. infected with DenV2 (1×10 6 ffu/mouse) were prepared at 48 h pi. Cytokine levels in culture supernatants were determined at indicated time after starting culture of splenocytes. (C) Levels of IL-6 and TNF-α in sera of BL/6 and TLR2 KO mice following DenV infection. Cytokine levels in sera collected at indicated times were determined by ELISA. (D) Dependence of phenotypic changes of Ag presentation-related markers on TLR2/MyD88 pathway in DenV2-infected DCs. BMDC derived from BL/6, TLR2 KO, and MyD88 KO mice were infected with DenV2 (5.0 moi) and used to stain activation markers at 24 h pi. (E) MFI ratio of phenotype markers to mock-infected DCs. Data denote the average ± SE of cytokines or MFI levels obtained from three independent experiments. * , p

    Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Ex Vivo, Derivative Assay, Staining, Activation Assay

    28) Product Images from "Structure of a bacterial type IV secretion core complex at subnanometre resolution"

    Article Title: Structure of a bacterial type IV secretion core complex at subnanometre resolution

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2013.58

    NB labelling of the FLCC, CC elastase and OL complexes. ( A ) Selection of NBs preferentially binding the CC elastase (see also Supplementary Figure 6 ). The diagram reports the comparative affinity of three NBs for the FLCC (green), the CC elastase (blue) and the OL (red) complexes. NBCA4271 and NBCA4304 target the IL while NBCA4296 binds to the OL, a region present in the three complexes used for the ELISA experiment. NBCA4304 was chosen for the labelling experiments. The right panel represents the schematic organisation of the complexes used in the ELISA experiments coloured correspondingly. ( B ) SDS–PAGE analysis and western blot of the labelled FLCC. The different lanes correspond to the FLCC, the NBCA4304, the purified complex containing the FLCC and the NB (FLCC+NBCA4304), and the final purified complex containing the FLCC, the NB and the anti-His antibody (CC+NBCA4304+anti-His). This final complex was used to prepare negative stain grids. The right panel shows a western blot of the FLCC+NBCA4304+anti-His fraction in the presence or absence (+/− DTT) of reducing agent. The western blot was performed using only the secondary antibody HRP-conjugated. Molecular weight standards (Std) are indicated in both cases. ( C ) Gallery of negatively stained single particles with a clear extra density emerging from the CC. ( D ) Representative top view class averages of the FLCC+NBCA4304+anti-His sample. The scale bar corresponds to 10 nm.
    Figure Legend Snippet: NB labelling of the FLCC, CC elastase and OL complexes. ( A ) Selection of NBs preferentially binding the CC elastase (see also Supplementary Figure 6 ). The diagram reports the comparative affinity of three NBs for the FLCC (green), the CC elastase (blue) and the OL (red) complexes. NBCA4271 and NBCA4304 target the IL while NBCA4296 binds to the OL, a region present in the three complexes used for the ELISA experiment. NBCA4304 was chosen for the labelling experiments. The right panel represents the schematic organisation of the complexes used in the ELISA experiments coloured correspondingly. ( B ) SDS–PAGE analysis and western blot of the labelled FLCC. The different lanes correspond to the FLCC, the NBCA4304, the purified complex containing the FLCC and the NB (FLCC+NBCA4304), and the final purified complex containing the FLCC, the NB and the anti-His antibody (CC+NBCA4304+anti-His). This final complex was used to prepare negative stain grids. The right panel shows a western blot of the FLCC+NBCA4304+anti-His fraction in the presence or absence (+/− DTT) of reducing agent. The western blot was performed using only the secondary antibody HRP-conjugated. Molecular weight standards (Std) are indicated in both cases. ( C ) Gallery of negatively stained single particles with a clear extra density emerging from the CC. ( D ) Representative top view class averages of the FLCC+NBCA4304+anti-His sample. The scale bar corresponds to 10 nm.

    Techniques Used: Selection, Binding Assay, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Purification, Staining, Molecular Weight

    29) Product Images from "Modulation of Antibody Responses to the V1V2 and V3 Regions of HIV-1 Envelope by Immune Complex Vaccines"

    Article Title: Modulation of Antibody Responses to the V1V2 and V3 Regions of HIV-1 Envelope by Immune Complex Vaccines

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02441

    Serum Ab responses induced by gp120 A244 /mAb complex vaccines. Mice were immunized with gp120 AE.A244 in complex with human IgG1 mAbs specific for V2i (2158), V2i (697), CD4bs (1331E), V3 (2219), or with no mAb in the presence of adjuvant MPL/DDA. (A–C) Sera collected 2 weeks after the fourth immunization were tested in ELISA for IgG reactivity to gp120 (A) , V3 (B) , and V1V2 (C) . (D) Sera were also evaluated for cross-reactivity with V3 and V1V2 from viruses of different HIV-1 subtypes. (E) Further mapping of V2 epitope was performed using overlapping V2 peptides P1 to P9. V2 sequence logo is shown to indicate amino-acid variability within the defined epitope region (orange-shaded box). (F) Sera were subjected to competition ELISA using V1V2 C.ZM109-1FD6 to assess the presence of V1V2-specific serum Abs able to compete with conformation-dependent V2i mAb 830A. **** p
    Figure Legend Snippet: Serum Ab responses induced by gp120 A244 /mAb complex vaccines. Mice were immunized with gp120 AE.A244 in complex with human IgG1 mAbs specific for V2i (2158), V2i (697), CD4bs (1331E), V3 (2219), or with no mAb in the presence of adjuvant MPL/DDA. (A–C) Sera collected 2 weeks after the fourth immunization were tested in ELISA for IgG reactivity to gp120 (A) , V3 (B) , and V1V2 (C) . (D) Sera were also evaluated for cross-reactivity with V3 and V1V2 from viruses of different HIV-1 subtypes. (E) Further mapping of V2 epitope was performed using overlapping V2 peptides P1 to P9. V2 sequence logo is shown to indicate amino-acid variability within the defined epitope region (orange-shaded box). (F) Sera were subjected to competition ELISA using V1V2 C.ZM109-1FD6 to assess the presence of V1V2-specific serum Abs able to compete with conformation-dependent V2i mAb 830A. **** p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Sequencing

    Serum Ab responses induced by vaccination with gp120 JRFL /mAb complexes. BALB/c mice were immunized with gp120 B.JRFL complexed with human IgG1 mAbs of defined specificity—C2 (1006-30D), V2i (2158), CD4bs (654), V3 (1006-15D)—or with no mAb. Immune complexes were administered 4 times subcutaneously in the presence of adjuvant MPL/DDA. Mice immunized with PBS and adjuvant (no gp120) served as negative controls. (A–C) Pooled sera collected 2 weeks after the last immunization were tested in ELISA for IgG reactivity against gp120 (A) , V1V2 (B) , or V3 (C) . (D) Sera from individual mice immunized with gp120 JRFL vs. gp120 JRFL /CD4bs mAb 654 were also tested for ELISA reactivity against V3. AUC: area under the titration curve of each serum sample; OD 405 : optical density at 405 nm obtained from designated serum dilution in ELISA with p-nitrophenyl phosphate substrate. * p
    Figure Legend Snippet: Serum Ab responses induced by vaccination with gp120 JRFL /mAb complexes. BALB/c mice were immunized with gp120 B.JRFL complexed with human IgG1 mAbs of defined specificity—C2 (1006-30D), V2i (2158), CD4bs (654), V3 (1006-15D)—or with no mAb. Immune complexes were administered 4 times subcutaneously in the presence of adjuvant MPL/DDA. Mice immunized with PBS and adjuvant (no gp120) served as negative controls. (A–C) Pooled sera collected 2 weeks after the last immunization were tested in ELISA for IgG reactivity against gp120 (A) , V1V2 (B) , or V3 (C) . (D) Sera from individual mice immunized with gp120 JRFL vs. gp120 JRFL /CD4bs mAb 654 were also tested for ELISA reactivity against V3. AUC: area under the titration curve of each serum sample; OD 405 : optical density at 405 nm obtained from designated serum dilution in ELISA with p-nitrophenyl phosphate substrate. * p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Titration

    V3-specific Ab responses induced by gp120 JRFL /mAb complexes vs. uncomplexed gp120 JRFL . (A) Sera from mice immunized with gp120 B.JRFL in complex with V2i mAb 2158, CD4bs mAb 654, or no mAb were tested in ELISA for IgG reactivity against V3 peptides of HIV-1 subtypes A, B, and C. (B) Overlapping V3 peptides P1 to P5 were used for epitope mapping. Sequence logo depicting V3 amino acid variations is included to show the central V3 regions that are targeted by Abs induced by gp120 JRFL (blue-shaded box) vs. gp120 JRFL /mAb complexes (orange-shaded box). (C) Sera from individual animals were tested for reactivity with overlapping V3 peptides P1 to P5 and demonstrated more uniform recognition of P2 and P3 peptides by all animals that received gp120 JRFL /CD4bs mAb 654 vs. uncomplexed gp120 JRFL. . ( D ) Sera from mice immunized with gp120 JRFL or gp120 JRFL /mAb complexes were also tested for IgG reactivity with different V1V2 antigens. AUC: area under the titration curve. PBS: Sera from control group that received PBS and adjuvant (no gp120).
    Figure Legend Snippet: V3-specific Ab responses induced by gp120 JRFL /mAb complexes vs. uncomplexed gp120 JRFL . (A) Sera from mice immunized with gp120 B.JRFL in complex with V2i mAb 2158, CD4bs mAb 654, or no mAb were tested in ELISA for IgG reactivity against V3 peptides of HIV-1 subtypes A, B, and C. (B) Overlapping V3 peptides P1 to P5 were used for epitope mapping. Sequence logo depicting V3 amino acid variations is included to show the central V3 regions that are targeted by Abs induced by gp120 JRFL (blue-shaded box) vs. gp120 JRFL /mAb complexes (orange-shaded box). (C) Sera from individual animals were tested for reactivity with overlapping V3 peptides P1 to P5 and demonstrated more uniform recognition of P2 and P3 peptides by all animals that received gp120 JRFL /CD4bs mAb 654 vs. uncomplexed gp120 JRFL. . ( D ) Sera from mice immunized with gp120 JRFL or gp120 JRFL /mAb complexes were also tested for IgG reactivity with different V1V2 antigens. AUC: area under the titration curve. PBS: Sera from control group that received PBS and adjuvant (no gp120).

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Titration

    30) Product Images from "Preclinical efficacy and safety of an anti-IL-1β vaccine for the treatment of type 2 diabetes"

    Article Title: Preclinical efficacy and safety of an anti-IL-1β vaccine for the treatment of type 2 diabetes

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2014.48

    Immunogenicity of Qβ-mIL-1b(D143K) in mice. ( a ) Induction of anti-mIL-1β antibody titers. Female C57BL/6 mice ( n = 5) were immunized twice (days 0 and 42, arrows) with 10 µg of Qβ-mIL-1b(D143K) in the presence of Alum. At the indicated time points, mouse IL-1β (wild type)-specific IgG antibody titers were measured by ELISA (filled black circles). At the same time points IL-1β neutralizing titers were determined ex vivo in the HeLa IL-6 secretion assay as described in Materials and Methods (open gray circles). Shown are mean titers ± SEM. ( b ) Ratio of IL-1β neutralizing versus ELISA titers over time. The ratios of IL-1β- neutralizing versus IL-1β-specific IgG ELISA titers were calculated for the group means of each time point of the experiment described in a . Vaccine injections are indicated by arrows. ( c ) Antibody responses in IL-1β-deficient mice. Groups ( n = 4) of female C57BL/6 IL-1β-deficient mice (-/-) or C57BL/6 control mice (+/+) were immunized on days 0, 14, and 28 with 10 µg of Qβ-mIL-1b(D143K) in the presence of Alum. On day 42, mouse IL-1β (wild type)- as well as Qβ-specific IgG antibody titers were measured by ELISA. Shown are mean titers ± SEM. ( d ) Induction of IL-1β-neutralizing titers in IL-1β-deficient mice. Mouse IL-1β (wild type)-neutralizing titers were determined in sera from day 42 of the experiment described in c . Shown are mean titers ± SEM. ( e ) In vivo neutralization of IL-1β in Qβ-mIL-1b(D143K)-immunized mice. Groups of female C57BL/6 mice ( n = 5) were immunized s.c. on days 0, 14, and 28 with 1 µg of Qβ-mIL-1b(D143K) or Qβ VLPs as control. On day 42, mice were challenged with an i.p. injection of 1 µg wild-type mouse IL-1β. Three hours after challenge, sera were collected and IL-6 levels were quantified with a Quantikine ELISA kit. Shown are data from individual mice and group means (* P
    Figure Legend Snippet: Immunogenicity of Qβ-mIL-1b(D143K) in mice. ( a ) Induction of anti-mIL-1β antibody titers. Female C57BL/6 mice ( n = 5) were immunized twice (days 0 and 42, arrows) with 10 µg of Qβ-mIL-1b(D143K) in the presence of Alum. At the indicated time points, mouse IL-1β (wild type)-specific IgG antibody titers were measured by ELISA (filled black circles). At the same time points IL-1β neutralizing titers were determined ex vivo in the HeLa IL-6 secretion assay as described in Materials and Methods (open gray circles). Shown are mean titers ± SEM. ( b ) Ratio of IL-1β neutralizing versus ELISA titers over time. The ratios of IL-1β- neutralizing versus IL-1β-specific IgG ELISA titers were calculated for the group means of each time point of the experiment described in a . Vaccine injections are indicated by arrows. ( c ) Antibody responses in IL-1β-deficient mice. Groups ( n = 4) of female C57BL/6 IL-1β-deficient mice (-/-) or C57BL/6 control mice (+/+) were immunized on days 0, 14, and 28 with 10 µg of Qβ-mIL-1b(D143K) in the presence of Alum. On day 42, mouse IL-1β (wild type)- as well as Qβ-specific IgG antibody titers were measured by ELISA. Shown are mean titers ± SEM. ( d ) Induction of IL-1β-neutralizing titers in IL-1β-deficient mice. Mouse IL-1β (wild type)-neutralizing titers were determined in sera from day 42 of the experiment described in c . Shown are mean titers ± SEM. ( e ) In vivo neutralization of IL-1β in Qβ-mIL-1b(D143K)-immunized mice. Groups of female C57BL/6 mice ( n = 5) were immunized s.c. on days 0, 14, and 28 with 1 µg of Qβ-mIL-1b(D143K) or Qβ VLPs as control. On day 42, mice were challenged with an i.p. injection of 1 µg wild-type mouse IL-1β. Three hours after challenge, sera were collected and IL-6 levels were quantified with a Quantikine ELISA kit. Shown are data from individual mice and group means (* P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Ex Vivo, In Vivo, Neutralization, Injection

    Safety assessment of the Qβ-mIL-1b(D143K) vaccine in mice. ( a ) Time course of anti-IL-1β antibody responses. Female C57BL/6 mice ( n = 4) were immunized s.c. on days 0, 14, and 35 (arrows) with 1 µg of Qβ-mIL-1b(D143K). On days 42, 70, 140, and 228, mice were challenged with i.p. injections of 1 µg wild-type mouse IL-1β. Three hours after challenge, sera were collected, and IL-6 levels were quantified by ELISA. Single data points (filled gray circles) represent individual animals. Antimouse IL-1β (wild type) IgG antibody titers were determined at the same time points by ELISA and are represented as group means ± SEM (black triangles). ( b ) Effect of increased IL-1β levels on antimouse IL-1β IgG antibody titers. Groups of female C57BL/6 mice ( n = 5) were immunized s.c. on day 0 with 50 µg Qβ-mIL-1b(D143K). On day 76, mice received either an i.v. injection of 100 ng wild-type mouse IL-1β (in 100 µl PBS), an i.p. injection of a mixture of 1 ng Escherichia coli lipopolysaccharide, and 20 mg N-galactosamine (Sigma-Aldrich), or a s.c. injection of 50 µg Qβ-mIL-1b(D143K). Control groups received either an i.v. injection of 100 µl PBS or a s.c. injection of 50 µg Qβ VLPs. Mice were bled on days 69, 76, 83, and 90 and mouse IL-1β (wild type)-specific IgG antibody titers were determined by ELISA. Shown are group means ± SEM. ( c ) Measurement of IL-1β-specific T cell responses after vaccination with Qβ-mIL-1b(D143K). Groups of mice were immunized with Qβ-mIL-1b(D143K)-p13 in the presence of CpG or Alum, respectively, as described in Materials and Methods . One group of female C57BL/6 mice was kept naive. After immunization, splenocytes were isolated from all mice and stimulated with BMDC that had been loaded either with synthetic p13 peptide or with wild-type mouse IL-1β. Nonspecific IFNγ-release from CD4 + T cells was determined by incubation of splenocytes with mock-pulsed BMDC (no antigen). Antigen-specific IFNγ-producing CD4 + T cells were determined by fluorescence-activated cell sorting. Shown are group means ± SEM. ( d ) Th cell dependence of anti-IL-1β antibody induction. Groups of mice received s.c. injections of either wild-type mouse IL-1β or mIL-1b(D143K)-p13, each in the presence of incomplete Freund’s adjuvant. A subgroup of mIL-1b(D143K)-p13-immunized mice were additionally injected with a depleting anti-CD4 antibody. Mouse IL-1β (wild type)-specific IgG antibody titers were analyzed by ELISA. Shown are group means ± SEM.
    Figure Legend Snippet: Safety assessment of the Qβ-mIL-1b(D143K) vaccine in mice. ( a ) Time course of anti-IL-1β antibody responses. Female C57BL/6 mice ( n = 4) were immunized s.c. on days 0, 14, and 35 (arrows) with 1 µg of Qβ-mIL-1b(D143K). On days 42, 70, 140, and 228, mice were challenged with i.p. injections of 1 µg wild-type mouse IL-1β. Three hours after challenge, sera were collected, and IL-6 levels were quantified by ELISA. Single data points (filled gray circles) represent individual animals. Antimouse IL-1β (wild type) IgG antibody titers were determined at the same time points by ELISA and are represented as group means ± SEM (black triangles). ( b ) Effect of increased IL-1β levels on antimouse IL-1β IgG antibody titers. Groups of female C57BL/6 mice ( n = 5) were immunized s.c. on day 0 with 50 µg Qβ-mIL-1b(D143K). On day 76, mice received either an i.v. injection of 100 ng wild-type mouse IL-1β (in 100 µl PBS), an i.p. injection of a mixture of 1 ng Escherichia coli lipopolysaccharide, and 20 mg N-galactosamine (Sigma-Aldrich), or a s.c. injection of 50 µg Qβ-mIL-1b(D143K). Control groups received either an i.v. injection of 100 µl PBS or a s.c. injection of 50 µg Qβ VLPs. Mice were bled on days 69, 76, 83, and 90 and mouse IL-1β (wild type)-specific IgG antibody titers were determined by ELISA. Shown are group means ± SEM. ( c ) Measurement of IL-1β-specific T cell responses after vaccination with Qβ-mIL-1b(D143K). Groups of mice were immunized with Qβ-mIL-1b(D143K)-p13 in the presence of CpG or Alum, respectively, as described in Materials and Methods . One group of female C57BL/6 mice was kept naive. After immunization, splenocytes were isolated from all mice and stimulated with BMDC that had been loaded either with synthetic p13 peptide or with wild-type mouse IL-1β. Nonspecific IFNγ-release from CD4 + T cells was determined by incubation of splenocytes with mock-pulsed BMDC (no antigen). Antigen-specific IFNγ-producing CD4 + T cells were determined by fluorescence-activated cell sorting. Shown are group means ± SEM. ( d ) Th cell dependence of anti-IL-1β antibody induction. Groups of mice received s.c. injections of either wild-type mouse IL-1β or mIL-1b(D143K)-p13, each in the presence of incomplete Freund’s adjuvant. A subgroup of mIL-1b(D143K)-p13-immunized mice were additionally injected with a depleting anti-CD4 antibody. Mouse IL-1β (wild type)-specific IgG antibody titers were analyzed by ELISA. Shown are group means ± SEM.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection, Isolation, Incubation, Fluorescence, FACS

    Characterization of the detoxified vaccine antigen hIL-1b(D145K). ( a ) hIL-1RI binding. Serial dilutions of wild-type hIL-1β or hIL-1b(D145K) were mixed with a constant amount of 1 nmol/l biotinylated hIL-1β and applied to ELISA plates that had been coated with hIL-1RI. Obtained OD values were reciprocally transformed to express % receptor binding of wild-type hIL-1β or hIL-1b(D145K), respectively. ( b ) Formation of the ternary hIL-1RI-hIL-1β-hIL-1RAcP signaling complex. Wild-type human IL-1β (0.4 µg/ml) or hIL-1b(D145K) (100 µg/ml) were incubated with hIL-1RAcP (1 µg/ml) and applied to ELISA plates that had been coated with 1 µg/ml of human IL-1RI. Formation of the ternary complex was detected with a hIL-1RAcP-specific antibody. ( c ) hIL-1RII binding. Experimental conditions were as in a but with hIL-1RII coated on the ELISA plate. ( d ) IL-6 secretion. HeLa cells were incubated with serial dilutions of wild-type hIL-1β or hIL-1b (D145K). After an incubation of 4 hours, IL-6 was quantified in supernatants by Sandwich ELISA. ( e ) Cytopathic effect. A375 cells were incubated with serial dilutions of either wild-type hIL-1β or hIL-1b(D145K). After 7 days, viable adherent cells were stained with crystal violet and quantified by measuring optical densities at 600 nm. Shown are mean values from triplicate measurements ± SEM. ( f ) In vivo inflammatory activity. Groups of female C57BL/6 mice ( n = 4) were injected i.p. with 1 µg of either wild-type hIL-1β or hIL-1b(D145K) or s.c. with 25 µg of the conjugate vaccine Qβ-hIL-1b(D145K). Three hours after injection, sera were collected, and IL-6 levels were quantified with a Quantikine ELISA kit (* P
    Figure Legend Snippet: Characterization of the detoxified vaccine antigen hIL-1b(D145K). ( a ) hIL-1RI binding. Serial dilutions of wild-type hIL-1β or hIL-1b(D145K) were mixed with a constant amount of 1 nmol/l biotinylated hIL-1β and applied to ELISA plates that had been coated with hIL-1RI. Obtained OD values were reciprocally transformed to express % receptor binding of wild-type hIL-1β or hIL-1b(D145K), respectively. ( b ) Formation of the ternary hIL-1RI-hIL-1β-hIL-1RAcP signaling complex. Wild-type human IL-1β (0.4 µg/ml) or hIL-1b(D145K) (100 µg/ml) were incubated with hIL-1RAcP (1 µg/ml) and applied to ELISA plates that had been coated with 1 µg/ml of human IL-1RI. Formation of the ternary complex was detected with a hIL-1RAcP-specific antibody. ( c ) hIL-1RII binding. Experimental conditions were as in a but with hIL-1RII coated on the ELISA plate. ( d ) IL-6 secretion. HeLa cells were incubated with serial dilutions of wild-type hIL-1β or hIL-1b (D145K). After an incubation of 4 hours, IL-6 was quantified in supernatants by Sandwich ELISA. ( e ) Cytopathic effect. A375 cells were incubated with serial dilutions of either wild-type hIL-1β or hIL-1b(D145K). After 7 days, viable adherent cells were stained with crystal violet and quantified by measuring optical densities at 600 nm. Shown are mean values from triplicate measurements ± SEM. ( f ) In vivo inflammatory activity. Groups of female C57BL/6 mice ( n = 4) were injected i.p. with 1 µg of either wild-type hIL-1β or hIL-1b(D145K) or s.c. with 25 µg of the conjugate vaccine Qβ-hIL-1b(D145K). Three hours after injection, sera were collected, and IL-6 levels were quantified with a Quantikine ELISA kit (* P

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay, Incubation, Sandwich ELISA, Staining, In Vivo, Activity Assay, Mouse Assay, Injection

    31) Product Images from "Anti‐β2‐glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression"

    Article Title: Anti‐β2‐glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.13229

    Dual‐reactivity of WB‐6 to cardiolipin‐β 2 ‐glycoprotein I (CL‐β 2 GPI) and DNA. (a) Direct‐binding enzyme‐linked immunosorbent assay (ELISA) to test the binding activity of WB‐6 to β 2 GPI, CL and CL‐β 2 GPI. (b) Direct‐binding ELISA to show the binding activity of WB‐6 and 2C10 to calf‐thymus (CT) native DNA. Mouse immunoglobulin (Ig)G2b, kappa monoclonal MPC‐11 is an isotype‐matched control without DNA‐binding activity. (c) Inhibition ELISA in which binding activity of WB‐6 to calf thymus (CT)‐DNA was inhibited by preincubation with poly(dT), poly(dA‐dT) or CT‐DNA.
    Figure Legend Snippet: Dual‐reactivity of WB‐6 to cardiolipin‐β 2 ‐glycoprotein I (CL‐β 2 GPI) and DNA. (a) Direct‐binding enzyme‐linked immunosorbent assay (ELISA) to test the binding activity of WB‐6 to β 2 GPI, CL and CL‐β 2 GPI. (b) Direct‐binding ELISA to show the binding activity of WB‐6 and 2C10 to calf‐thymus (CT) native DNA. Mouse immunoglobulin (Ig)G2b, kappa monoclonal MPC‐11 is an isotype‐matched control without DNA‐binding activity. (c) Inhibition ELISA in which binding activity of WB‐6 to calf thymus (CT)‐DNA was inhibited by preincubation with poly(dT), poly(dA‐dT) or CT‐DNA.

    Techniques Used: Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Inhibition

    32) Product Images from "Effects of Low-level Brodifacoum Exposure on the Feline Immune Response"

    Article Title: Effects of Low-level Brodifacoum Exposure on the Feline Immune Response

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26558-3

    Brodifacoum- and sham-treated cats have similar antibody responses to ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). Mean ELISA endpoint dilution, indicating last positive dilution shown. Standard deviation shown for each group. No significant differences noted between sham- and brodifacoum-treated groups. There was a trend towards lower anti-KLH antibody titers in brodifacoum-treated cats at week 6 (p = 0.17).
    Figure Legend Snippet: Brodifacoum- and sham-treated cats have similar antibody responses to ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). Mean ELISA endpoint dilution, indicating last positive dilution shown. Standard deviation shown for each group. No significant differences noted between sham- and brodifacoum-treated groups. There was a trend towards lower anti-KLH antibody titers in brodifacoum-treated cats at week 6 (p = 0.17).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

    33) Product Images from "Identification of a Continuous Neutralizing Epitope within UL128 of Human Cytomegalovirus"

    Article Title: Identification of a Continuous Neutralizing Epitope within UL128 of Human Cytomegalovirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.01857-16

    Binding of human serum antibodies to the UL128 peptide library. An ELISA was performed to determine the reactivity of the UL128 peptide library with commercial serum products from HCMV + (S4360, S4234, and S4371; SeraCare) or HCMV − (SNeg; SeraCare)
    Figure Legend Snippet: Binding of human serum antibodies to the UL128 peptide library. An ELISA was performed to determine the reactivity of the UL128 peptide library with commercial serum products from HCMV + (S4360, S4234, and S4371; SeraCare) or HCMV − (SNeg; SeraCare)

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    Mapping of the NAb 13B5 minimal binding site. (A and B) C- and N-terminally truncated peptides based on library peptide 40 were used in an ELISA to identify the shortest amino acid sequence needed for binding of NAb 13B5. (C) ELISA to compare 13B5 binding
    Figure Legend Snippet: Mapping of the NAb 13B5 minimal binding site. (A and B) C- and N-terminally truncated peptides based on library peptide 40 were used in an ELISA to identify the shortest amino acid sequence needed for binding of NAb 13B5. (C) ELISA to compare 13B5 binding

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing

    UL128 peptide library binding of 13B5 and Z9G11. An ELISA was used to detect the binding of PC-specific NAb 13B5 and nonneutralizing Ab Z9G11 to a UL128 peptide library consisting of 41 overlapping 15-mers with an offset of 4. OD, optical density.
    Figure Legend Snippet: UL128 peptide library binding of 13B5 and Z9G11. An ELISA was used to detect the binding of PC-specific NAb 13B5 and nonneutralizing Ab Z9G11 to a UL128 peptide library consisting of 41 overlapping 15-mers with an offset of 4. OD, optical density.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    34) Product Images from "Identification of a Chrysanthemic Ester as an Apolipoprotein E Inducer in Astrocytes"

    Article Title: Identification of a Chrysanthemic Ester as an Apolipoprotein E Inducer in Astrocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0162384

    Compound 82879 upregulates apoE secretion in primary human astrocytes. Primary human astrocytes from three donors genotyped for APOE ( A ) D1: E3/E4 , ( B ) D2: E3/E4 , ( C ) D3: E3/E3 ) were treated with DMSO alone, GW3965, or compound 82879 for 96 h in two independent experiments and apoE secretion levels were measured by ELISA. Graphs represent mean and SD of media apoE concentration normalized against total cellular protein content. Statistics were determined by a one-way ANOVA with a blocking factor (Experiment) and a Dunnett’s post-test (* p
    Figure Legend Snippet: Compound 82879 upregulates apoE secretion in primary human astrocytes. Primary human astrocytes from three donors genotyped for APOE ( A ) D1: E3/E4 , ( B ) D2: E3/E4 , ( C ) D3: E3/E3 ) were treated with DMSO alone, GW3965, or compound 82879 for 96 h in two independent experiments and apoE secretion levels were measured by ELISA. Graphs represent mean and SD of media apoE concentration normalized against total cellular protein content. Statistics were determined by a one-way ANOVA with a blocking factor (Experiment) and a Dunnett’s post-test (* p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Blocking Assay

    35) Product Images from "IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity"

    Article Title: IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151722

    IFN-γR signaling is required for Spt-GC formation and IgG production. (A and B) The percentages of B220 + Fas hi PNA hi GC B cells (A) and CD4 + CXCR5 hi PD-1 hi Tfh cells (B) were obtained from flow cytometric analysis of spleen cells of 3- and 6-mo-old B6 and B6.IFN-γR 1 −/− mice. Each symbol represents a mouse ( n = 11–15). (C) Representative histological images of spleen sections from 6-mo-old mice ( n = 5 per group) stained with the GC B cell marker PNA and anti-IgD. Bars, 150 µm. (D) Flow cytometric analysis of CD86 expression (MFI) on total B220 + B cells at 3 and 6 mo of age ( n = 5 mice per group). Error bars are mean ± SD. (E and F) Numbers of IgG + (E) and IgM + (F) splenic AFCs in 6-mo-old mice of indicated strains ( n = 5–9). (G–K) Analysis of serum titers of IgG, IgG 1 , IgG 2b, IgG 2c , and IgM Abs in 6-mo-old mice by ELISA. Each symbol represents a mouse ( n = 6–8). The data shown are the cumulative results of two or three independent experiments. Statistical values were determined using an unpaired, nonparametric, Mann–Whitney Student’s t test. Horizontal lines indicate mean values. *, P
    Figure Legend Snippet: IFN-γR signaling is required for Spt-GC formation and IgG production. (A and B) The percentages of B220 + Fas hi PNA hi GC B cells (A) and CD4 + CXCR5 hi PD-1 hi Tfh cells (B) were obtained from flow cytometric analysis of spleen cells of 3- and 6-mo-old B6 and B6.IFN-γR 1 −/− mice. Each symbol represents a mouse ( n = 11–15). (C) Representative histological images of spleen sections from 6-mo-old mice ( n = 5 per group) stained with the GC B cell marker PNA and anti-IgD. Bars, 150 µm. (D) Flow cytometric analysis of CD86 expression (MFI) on total B220 + B cells at 3 and 6 mo of age ( n = 5 mice per group). Error bars are mean ± SD. (E and F) Numbers of IgG + (E) and IgM + (F) splenic AFCs in 6-mo-old mice of indicated strains ( n = 5–9). (G–K) Analysis of serum titers of IgG, IgG 1 , IgG 2b, IgG 2c , and IgM Abs in 6-mo-old mice by ELISA. Each symbol represents a mouse ( n = 6–8). The data shown are the cumulative results of two or three independent experiments. Statistical values were determined using an unpaired, nonparametric, Mann–Whitney Student’s t test. Horizontal lines indicate mean values. *, P

    Techniques Used: Single-particle Tracking, Flow Cytometry, Mouse Assay, Staining, Marker, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Critical role of T-bet in Spt-GC development and Th1 Ab responses. (A and B) Percentages of B220 + Fas hi PNA hi GC B cells (A) and CD4 + CXCR5 hi PD-1 hi Tfh cells (B) obtained from flow cytometric analysis of spleen cells of 3-mo-old B6 and B6. T-bet −/− mice. Each symbol represents a mouse ( n = 3–4). (C) Representative images of spleen sections from 3-mo-old B6 and B6. T-bet −/− mice stained with GL7, anti-CD4, and anti-IgD. GC area measurement is shown in the right panel ( n = 3–4 mice per group). Bars, 150 µm. (D–F) Total serum Ab titers of IgG (D), IgG 2b (E), and IgG 2c (F) measured by ELISA ( n = 3–4 per group). (G) Maintenance of ex vivo–generated GC B cell phenotype of B6 and B6. T-bet −/− mice in media alone or with IFN-γ. (H) Quantitative RT-PCR analysis of IFN-γ transcripts in ex vivo–generated GC B cells (as described in G) from B6 and B6. T-bet −/− mice ( n = 3–4 mice per group). The data shown in G and H are representative of three independent experiments. In D–F, statistical analysis was performed by one-way ANOVA with a follow-up Tukey multiple-comparison test. In A–C, G, and H, statistical values were determined using an unpaired, nonparametric, Mann–Whitney Student’s t test. Error bars are mean ± SD. *, P
    Figure Legend Snippet: Critical role of T-bet in Spt-GC development and Th1 Ab responses. (A and B) Percentages of B220 + Fas hi PNA hi GC B cells (A) and CD4 + CXCR5 hi PD-1 hi Tfh cells (B) obtained from flow cytometric analysis of spleen cells of 3-mo-old B6 and B6. T-bet −/− mice. Each symbol represents a mouse ( n = 3–4). (C) Representative images of spleen sections from 3-mo-old B6 and B6. T-bet −/− mice stained with GL7, anti-CD4, and anti-IgD. GC area measurement is shown in the right panel ( n = 3–4 mice per group). Bars, 150 µm. (D–F) Total serum Ab titers of IgG (D), IgG 2b (E), and IgG 2c (F) measured by ELISA ( n = 3–4 per group). (G) Maintenance of ex vivo–generated GC B cell phenotype of B6 and B6. T-bet −/− mice in media alone or with IFN-γ. (H) Quantitative RT-PCR analysis of IFN-γ transcripts in ex vivo–generated GC B cells (as described in G) from B6 and B6. T-bet −/− mice ( n = 3–4 mice per group). The data shown in G and H are representative of three independent experiments. In D–F, statistical analysis was performed by one-way ANOVA with a follow-up Tukey multiple-comparison test. In A–C, G, and H, statistical values were determined using an unpaired, nonparametric, Mann–Whitney Student’s t test. Error bars are mean ± SD. *, P

    Techniques Used: Single-particle Tracking, Flow Cytometry, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Ex Vivo, Generated, Quantitative RT-PCR, MANN-WHITNEY

    36) Product Images from "Molecular Signatures of the Evolving Immune Response in Mice following a Bordetella pertussis Infection"

    Article Title: Molecular Signatures of the Evolving Immune Response in Mice following a Bordetella pertussis Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104548

    Antibody profiling after B. pertussis infection. Antibody responses were determined in mouse sera after intranasal infection by whole cell B. pertussis ELISA or MIA and expressed as OD450 or Fluorescent Intensity (F.I.) respectively. (A) IgM against OMV B1917 were absent. (B) Whole cell B. pertussis ELISA indicated IgG antibody formation 10–14 days p.i., which increased until day 28 days p.i. (C–G) Levels of total IgG and individual subtypes (IgG1, IgG2a, IgG2b and IgG3) against OMV B1917 indicated presence of multiple subclasses. (H) Anti-OMV IgA antibodies in serum were induced after 14–28 days p.i. (I) In lung lysates, anti-OMV IgA antibodies were detected 14 and 28 days p.i. I p -values were determined by one-way ANOVA with multiple comparison compared to naive mice (* = p
    Figure Legend Snippet: Antibody profiling after B. pertussis infection. Antibody responses were determined in mouse sera after intranasal infection by whole cell B. pertussis ELISA or MIA and expressed as OD450 or Fluorescent Intensity (F.I.) respectively. (A) IgM against OMV B1917 were absent. (B) Whole cell B. pertussis ELISA indicated IgG antibody formation 10–14 days p.i., which increased until day 28 days p.i. (C–G) Levels of total IgG and individual subtypes (IgG1, IgG2a, IgG2b and IgG3) against OMV B1917 indicated presence of multiple subclasses. (H) Anti-OMV IgA antibodies in serum were induced after 14–28 days p.i. (I) In lung lysates, anti-OMV IgA antibodies were detected 14 and 28 days p.i. I p -values were determined by one-way ANOVA with multiple comparison compared to naive mice (* = p

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay

    37) Product Images from "Assessment of ELISA as Endpoint in Neuronal Cell-based Assay for BoNT Detection using hiPSC derived neurons"

    Article Title: Assessment of ELISA as Endpoint in Neuronal Cell-based Assay for BoNT Detection using hiPSC derived neurons

    Journal: Journal of pharmacological and toxicological methods

    doi: 10.1016/j.vascn.2017.04.013

    Plate selection for ELISA. HIP Neurons were exposed to serial dilutions of BoNT/A1, and cell lysates were analyzed for cleaved SNAP-25 by ELISA using either a Nunc Polystyrene Maxisorp plate (Nunc) or a Thermo Scientific Immulon Immunoassay plate (Immulon). The signal was detected with ultra TMB. The average and standard deviations of 4 replicates is shown. Dose response curves were fitted using Prims 6 software and a nonlinear regression (variable slope, four parameter).
    Figure Legend Snippet: Plate selection for ELISA. HIP Neurons were exposed to serial dilutions of BoNT/A1, and cell lysates were analyzed for cleaved SNAP-25 by ELISA using either a Nunc Polystyrene Maxisorp plate (Nunc) or a Thermo Scientific Immulon Immunoassay plate (Immulon). The signal was detected with ultra TMB. The average and standard deviations of 4 replicates is shown. Dose response curves were fitted using Prims 6 software and a nonlinear regression (variable slope, four parameter).

    Techniques Used: Selection, Enzyme-linked Immunosorbent Assay, Software

    38) Product Images from "Innate immunity and PBC: Activated invariant NKT cells exacerbate murine autoimmune cholangitis and fibrosis"

    Article Title: Innate immunity and PBC: Activated invariant NKT cells exacerbate murine autoimmune cholangitis and fibrosis

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.24113

    α-GalCer induced IFN-γ and IL-4 production of iNKT cells and maturation of dendritic cells. C57BL/6 mice were intravenously injected with α-GalCer or PBS. (A) Serum samples were collected at 0, 2, 6, 10, 24, and 48 hours after α-GalCer or PBS injection. IFN-γ and IL-4 were measured by ELISA. n=5 mice per group. (B) IFN-γ and IL-4 secretion from liver mononuclear cells 24 hours after α-GalCer injection was assayed by staining with surface markers and intracellular anti-mouse IFN-γ and IL-4 Abs. (C) Surface molecule expression on DCs was determined by flow cytometry. Data shown are representative flow cytometric profiles of MHC class I (H-2K b ), MHC class II (I-A b ), CD1d, CD40, CD80, and CD86 expression on the gated DCs (CD11c + NK1.1 − cells) in liver and spleen. The shadow area is an isotype control; the non-shadow area reflects surface molecule expression. The quantity of surface molecules per cell is indicated by the mean fluorescence intensity (MFI). Results are representative of three independent experiments.
    Figure Legend Snippet: α-GalCer induced IFN-γ and IL-4 production of iNKT cells and maturation of dendritic cells. C57BL/6 mice were intravenously injected with α-GalCer or PBS. (A) Serum samples were collected at 0, 2, 6, 10, 24, and 48 hours after α-GalCer or PBS injection. IFN-γ and IL-4 were measured by ELISA. n=5 mice per group. (B) IFN-γ and IL-4 secretion from liver mononuclear cells 24 hours after α-GalCer injection was assayed by staining with surface markers and intracellular anti-mouse IFN-γ and IL-4 Abs. (C) Surface molecule expression on DCs was determined by flow cytometry. Data shown are representative flow cytometric profiles of MHC class I (H-2K b ), MHC class II (I-A b ), CD1d, CD40, CD80, and CD86 expression on the gated DCs (CD11c + NK1.1 − cells) in liver and spleen. The shadow area is an isotype control; the non-shadow area reflects surface molecule expression. The quantity of surface molecules per cell is indicated by the mean fluorescence intensity (MFI). Results are representative of three independent experiments.

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence

    39) Product Images from "Incorporation of a GPI-anchored engineered cytokine as a molecular adjuvant enhances the immunogenicity of HIV VLPs"

    Article Title: Incorporation of a GPI-anchored engineered cytokine as a molecular adjuvant enhances the immunogenicity of HIV VLPs

    Journal: Scientific Reports

    doi: 10.1038/srep11856

    Mucosal antibody endpoint titers. Mucosal samples were collected at week 12, 4 weeks after the last boosting immunization. Env-specific IgG and IgA endpoint titers were detected by ELISA as described in Materials and Methods. a , saliva IgG; b , saliva IgA; c , v a ginal IgG; d , Vaginal IgA. Data were depicte d as means ± standard deviations. *P
    Figure Legend Snippet: Mucosal antibody endpoint titers. Mucosal samples were collected at week 12, 4 weeks after the last boosting immunization. Env-specific IgG and IgA endpoint titers were detected by ELISA as described in Materials and Methods. a , saliva IgG; b , saliva IgA; c , v a ginal IgG; d , Vaginal IgA. Data were depicte d as means ± standard deviations. *P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    40) Product Images from "Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs"

    Article Title: Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs

    Journal: Journal of Virology

    doi: 10.1128/JVI.00369-18

    Vaccination regimens and characterization of binding antibody responses. (A) Vaccination regimens for guinea pigs immunized with a longitudinal prime/boost vaccination schedule. Animals were vaccinated at weeks 0, 4, 8, 12, 62, 66, 70, 74, 104, 108, 112, and 116 utilizing the listed vaccination regimens and bled 4 weeks after each vaccination, as well as at weeks 138 and 200. C97, C97ZA012 gp140; 92UG, 92UG037 gp140; Mos, mosaic gp140. Error bars represent the standard deviations. (B) Binding antibody titers for HIV-1 Env gp140s of different clades as measured by endpoint ELISAs. (C) Binding antibody titers for HIV-1 C97ZA012 gp140, showing specific isotype and subclass responses, as measured utilizing endpoint ELISAs. Error bars represent the standard deviations. (D) Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) × 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and > 50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.
    Figure Legend Snippet: Vaccination regimens and characterization of binding antibody responses. (A) Vaccination regimens for guinea pigs immunized with a longitudinal prime/boost vaccination schedule. Animals were vaccinated at weeks 0, 4, 8, 12, 62, 66, 70, 74, 104, 108, 112, and 116 utilizing the listed vaccination regimens and bled 4 weeks after each vaccination, as well as at weeks 138 and 200. C97, C97ZA012 gp140; 92UG, 92UG037 gp140; Mos, mosaic gp140. Error bars represent the standard deviations. (B) Binding antibody titers for HIV-1 Env gp140s of different clades as measured by endpoint ELISAs. (C) Binding antibody titers for HIV-1 C97ZA012 gp140, showing specific isotype and subclass responses, as measured utilizing endpoint ELISAs. Error bars represent the standard deviations. (D) Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) × 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and > 50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    41) Product Images from "Evaluation of thermo-stability of bluetongue virus recombinant VP7 antigen in indirect ELISA"

    Article Title: Evaluation of thermo-stability of bluetongue virus recombinant VP7 antigen in indirect ELISA

    Journal: VirusDisease

    doi: 10.1007/s13337-014-0244-6

    a Thermo-stability of rVP7 protein of different groups exposed at 45 °C; b ratio of positive to negative (P/N) serum reactivity in Indirect ELISA
    Figure Legend Snippet: a Thermo-stability of rVP7 protein of different groups exposed at 45 °C; b ratio of positive to negative (P/N) serum reactivity in Indirect ELISA

    Techniques Used: Indirect ELISA

    a Thermo-stability of rVP7 protein of different groups exposed at 4 °C; b ratio of positive to negative (P/N) serum reactivity in Indirect ELISA
    Figure Legend Snippet: a Thermo-stability of rVP7 protein of different groups exposed at 4 °C; b ratio of positive to negative (P/N) serum reactivity in Indirect ELISA

    Techniques Used: Indirect ELISA

    a Thermo-stability of rVP7 protein of different groups exposed at 37 °C; b ratio of positive to negative (P/N) serum reactivity in Indirect ELISA
    Figure Legend Snippet: a Thermo-stability of rVP7 protein of different groups exposed at 37 °C; b ratio of positive to negative (P/N) serum reactivity in Indirect ELISA

    Techniques Used: Indirect ELISA

    a Thermo-stability of rVP7 protein of different groups exposed at 25 °C; b ratio of positive to negative (P/N) serum reactivity in Indirect ELISA
    Figure Legend Snippet: a Thermo-stability of rVP7 protein of different groups exposed at 25 °C; b ratio of positive to negative (P/N) serum reactivity in Indirect ELISA

    Techniques Used: Indirect ELISA

    42) Product Images from "Molecular Landscape of Anti-Drug Antibodies Reveals the Mechanism of the Immune Response Following Treatment With TNFα Antagonists"

    Article Title: Molecular Landscape of Anti-Drug Antibodies Reveals the Mechanism of the Immune Response Following Treatment With TNFα Antagonists

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02921

    AHLC and the newly developed mAb-F(ab') 2 based bio-immunoassay configuration and their application on serum samples from patients treated with IFX. (A) AHLC assay is based on an ELISA where TNFα is used as the coating agent, following the incubation with the mAb drug followed by serial dilutions of the tested sera. anti-λ HRP conjugate is used at the detection phase. (B) The newly developed mAb-F(ab') 2 based bio-immunoassay configuration. The assay is based on an ELISA where mAb-F(ab') 2 is used as the coating agent followed by serial dilutions of the tested sera. Anti-Fc HRP conjugate is used at the detection phase. (C) ELISA obtained by utilizing the AHLC assay on two serum samples. Using this assay, one of the tested sera showed detectable levels of ADA [AHLC (+) ] and one had no detectable levels of ADA [AHLC (−) ]. (D) Both serum samples were tested by the newly developed mAb-F(ab') 2 based bio-immunoassay. This assay was able to detect ADA in both sera. For (C,D) , averages were calculated as mean from triplicates, with error bars indicating s.d.
    Figure Legend Snippet: AHLC and the newly developed mAb-F(ab') 2 based bio-immunoassay configuration and their application on serum samples from patients treated with IFX. (A) AHLC assay is based on an ELISA where TNFα is used as the coating agent, following the incubation with the mAb drug followed by serial dilutions of the tested sera. anti-λ HRP conjugate is used at the detection phase. (B) The newly developed mAb-F(ab') 2 based bio-immunoassay configuration. The assay is based on an ELISA where mAb-F(ab') 2 is used as the coating agent followed by serial dilutions of the tested sera. Anti-Fc HRP conjugate is used at the detection phase. (C) ELISA obtained by utilizing the AHLC assay on two serum samples. Using this assay, one of the tested sera showed detectable levels of ADA [AHLC (+) ] and one had no detectable levels of ADA [AHLC (−) ]. (D) Both serum samples were tested by the newly developed mAb-F(ab') 2 based bio-immunoassay. This assay was able to detect ADA in both sera. For (C,D) , averages were calculated as mean from triplicates, with error bars indicating s.d.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation

    Configuration of the assay for determining the neutralization index of ADA in patient sera and competitive ELISA between ADA and rhTNFα. (A) The newly developed mAb-F(ab') 2 based bio-immunoassay configuration (left) and the modified configuration where mAb-F(ab') 2 binding site is blocked by saturating the assay with rhTNFα (right). (B) Competitive effect of rhTNFα on ADA binding to IFX-F(ab') 2 . ELISA plate was coated with 5 μg/ml of IFX-F(ab') 2 . ADA standard was diluted 3-fold in blocking solution supplemented with 5 nM rhTNFα. ADA diluted 3-fold in blocking solution without the presence of rhTNFα served as a control.
    Figure Legend Snippet: Configuration of the assay for determining the neutralization index of ADA in patient sera and competitive ELISA between ADA and rhTNFα. (A) The newly developed mAb-F(ab') 2 based bio-immunoassay configuration (left) and the modified configuration where mAb-F(ab') 2 binding site is blocked by saturating the assay with rhTNFα (right). (B) Competitive effect of rhTNFα on ADA binding to IFX-F(ab') 2 . ELISA plate was coated with 5 μg/ml of IFX-F(ab') 2 . ADA standard was diluted 3-fold in blocking solution supplemented with 5 nM rhTNFα. ADA diluted 3-fold in blocking solution without the presence of rhTNFα served as a control.

    Techniques Used: Neutralization, Competitive ELISA, Modification, Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

    43) Product Images from "Hormonal modulators of glial ABCA1 and apoE levels [S]"

    Article Title: Hormonal modulators of glial ABCA1 and apoE levels [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M042473

    Estrogens do not increase apoE secretion from CCF-STTG1 cells, whereas lynestrenol and progesterone increase apoE secretion in a dose-dependent manner. CCF-STTG1 cells were seeded in 384-well plates at 17.5 K cells/well and treated with 0.3125–40.0 µM of (A) estrone, (B) 17α-estradiol, (C) 17β-estradiol, (D) estriol, (E) lynestrenol, or (F) progesterone for 96 h. Data represent fold difference of secreted media apoE measured by ELISA compared with the vehicle control (not shown). Graphs represent mean and SD of quadruplicate wells, analyzed by one-way ANOVA with Tukey's post-test. * P
    Figure Legend Snippet: Estrogens do not increase apoE secretion from CCF-STTG1 cells, whereas lynestrenol and progesterone increase apoE secretion in a dose-dependent manner. CCF-STTG1 cells were seeded in 384-well plates at 17.5 K cells/well and treated with 0.3125–40.0 µM of (A) estrone, (B) 17α-estradiol, (C) 17β-estradiol, (D) estriol, (E) lynestrenol, or (F) progesterone for 96 h. Data represent fold difference of secreted media apoE measured by ELISA compared with the vehicle control (not shown). Graphs represent mean and SD of quadruplicate wells, analyzed by one-way ANOVA with Tukey's post-test. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    44) Product Images from "Autoimmunity against Fibrinogen Mediates Inflammatory Arthritis in Mice"

    Article Title: Autoimmunity against Fibrinogen Mediates Inflammatory Arthritis in Mice

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0901639

    Anti-CCP–positive RA patients possess Abs that recognize in vitro citrullinated fibrinogen. IgG ( A ) and IgM ( B ) autoantibodies targeting in vitro citrullinated fibrinogen were measured by ELISA in plasma samples derived from healthy individuals
    Figure Legend Snippet: Anti-CCP–positive RA patients possess Abs that recognize in vitro citrullinated fibrinogen. IgG ( A ) and IgM ( B ) autoantibodies targeting in vitro citrullinated fibrinogen were measured by ELISA in plasma samples derived from healthy individuals

    Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay, Derivative Assay

    45) Product Images from "Spontaneous and tetracosactide‐induced anti‐ACTH antibodies in man"

    Article Title: Spontaneous and tetracosactide‐induced anti‐ACTH antibodies in man

    Journal: Clinical Endocrinology

    doi: 10.1111/cen.12795

    Antitetracosactide immunoreactivity detected with immunoblotting and ELISA. Panel A: Detection of serum binding activity to immunoblotted ACTH1–24, at baseline and week 20 for participants 1, 6, 7 and 10, as assessed by the band of approximately 4 kDa, the expected molecular weight of ACTH1–24. Sera from healthy subjects were used as a negative control. Panel B: Immunoblotting was performed with blots made using both tetracosactide (ACTH1–24) peptide and full‐length human ACTH1–39, both 2·5 μg per well. Representative positive sera from two participants (sera A and B), a monoclonal anti‐ACTH1–24 antibody (C) and serum from a healthy control (serum D) are shown. The monoclonal anti‐ACTH antibody recognizes bands of the same molecular mass as the positive participant sera for both ACTH1–24 and ACTH1–39 peptides, which was not seen with control sera. Panel C. Anti‐tetracosactide binding activity on immunoblotting from all the patients of RoSA study was quantified using ELISA. Results (±SEM) are expressed as arbitrary units of absorbance at 450 n m , having subtracted the background absorbance (mean of three wells without serum). A total of 11 females and two males (04 and 08) participated in this study. All except five patients had relative absorbance units of less than 0·21 at baseline. A positive control was run on each occasion to check for interassay variation, which was within 10% on successive plates.
    Figure Legend Snippet: Antitetracosactide immunoreactivity detected with immunoblotting and ELISA. Panel A: Detection of serum binding activity to immunoblotted ACTH1–24, at baseline and week 20 for participants 1, 6, 7 and 10, as assessed by the band of approximately 4 kDa, the expected molecular weight of ACTH1–24. Sera from healthy subjects were used as a negative control. Panel B: Immunoblotting was performed with blots made using both tetracosactide (ACTH1–24) peptide and full‐length human ACTH1–39, both 2·5 μg per well. Representative positive sera from two participants (sera A and B), a monoclonal anti‐ACTH1–24 antibody (C) and serum from a healthy control (serum D) are shown. The monoclonal anti‐ACTH antibody recognizes bands of the same molecular mass as the positive participant sera for both ACTH1–24 and ACTH1–39 peptides, which was not seen with control sera. Panel C. Anti‐tetracosactide binding activity on immunoblotting from all the patients of RoSA study was quantified using ELISA. Results (±SEM) are expressed as arbitrary units of absorbance at 450 n m , having subtracted the background absorbance (mean of three wells without serum). A total of 11 females and two males (04 and 08) participated in this study. All except five patients had relative absorbance units of less than 0·21 at baseline. A positive control was run on each occasion to check for interassay variation, which was within 10% on successive plates.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Molecular Weight, Negative Control, Positive Control

    Anti‐tetracosactide binding activity detected by ELISA and immunoblotting. Panel A. Relative quantitation of anti‐tetracosactide binding activity using ELISA with sera from 102 controls, 131 unrelated patients with AAD, 92 patients with Graves’ disease and 15 patients with isolated ACTH deficiency. Tetracosactide (ACTH1–24) 1 μg/ml was bound to solid phase ELISA plates (NUNC maxisorp). Results (±SEM) are expressed as arbitrary units of absorbance at 450 n m , having subtracted the background absorbance (mean of three wells without serum). A positive control was run on each occasion to check for interassay variation, which was within 10% on successive plates. ELISA of the 102 control sera (from anonymous hospital attendees with negative autoantibody status) showed absorbance readings of ≤0·2 units in 101 sera, with 1 serum showing an absorbance of 0·6 units. Panel B. All positive samples were run against an identical ELISA protocol, with plates coated with octreotide (somatostatin 1–8) 1 μg/ml. None of the sera gave a positive signal in the assay with octreotide. Panel C. All positive patient sera in the ELISA were tested against the tetracosactide peptide on immunoblotting, and all 41 showed specific 4 kDa binding. Representative positive sera from six patients (A–F) are shown.
    Figure Legend Snippet: Anti‐tetracosactide binding activity detected by ELISA and immunoblotting. Panel A. Relative quantitation of anti‐tetracosactide binding activity using ELISA with sera from 102 controls, 131 unrelated patients with AAD, 92 patients with Graves’ disease and 15 patients with isolated ACTH deficiency. Tetracosactide (ACTH1–24) 1 μg/ml was bound to solid phase ELISA plates (NUNC maxisorp). Results (±SEM) are expressed as arbitrary units of absorbance at 450 n m , having subtracted the background absorbance (mean of three wells without serum). A positive control was run on each occasion to check for interassay variation, which was within 10% on successive plates. ELISA of the 102 control sera (from anonymous hospital attendees with negative autoantibody status) showed absorbance readings of ≤0·2 units in 101 sera, with 1 serum showing an absorbance of 0·6 units. Panel B. All positive samples were run against an identical ELISA protocol, with plates coated with octreotide (somatostatin 1–8) 1 μg/ml. None of the sera gave a positive signal in the assay with octreotide. Panel C. All positive patient sera in the ELISA were tested against the tetracosactide peptide on immunoblotting, and all 41 showed specific 4 kDa binding. Representative positive sera from six patients (A–F) are shown.

    Techniques Used: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Isolation, Positive Control

    46) Product Images from "Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs"

    Article Title: Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs

    Journal: Journal of Virology

    doi: 10.1128/JVI.00369-18

    Vaccination regimens and characterization of binding antibody responses. (A) Vaccination regimens for guinea pigs immunized with a longitudinal prime/boost vaccination schedule. Animals were vaccinated at weeks 0, 4, 8, 12, 62, 66, 70, 74, 104, 108, 112, and 116 utilizing the listed vaccination regimens and bled 4 weeks after each vaccination, as well as at weeks 138 and 200. C97, C97ZA012 gp140; 92UG, 92UG037 gp140; Mos, mosaic gp140. Error bars represent the standard deviations. (B) Binding antibody titers for HIV-1 Env gp140s of different clades as measured by endpoint ELISAs. (C) Binding antibody titers for HIV-1 C97ZA012 gp140, showing specific isotype and subclass responses, as measured utilizing endpoint ELISAs. Error bars represent the standard deviations. (D) Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) × 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and > 50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.
    Figure Legend Snippet: Vaccination regimens and characterization of binding antibody responses. (A) Vaccination regimens for guinea pigs immunized with a longitudinal prime/boost vaccination schedule. Animals were vaccinated at weeks 0, 4, 8, 12, 62, 66, 70, 74, 104, 108, 112, and 116 utilizing the listed vaccination regimens and bled 4 weeks after each vaccination, as well as at weeks 138 and 200. C97, C97ZA012 gp140; 92UG, 92UG037 gp140; Mos, mosaic gp140. Error bars represent the standard deviations. (B) Binding antibody titers for HIV-1 Env gp140s of different clades as measured by endpoint ELISAs. (C) Binding antibody titers for HIV-1 C97ZA012 gp140, showing specific isotype and subclass responses, as measured utilizing endpoint ELISAs. Error bars represent the standard deviations. (D) Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) × 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and > 50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    47) Product Images from "Ultrasensitive isolation, identification and quantification of DNA–protein adducts by ELISA-based RADAR assay"

    Article Title: Ultrasensitive isolation, identification and quantification of DNA–protein adducts by ELISA-based RADAR assay

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku490

    Reproducibility of detection of Top1-DPCC by ELISA-based RADAR assay. ( A ) Specificity of detection by ELISA assay using an anti-Top1 antibody. ELISA assays were performed on DNA isolates of CCRF-CEM treated with cognate (TPT) versus non-cognate (VP16) topoisomerase poison, and compared to an untreated control (NT). ( B ) Comparison of ELISA and slot-blot immunoassays of fold induction of Top1-DPCC, assayed in samples from HCT116 cells treated with CPT, containing indicated initial amounts of DNA. ( C ) Z’ factor of Top1 DPCC detection by ELISA-based RADAR assay. Z′ factor was calculated for each DNA dilution indicated using replicates of HCT116 cells treated with CPT and untreated controls.
    Figure Legend Snippet: Reproducibility of detection of Top1-DPCC by ELISA-based RADAR assay. ( A ) Specificity of detection by ELISA assay using an anti-Top1 antibody. ELISA assays were performed on DNA isolates of CCRF-CEM treated with cognate (TPT) versus non-cognate (VP16) topoisomerase poison, and compared to an untreated control (NT). ( B ) Comparison of ELISA and slot-blot immunoassays of fold induction of Top1-DPCC, assayed in samples from HCT116 cells treated with CPT, containing indicated initial amounts of DNA. ( C ) Z’ factor of Top1 DPCC detection by ELISA-based RADAR assay. Z′ factor was calculated for each DNA dilution indicated using replicates of HCT116 cells treated with CPT and untreated controls.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Dot Blot, Cycling Probe Technology

    Detection of Top1-DPCC by a direct ELISA immunoassay. ( A ) Comparison of ELISA immunoassays for Top1-DPCC using DNA isolates (80 ng DNA per well) from untreated (NT) or TPT-treated HCT116 cells that were adsorbed to standard plates following digestion with Benzonase nuclease (left) or to Reacti-Bind-coated plates (right). ( B ) Linearity range and sensitivity of ELISA immunoassay for detection of Top1-DPCC in lysates from untreated (NT) or TPT-treated HCT116 cells, assayed in samples containing indicated initial amounts of DNA digested with Benzonase prior to adsorption to plates. ( C ) Fold induction of Top1-DPCC (ratio of treated to untreated samples) in HCT116 cells treated with CPT or TPT and GM639 cells treated with CPT, assayed in samples containing indicated initial amounts of DNA digested with Benzonase prior to adsorption to plates.
    Figure Legend Snippet: Detection of Top1-DPCC by a direct ELISA immunoassay. ( A ) Comparison of ELISA immunoassays for Top1-DPCC using DNA isolates (80 ng DNA per well) from untreated (NT) or TPT-treated HCT116 cells that were adsorbed to standard plates following digestion with Benzonase nuclease (left) or to Reacti-Bind-coated plates (right). ( B ) Linearity range and sensitivity of ELISA immunoassay for detection of Top1-DPCC in lysates from untreated (NT) or TPT-treated HCT116 cells, assayed in samples containing indicated initial amounts of DNA digested with Benzonase prior to adsorption to plates. ( C ) Fold induction of Top1-DPCC (ratio of treated to untreated samples) in HCT116 cells treated with CPT or TPT and GM639 cells treated with CPT, assayed in samples containing indicated initial amounts of DNA digested with Benzonase prior to adsorption to plates.

    Techniques Used: Direct ELISA, Enzyme-linked Immunosorbent Assay, Adsorption, Cycling Probe Technology

    Detection of GyrA–DNA adducts by ELISA-based RADAR assay. DNA and DNA–protein adducts were recovered and GyrA quantified in samples containing 0.2 μg DNA from untreated E. coli (gray) or E. coli treated with nalidixic acid (100 μg/ml) or ciprofloxacin (20 μg/ml) for 45 min. ( A ) Relative DNA recovery expressed as percentage of untreated sample. ( B ) Averages of fold GyrA-DNA DPCC induction calculated from triplicates of a representative ELISA experiment with anti-GyrA antibodies. Error bars represent standard deviation.
    Figure Legend Snippet: Detection of GyrA–DNA adducts by ELISA-based RADAR assay. DNA and DNA–protein adducts were recovered and GyrA quantified in samples containing 0.2 μg DNA from untreated E. coli (gray) or E. coli treated with nalidixic acid (100 μg/ml) or ciprofloxacin (20 μg/ml) for 45 min. ( A ) Relative DNA recovery expressed as percentage of untreated sample. ( B ) Averages of fold GyrA-DNA DPCC induction calculated from triplicates of a representative ELISA experiment with anti-GyrA antibodies. Error bars represent standard deviation.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

    Detection of Top2a–DNA adducts by ELISA-based RADAR assay. ( A ) Specificity of detection by ELISA assay using an anti-Top2a antibody. ELISA assays were performed with DNA isolates of CCRF-CEM cells treated with cognate (VP16) or non-cognate (TPT) topoisomerase poison, and compared to an untreated control (NT). ( B ) Kinetics of Top2a–DPCC formation. Top2a-DPCC were detected by ELISA-based RADAR of DNA isolates (0.5 μg DNA per well) from CCRF-CEM and MOLT4 cells treated with 50 mM VP16 for indicated times and lysed in LS2. Left panel shows averages of replicates of A 450 reading from a representative ELISA experiment with anti-Top2a antibodies. Right panel shows averages of fold Top2a-DPCC induction calculated from two ELISA experiments performed on different days. Error bars represent standard deviation.
    Figure Legend Snippet: Detection of Top2a–DNA adducts by ELISA-based RADAR assay. ( A ) Specificity of detection by ELISA assay using an anti-Top2a antibody. ELISA assays were performed with DNA isolates of CCRF-CEM cells treated with cognate (VP16) or non-cognate (TPT) topoisomerase poison, and compared to an untreated control (NT). ( B ) Kinetics of Top2a–DPCC formation. Top2a-DPCC were detected by ELISA-based RADAR of DNA isolates (0.5 μg DNA per well) from CCRF-CEM and MOLT4 cells treated with 50 mM VP16 for indicated times and lysed in LS2. Left panel shows averages of replicates of A 450 reading from a representative ELISA experiment with anti-Top2a antibodies. Right panel shows averages of fold Top2a-DPCC induction calculated from two ELISA experiments performed on different days. Error bars represent standard deviation.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

    48) Product Images from "The Identification and Characterization of Two Novel Epitopes on the Nucleocapsid Protein of the Porcine Epidemic Diarrhea Virus"

    Article Title: The Identification and Characterization of Two Novel Epitopes on the Nucleocapsid Protein of the Porcine Epidemic Diarrhea Virus

    Journal: Scientific Reports

    doi: 10.1038/srep39010

    Mapping of PEDV N protein epitopes. ( a ) Schematic diagram of the epitope mapping. The segments that could be recognized by both PN-D4 and PN-D6 are highlighted in red; the segments that could only be recognized by PN-D4 are highlighted in blue; the segments that could only be recognized by PN-D6 are highlighted in green; and the segments in gray are those that could not be recognized by either PN-D4 or PN-D6. According to ( a ), the N gene was divided into mutually overlapping N1 and N2 fragments and was directionally cloned into the pGEX-6p-1 vector. ( b ) Six hours after the addition of 1 mM IPTG, the induced products of GST-N, GST-N1 and GST-N2 were processed and analyzed using SDS-PAGE (12% separating gel and 5% stacking gel). ( c ) Next, the GST fusion proteins GST-N, GST-N1 and GST-N2 were coated onto ELISA plates (0.5 μg/well) and were probed with MAbs PN-D4 and PN-D6. After the first round of identification, N1 and N2 were further divided and expressed in two series of fusion proteins, GST-N1-1-GST-N1-11 and GST-N2-1-GST-N2-11 ( d , h ), which were analyzed by Western blotting and ELISA using PN-D4 ( e , g ), PN-D6 ( i , k ), and anti-GST Tag antibody ( f , j ), respectively.
    Figure Legend Snippet: Mapping of PEDV N protein epitopes. ( a ) Schematic diagram of the epitope mapping. The segments that could be recognized by both PN-D4 and PN-D6 are highlighted in red; the segments that could only be recognized by PN-D4 are highlighted in blue; the segments that could only be recognized by PN-D6 are highlighted in green; and the segments in gray are those that could not be recognized by either PN-D4 or PN-D6. According to ( a ), the N gene was divided into mutually overlapping N1 and N2 fragments and was directionally cloned into the pGEX-6p-1 vector. ( b ) Six hours after the addition of 1 mM IPTG, the induced products of GST-N, GST-N1 and GST-N2 were processed and analyzed using SDS-PAGE (12% separating gel and 5% stacking gel). ( c ) Next, the GST fusion proteins GST-N, GST-N1 and GST-N2 were coated onto ELISA plates (0.5 μg/well) and were probed with MAbs PN-D4 and PN-D6. After the first round of identification, N1 and N2 were further divided and expressed in two series of fusion proteins, GST-N1-1-GST-N1-11 and GST-N2-1-GST-N2-11 ( d , h ), which were analyzed by Western blotting and ELISA using PN-D4 ( e , g ), PN-D6 ( i , k ), and anti-GST Tag antibody ( f , j ), respectively.

    Techniques Used: Clone Assay, Plasmid Preparation, SDS Page, Enzyme-linked Immunosorbent Assay, Western Blot

    Selection of MAbs against PEDV N protein. ( a ) Six MAbs were proven to be positive by IFA using PEDV inoculated cells. ( b ) The six positive clones were then tested by ELISA against GST-fusion N protein and GST-tag expressed in Escherichia coli .
    Figure Legend Snippet: Selection of MAbs against PEDV N protein. ( a ) Six MAbs were proven to be positive by IFA using PEDV inoculated cells. ( b ) The six positive clones were then tested by ELISA against GST-fusion N protein and GST-tag expressed in Escherichia coli .

    Techniques Used: Selection, Immunofluorescence, Clone Assay, Enzyme-linked Immunosorbent Assay

    49) Product Images from "Effects of Low-level Brodifacoum Exposure on the Feline Immune Response"

    Article Title: Effects of Low-level Brodifacoum Exposure on the Feline Immune Response

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26558-3

    Brodifacoum- and sham-treated cats have similar antibody responses to ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). Mean ELISA endpoint dilution, indicating last positive dilution shown. Standard deviation shown for each group. No significant differences noted between sham- and brodifacoum-treated groups. There was a trend towards lower anti-KLH antibody titers in brodifacoum-treated cats at week 6 (p = 0.17).
    Figure Legend Snippet: Brodifacoum- and sham-treated cats have similar antibody responses to ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). Mean ELISA endpoint dilution, indicating last positive dilution shown. Standard deviation shown for each group. No significant differences noted between sham- and brodifacoum-treated groups. There was a trend towards lower anti-KLH antibody titers in brodifacoum-treated cats at week 6 (p = 0.17).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

    50) Product Images from "Inverse Immunological Responses Induced by Allergic Rhinitis and Head and Neck Squamous Cell Carcinoma"

    Article Title: Inverse Immunological Responses Induced by Allergic Rhinitis and Head and Neck Squamous Cell Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086796

    Increased activation of polymorphonuclear leukocytes (PMN) in patients with head and neck squamous cell carcinoma (HNSCC). Blood was obtained from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (ELISA n = 9; FACS n = 10). PMN were isolated and cultured in the presence or absence of Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 4 and 24 h. ( A ) The cell free supernatants were then analyzed for IL-6 and IL-8 with ELISA, ( B ) and the CD16 positive cells were investigated for the expression of CD11b and CD69 using flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. ( C ) PMN from healthy individuals (n = 9) were incubated with or without culture medium supernatant from the HNSCC cell line FaDu, and stimulated with or without 1 µg/ml LPS for 4 h, and analyzed for IL-6 and IL-8 secretion with ELISA. The basal cytokine levels in the media were subtracted from the concentrations present in the PMN cultures. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.
    Figure Legend Snippet: Increased activation of polymorphonuclear leukocytes (PMN) in patients with head and neck squamous cell carcinoma (HNSCC). Blood was obtained from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (ELISA n = 9; FACS n = 10). PMN were isolated and cultured in the presence or absence of Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 4 and 24 h. ( A ) The cell free supernatants were then analyzed for IL-6 and IL-8 with ELISA, ( B ) and the CD16 positive cells were investigated for the expression of CD11b and CD69 using flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. ( C ) PMN from healthy individuals (n = 9) were incubated with or without culture medium supernatant from the HNSCC cell line FaDu, and stimulated with or without 1 µg/ml LPS for 4 h, and analyzed for IL-6 and IL-8 secretion with ELISA. The basal cytokine levels in the media were subtracted from the concentrations present in the PMN cultures. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, FACS, Isolation, Cell Culture, Expressing, Flow Cytometry, Cytometry, Incubation, Fluorescence

    51) Product Images from "Haematopoietic development and immunological function in the absence of cathepsin D"

    Article Title: Haematopoietic development and immunological function in the absence of cathepsin D

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-8-22

    Antibody responses to soluble and particulate antigens in CCDWT and CCDD . a) ELISA from chimaeric mice immunised with 6 μg DNP-BIg/alum as described in the text. IgM responses were measured five days post priming; the highest concentration of plasma corresponds to a dilution of 1: 30. IgG1 and IgG 2b responses were measured six days post boost; the highest concentration of plasma corresponds to a dilution of 1: 100. Each point represents average and standard error of the mean of three mice. Preimmune sera all gave OD values of less than 0.2 at the highest concentration shown. b) ELISA from plasma of chimaeric mice immunised with SRBC (10 8 cells/mouse). IgM responses were measured five days post priming; the highest concentration of plasma corresponds to a dilution of 1: 300. IgG1 and IgG 2b responses were measured six days post boost; the highest concentration of plasma corresponds to a dilution of 1: 900. Each point represents average of at least six mice. Preimmune sera all gave OD values of less than 0.1 at the highest concentration shown.
    Figure Legend Snippet: Antibody responses to soluble and particulate antigens in CCDWT and CCDD . a) ELISA from chimaeric mice immunised with 6 μg DNP-BIg/alum as described in the text. IgM responses were measured five days post priming; the highest concentration of plasma corresponds to a dilution of 1: 30. IgG1 and IgG 2b responses were measured six days post boost; the highest concentration of plasma corresponds to a dilution of 1: 100. Each point represents average and standard error of the mean of three mice. Preimmune sera all gave OD values of less than 0.2 at the highest concentration shown. b) ELISA from plasma of chimaeric mice immunised with SRBC (10 8 cells/mouse). IgM responses were measured five days post priming; the highest concentration of plasma corresponds to a dilution of 1: 300. IgG1 and IgG 2b responses were measured six days post boost; the highest concentration of plasma corresponds to a dilution of 1: 900. Each point represents average of at least six mice. Preimmune sera all gave OD values of less than 0.1 at the highest concentration shown.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Concentration Assay

    52) Product Images from "B-Cell-Intrinsic Type 1 Interferon Signaling Is Crucial for Loss of Tolerance and the Development of Autoreactive B Cells"

    Article Title: B-Cell-Intrinsic Type 1 Interferon Signaling Is Crucial for Loss of Tolerance and the Development of Autoreactive B Cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.06.046

    IFNαR 1 Is Required for Dysregulated Spt-AFC and Spt-GC Formation in B6. Sle1b Mice (A) Flow cytometric analysis of surface expression of IFNαR 1 on B220 + Fas low GL-7 low non-GC B cells (red), B220 + Fas h GL-7 hi GC B cells (blue), CD4 + PD-1 low CXCR5 low non-Tfh (orange), and CD4 + PD-1 hi CXCR5 hi Tfh (green) in female B6 and B6. Sle1b mice. Error bars represent mean ± SEM. (B and C) The percentages of GC B cells (B) and Tfh cells (C) in splenocytes from 3- and 6-month-old female mice. (D) Spleen sections from 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice (5 mice per group) were stained with anti-CD4 Ab (red) GL-7 (green) and anti-IgD Ab (blue), and GC areas were measured for 10 GCs per spleen section (right panels). The scale bars represent 50 μm. (E and F) Each symbol represents a measured GC. IgM + (E) and IgG + (F) AFCs in the spleens of 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice are shown. (G-J) Analysis of serum titers of IgM (G), IgG (H), IgG1 (I), and IgG2c (J) antibodies in 6-month-old mice by ELISA. Each symbol represents a mouse, and these data represent at least two-three independent experiments. Statistical significance was determined by one-way ANOVA with a follow-up Tukey multiple-comparison test (NS, not significant, *p
    Figure Legend Snippet: IFNαR 1 Is Required for Dysregulated Spt-AFC and Spt-GC Formation in B6. Sle1b Mice (A) Flow cytometric analysis of surface expression of IFNαR 1 on B220 + Fas low GL-7 low non-GC B cells (red), B220 + Fas h GL-7 hi GC B cells (blue), CD4 + PD-1 low CXCR5 low non-Tfh (orange), and CD4 + PD-1 hi CXCR5 hi Tfh (green) in female B6 and B6. Sle1b mice. Error bars represent mean ± SEM. (B and C) The percentages of GC B cells (B) and Tfh cells (C) in splenocytes from 3- and 6-month-old female mice. (D) Spleen sections from 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice (5 mice per group) were stained with anti-CD4 Ab (red) GL-7 (green) and anti-IgD Ab (blue), and GC areas were measured for 10 GCs per spleen section (right panels). The scale bars represent 50 μm. (E and F) Each symbol represents a measured GC. IgM + (E) and IgG + (F) AFCs in the spleens of 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice are shown. (G-J) Analysis of serum titers of IgM (G), IgG (H), IgG1 (I), and IgG2c (J) antibodies in 6-month-old mice by ELISA. Each symbol represents a mouse, and these data represent at least two-three independent experiments. Statistical significance was determined by one-way ANOVA with a follow-up Tukey multiple-comparison test (NS, not significant, *p

    Techniques Used: Single-particle Tracking, Mouse Assay, Flow Cytometry, Expressing, Staining, Enzyme-linked Immunosorbent Assay

    53) Product Images from "Assessment of ELISA as Endpoint in Neuronal Cell-based Assay for BoNT Detection using hiPSC derived neurons"

    Article Title: Assessment of ELISA as Endpoint in Neuronal Cell-based Assay for BoNT Detection using hiPSC derived neurons

    Journal: Journal of pharmacological and toxicological methods

    doi: 10.1016/j.vascn.2017.04.013

    Plate selection for ELISA. HIP Neurons were exposed to serial dilutions of BoNT/A1, and cell lysates were analyzed for cleaved SNAP-25 by ELISA using either a Nunc Polystyrene Maxisorp plate (Nunc) or a Thermo Scientific Immulon Immunoassay plate (Immulon). The signal was detected with ultra TMB. The average and standard deviations of 4 replicates is shown. Dose response curves were fitted using Prims 6 software and a nonlinear regression (variable slope, four parameter).
    Figure Legend Snippet: Plate selection for ELISA. HIP Neurons were exposed to serial dilutions of BoNT/A1, and cell lysates were analyzed for cleaved SNAP-25 by ELISA using either a Nunc Polystyrene Maxisorp plate (Nunc) or a Thermo Scientific Immulon Immunoassay plate (Immulon). The signal was detected with ultra TMB. The average and standard deviations of 4 replicates is shown. Dose response curves were fitted using Prims 6 software and a nonlinear regression (variable slope, four parameter).

    Techniques Used: Selection, Enzyme-linked Immunosorbent Assay, Software

    54) Product Images from "Genetic and pharmacological validation of TAK1 inhibition in macrophages as a therapeutic strategy to effectively inhibit TNF secretion"

    Article Title: Genetic and pharmacological validation of TAK1 inhibition in macrophages as a therapeutic strategy to effectively inhibit TNF secretion

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-35189-7

    Takinib reduces TNF in a murine LPS challenge. FVB mice treated i.p. with varying concentrations of Takinib, once (qw) or twice weekly (qbw), caused no significant adverse events or weight change over 8 weeks of chronic treatment ( a ). Bone marrow derived macrophages (BMDM’s) were plated at (2 × 10 6 /well) in a 24-well plate and treated with M-CSF (20 ng/mL) for 72 hours followed by a 24 hour rest period prior to activation with LPS (10 ng/mL) and IFNγ (50 ng/mL) and treatment with Takinib, at the indicated concentrations, or vehicle for 24 hours. Cytokine production was sampled from cell supernatant and examined by ELISA ( b ) (One-way ANOVA with Dunnett’s, n = 3± SEM). C57/bl6 male and female mice were sacrificed prior to LPS challenge and baseline TNF and IL-6 serum concentrations were examined by ELISA ( c,d ) (Student t-test, n = 3± SEM, 3 males, 3 females). C57/bl6 male and female mice were injected intraperitoneally (i.p.) with LPS (100 μg/kg in PBS) followed immediately by vehicle or Takinib (50 mg/kg in DMSO). 1 hour later, mice were sacrificed and blood samples clotted in centrifuge for 10 minutes at 3,000 rpm. Concentration of serum cytokines TNF and IL-6 determined by ELISA ( c,d ) (Student t-test, n = 5± SEM).
    Figure Legend Snippet: Takinib reduces TNF in a murine LPS challenge. FVB mice treated i.p. with varying concentrations of Takinib, once (qw) or twice weekly (qbw), caused no significant adverse events or weight change over 8 weeks of chronic treatment ( a ). Bone marrow derived macrophages (BMDM’s) were plated at (2 × 10 6 /well) in a 24-well plate and treated with M-CSF (20 ng/mL) for 72 hours followed by a 24 hour rest period prior to activation with LPS (10 ng/mL) and IFNγ (50 ng/mL) and treatment with Takinib, at the indicated concentrations, or vehicle for 24 hours. Cytokine production was sampled from cell supernatant and examined by ELISA ( b ) (One-way ANOVA with Dunnett’s, n = 3± SEM). C57/bl6 male and female mice were sacrificed prior to LPS challenge and baseline TNF and IL-6 serum concentrations were examined by ELISA ( c,d ) (Student t-test, n = 3± SEM, 3 males, 3 females). C57/bl6 male and female mice were injected intraperitoneally (i.p.) with LPS (100 μg/kg in PBS) followed immediately by vehicle or Takinib (50 mg/kg in DMSO). 1 hour later, mice were sacrificed and blood samples clotted in centrifuge for 10 minutes at 3,000 rpm. Concentration of serum cytokines TNF and IL-6 determined by ELISA ( c,d ) (Student t-test, n = 5± SEM).

    Techniques Used: Mouse Assay, Derivative Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Injection, Concentration Assay

    55) Product Images from "Serological evidence of ebolavirus infection in bats, China"

    Article Title: Serological evidence of ebolavirus infection in bats, China

    Journal: Virology Journal

    doi: 10.1186/1743-422X-9-236

    Characterization of the truncated recombinant nucleocapsid protein of RESTV (Reston-NP) and EBOV (Zaire-NP). Coomassie staining ( A ) and western blot analysis ( B ) of purified proteins separated by 10% SDS-PAGE. Lane M: molecular weight markers from Fermentas; Lane 1: Reston-NP; Lane 2: Zaire-NP. ( C ) ELISA analysis of Reston-NP and Zaire-NP using hyperimmune rabbit sera raised against the full-length Reston-NP.
    Figure Legend Snippet: Characterization of the truncated recombinant nucleocapsid protein of RESTV (Reston-NP) and EBOV (Zaire-NP). Coomassie staining ( A ) and western blot analysis ( B ) of purified proteins separated by 10% SDS-PAGE. Lane M: molecular weight markers from Fermentas; Lane 1: Reston-NP; Lane 2: Zaire-NP. ( C ) ELISA analysis of Reston-NP and Zaire-NP using hyperimmune rabbit sera raised against the full-length Reston-NP.

    Techniques Used: Recombinant, Staining, Western Blot, Purification, SDS Page, Molecular Weight, Enzyme-linked Immunosorbent Assay

    Western-blot analysis of ELISA positive serum samples with recombinant Reston-NP and Zaire-NP expressed in E. coli . The polyclonal antibody against the full-length nucleocapsid protein of RESTV was used as positive control. Sample no. 2195 was ELISA negative and used as negative control. The other 5 samples were western blot positive. Note: western blot no.1487, 1552, 1689, 1973 and 2166 is not presented due to the absence of signal in the scanned photograph.
    Figure Legend Snippet: Western-blot analysis of ELISA positive serum samples with recombinant Reston-NP and Zaire-NP expressed in E. coli . The polyclonal antibody against the full-length nucleocapsid protein of RESTV was used as positive control. Sample no. 2195 was ELISA negative and used as negative control. The other 5 samples were western blot positive. Note: western blot no.1487, 1552, 1689, 1973 and 2166 is not presented due to the absence of signal in the scanned photograph.

    Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Negative Control

    56) Product Images from "Macrophage migration inhibitory factor mediates peripheral nerve injury-induced hypersensitivity by curbing dopaminergic descending inhibition"

    Article Title: Macrophage migration inhibitory factor mediates peripheral nerve injury-induced hypersensitivity by curbing dopaminergic descending inhibition

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2017.271

    Effects of ISO-1 on migration inhibitory factor (MIF) and monoamine content in L-SC and VTA following chronic constriction injury (CCI). The MIF inhibitor ISO-1 was administered i.t. or i.c.v. for 2 weeks from day 7 post-nerve injury. In L-SC and VTA, the MIF and monoamine content was detected with ELISA and HPLC, respectively, at post-injury day 21. The turnover effect of i.t. ISO-1 for MIF and dopamine in L-SC is shown in a and b (* P
    Figure Legend Snippet: Effects of ISO-1 on migration inhibitory factor (MIF) and monoamine content in L-SC and VTA following chronic constriction injury (CCI). The MIF inhibitor ISO-1 was administered i.t. or i.c.v. for 2 weeks from day 7 post-nerve injury. In L-SC and VTA, the MIF and monoamine content was detected with ELISA and HPLC, respectively, at post-injury day 21. The turnover effect of i.t. ISO-1 for MIF and dopamine in L-SC is shown in a and b (* P

    Techniques Used: Migration, Enzyme-linked Immunosorbent Assay, High Performance Liquid Chromatography

    Migration inhibitory factor (MIF) and dopamine content in L-SC and VTA in CCI mice over the 70-day observation period. MIF and dopamine content in microdialysate obtained from L-SC and VTA were detected with ELISA and HPLC, respectively, on days 0, 1, 2, 4, 7, 14, 21, 35, 49 and 70 post CCI. Time-dependent MIF content upregulation in L-SC and VTA, as well as inversed dopamine content downregulation, are shown in a and b (* P
    Figure Legend Snippet: Migration inhibitory factor (MIF) and dopamine content in L-SC and VTA in CCI mice over the 70-day observation period. MIF and dopamine content in microdialysate obtained from L-SC and VTA were detected with ELISA and HPLC, respectively, on days 0, 1, 2, 4, 7, 14, 21, 35, 49 and 70 post CCI. Time-dependent MIF content upregulation in L-SC and VTA, as well as inversed dopamine content downregulation, are shown in a and b (* P

    Techniques Used: Migration, Mouse Assay, Enzyme-linked Immunosorbent Assay, High Performance Liquid Chromatography

    57) Product Images from "TH17-Induced Neutrophils Enhance the Pulmonary Allergic Response Following BALB/c Exposure to House Dust Mite Allergen and Fine Particulate Matter From California and China"

    Article Title: TH17-Induced Neutrophils Enhance the Pulmonary Allergic Response Following BALB/c Exposure to House Dust Mite Allergen and Fine Particulate Matter From California and China

    Journal: Toxicological Sciences

    doi: 10.1093/toxsci/kfy127

    Ig protein levels measured in blood serum by ELISA. Panels show nonspecific IgE levels (A), HDM-specific IgE levels (B), and HDM-specific IgG 1 levels (C). Protein levels were analyzed in duplicate for each animal and averaged for each treatment group. Data were analyzed by ANOVAs considering the effect of treatment, and are shown as the mean ± SEM. Brackets indicate a significant ( p
    Figure Legend Snippet: Ig protein levels measured in blood serum by ELISA. Panels show nonspecific IgE levels (A), HDM-specific IgE levels (B), and HDM-specific IgG 1 levels (C). Protein levels were analyzed in duplicate for each animal and averaged for each treatment group. Data were analyzed by ANOVAs considering the effect of treatment, and are shown as the mean ± SEM. Brackets indicate a significant ( p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Exposure to California PM and HDM allergen (PM CA +HDM) significantly increases the protein and gene expression profiles of T-helper cell type 17 (T H -17)- cytokines above other treatments. Graphs show results obtained from analysis of mouse lung proteins via ELISA and genes (mRNA) via PCR. Analyzed proteins included IL-17A (A) and IL-17F (B). Protein levels were analyzed in duplicate for each animal and averaged for each treatment group. Analyzed genes included IL-17A (C), IL-17F (D), IL-21 (E), and IL-27 (F). Gene expression is shown as relative expression to the housekeeping gene, GAPDH. Gene levels were analyzed for each animal and averaged for each treatment group. Data are shown as the mean ± SEM. Brackets indicate a significant ( p
    Figure Legend Snippet: Exposure to California PM and HDM allergen (PM CA +HDM) significantly increases the protein and gene expression profiles of T-helper cell type 17 (T H -17)- cytokines above other treatments. Graphs show results obtained from analysis of mouse lung proteins via ELISA and genes (mRNA) via PCR. Analyzed proteins included IL-17A (A) and IL-17F (B). Protein levels were analyzed in duplicate for each animal and averaged for each treatment group. Analyzed genes included IL-17A (C), IL-17F (D), IL-21 (E), and IL-27 (F). Gene expression is shown as relative expression to the housekeeping gene, GAPDH. Gene levels were analyzed for each animal and averaged for each treatment group. Data are shown as the mean ± SEM. Brackets indicate a significant ( p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction

    58) Product Images from "Modulation of Antibody Responses to the V1V2 and V3 Regions of HIV-1 Envelope by Immune Complex Vaccines"

    Article Title: Modulation of Antibody Responses to the V1V2 and V3 Regions of HIV-1 Envelope by Immune Complex Vaccines

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02441

    Serum Ab responses induced by gp120 A244 /mAb complex vaccines. Mice were immunized with gp120 AE.A244 in complex with human IgG1 mAbs specific for V2i (2158), V2i (697), CD4bs (1331E), V3 (2219), or with no mAb in the presence of adjuvant MPL/DDA. (A–C) Sera collected 2 weeks after the fourth immunization were tested in ELISA for IgG reactivity to gp120 (A) , V3 (B) , and V1V2 (C) . (D) Sera were also evaluated for cross-reactivity with V3 and V1V2 from viruses of different HIV-1 subtypes. (E) Further mapping of V2 epitope was performed using overlapping V2 peptides P1 to P9. V2 sequence logo is shown to indicate amino-acid variability within the defined epitope region (orange-shaded box). (F) Sera were subjected to competition ELISA using V1V2 C.ZM109-1FD6 to assess the presence of V1V2-specific serum Abs able to compete with conformation-dependent V2i mAb 830A. **** p
    Figure Legend Snippet: Serum Ab responses induced by gp120 A244 /mAb complex vaccines. Mice were immunized with gp120 AE.A244 in complex with human IgG1 mAbs specific for V2i (2158), V2i (697), CD4bs (1331E), V3 (2219), or with no mAb in the presence of adjuvant MPL/DDA. (A–C) Sera collected 2 weeks after the fourth immunization were tested in ELISA for IgG reactivity to gp120 (A) , V3 (B) , and V1V2 (C) . (D) Sera were also evaluated for cross-reactivity with V3 and V1V2 from viruses of different HIV-1 subtypes. (E) Further mapping of V2 epitope was performed using overlapping V2 peptides P1 to P9. V2 sequence logo is shown to indicate amino-acid variability within the defined epitope region (orange-shaded box). (F) Sera were subjected to competition ELISA using V1V2 C.ZM109-1FD6 to assess the presence of V1V2-specific serum Abs able to compete with conformation-dependent V2i mAb 830A. **** p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Sequencing

    Serum Ab responses induced by vaccination with gp120 JRFL /mAb complexes. BALB/c mice were immunized with gp120 B.JRFL complexed with human IgG1 mAbs of defined specificity—C2 (1006-30D), V2i (2158), CD4bs (654), V3 (1006-15D)—or with no mAb. Immune complexes were administered 4 times subcutaneously in the presence of adjuvant MPL/DDA. Mice immunized with PBS and adjuvant (no gp120) served as negative controls. (A–C) Pooled sera collected 2 weeks after the last immunization were tested in ELISA for IgG reactivity against gp120 (A) , V1V2 (B) , or V3 (C) . (D) Sera from individual mice immunized with gp120 JRFL vs. gp120 JRFL /CD4bs mAb 654 were also tested for ELISA reactivity against V3. AUC: area under the titration curve of each serum sample; OD 405 : optical density at 405 nm obtained from designated serum dilution in ELISA with p-nitrophenyl phosphate substrate. * p
    Figure Legend Snippet: Serum Ab responses induced by vaccination with gp120 JRFL /mAb complexes. BALB/c mice were immunized with gp120 B.JRFL complexed with human IgG1 mAbs of defined specificity—C2 (1006-30D), V2i (2158), CD4bs (654), V3 (1006-15D)—or with no mAb. Immune complexes were administered 4 times subcutaneously in the presence of adjuvant MPL/DDA. Mice immunized with PBS and adjuvant (no gp120) served as negative controls. (A–C) Pooled sera collected 2 weeks after the last immunization were tested in ELISA for IgG reactivity against gp120 (A) , V1V2 (B) , or V3 (C) . (D) Sera from individual mice immunized with gp120 JRFL vs. gp120 JRFL /CD4bs mAb 654 were also tested for ELISA reactivity against V3. AUC: area under the titration curve of each serum sample; OD 405 : optical density at 405 nm obtained from designated serum dilution in ELISA with p-nitrophenyl phosphate substrate. * p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Titration

    V3-specific Ab responses induced by gp120 JRFL /mAb complexes vs. uncomplexed gp120 JRFL . (A) Sera from mice immunized with gp120 B.JRFL in complex with V2i mAb 2158, CD4bs mAb 654, or no mAb were tested in ELISA for IgG reactivity against V3 peptides of HIV-1 subtypes A, B, and C. (B) Overlapping V3 peptides P1 to P5 were used for epitope mapping. Sequence logo depicting V3 amino acid variations is included to show the central V3 regions that are targeted by Abs induced by gp120 JRFL (blue-shaded box) vs. gp120 JRFL /mAb complexes (orange-shaded box). (C) Sera from individual animals were tested for reactivity with overlapping V3 peptides P1 to P5 and demonstrated more uniform recognition of P2 and P3 peptides by all animals that received gp120 JRFL /CD4bs mAb 654 vs. uncomplexed gp120 JRFL. . ( D ) Sera from mice immunized with gp120 JRFL or gp120 JRFL /mAb complexes were also tested for IgG reactivity with different V1V2 antigens. AUC: area under the titration curve. PBS: Sera from control group that received PBS and adjuvant (no gp120).
    Figure Legend Snippet: V3-specific Ab responses induced by gp120 JRFL /mAb complexes vs. uncomplexed gp120 JRFL . (A) Sera from mice immunized with gp120 B.JRFL in complex with V2i mAb 2158, CD4bs mAb 654, or no mAb were tested in ELISA for IgG reactivity against V3 peptides of HIV-1 subtypes A, B, and C. (B) Overlapping V3 peptides P1 to P5 were used for epitope mapping. Sequence logo depicting V3 amino acid variations is included to show the central V3 regions that are targeted by Abs induced by gp120 JRFL (blue-shaded box) vs. gp120 JRFL /mAb complexes (orange-shaded box). (C) Sera from individual animals were tested for reactivity with overlapping V3 peptides P1 to P5 and demonstrated more uniform recognition of P2 and P3 peptides by all animals that received gp120 JRFL /CD4bs mAb 654 vs. uncomplexed gp120 JRFL. . ( D ) Sera from mice immunized with gp120 JRFL or gp120 JRFL /mAb complexes were also tested for IgG reactivity with different V1V2 antigens. AUC: area under the titration curve. PBS: Sera from control group that received PBS and adjuvant (no gp120).

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Titration

    59) Product Images from "Cutaneous exposure to agglomerates of silica nanoparticles and allergen results in IgE-biased immune response and increased sensitivity to anaphylaxis in mice"

    Article Title: Cutaneous exposure to agglomerates of silica nanoparticles and allergen results in IgE-biased immune response and increased sensitivity to anaphylaxis in mice

    Journal: Particle and Fibre Toxicology

    doi: 10.1186/s12989-015-0095-3

    Effects of Dp + nSP30 aggregates compared with Dp and nSP30 administered on alternate days. a Effect of topical application of Dp + nSP30 and PBS (Dp + nSP30/PBS) compared with Dp and nSP30 (Dp/nSP30) on alternate days on ear thickness in NC/Nga mice. b–e Plasma levels of ( b ) total IgE, ( c ) Dp-specific IgE, ( d ) IgG and ( e ) IgG1 and IgG2a 24 h after final treatment, as analyzed by ELISA. f Numbers of Dp-specific IFN-γ- and IL-4-producing splenocytes after re-stimulation with 100 μg mL −1 Dp, as determined by ELISPOT assays specific for each cytokine. Data are presented as means ± SEMs (n = 5). * P
    Figure Legend Snippet: Effects of Dp + nSP30 aggregates compared with Dp and nSP30 administered on alternate days. a Effect of topical application of Dp + nSP30 and PBS (Dp + nSP30/PBS) compared with Dp and nSP30 (Dp/nSP30) on alternate days on ear thickness in NC/Nga mice. b–e Plasma levels of ( b ) total IgE, ( c ) Dp-specific IgE, ( d ) IgG and ( e ) IgG1 and IgG2a 24 h after final treatment, as analyzed by ELISA. f Numbers of Dp-specific IFN-γ- and IL-4-producing splenocytes after re-stimulation with 100 μg mL −1 Dp, as determined by ELISPOT assays specific for each cytokine. Data are presented as means ± SEMs (n = 5). * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

    Prevention of Dp + nSP30-mediated IgE-biased immune response by surface modification of nSP30. a – c Plasma levels of Dp-specific ( a ) IgE, ( b ) IgG, and ( c ) IgG1 and IgG2a at 24 h after final treatment of NC/Nga mice with Dp alone, Dp + nSP30, or Dp + nSP30C, as analyzed by ELISA. Data are given as means ± SEMs ( n = 5). * P
    Figure Legend Snippet: Prevention of Dp + nSP30-mediated IgE-biased immune response by surface modification of nSP30. a – c Plasma levels of Dp-specific ( a ) IgE, ( b ) IgG, and ( c ) IgG1 and IgG2a at 24 h after final treatment of NC/Nga mice with Dp alone, Dp + nSP30, or Dp + nSP30C, as analyzed by ELISA. Data are given as means ± SEMs ( n = 5). * P

    Techniques Used: Modification, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Effect of topical application of dispersed mixture of Dp + nSP30 on antibody response. a–c Plasma levels of Dp-specific ( a ) IgE, ( b ) IgG, and ( c ) IgG1 and IgG2a at 24 h after final topical treatment of NC/Nga mice with Dp alone or Dp + nSP30 in water, as analyzed by ELISA. Data are given as means ± SEMs ( n = 5 or 6). * P
    Figure Legend Snippet: Effect of topical application of dispersed mixture of Dp + nSP30 on antibody response. a–c Plasma levels of Dp-specific ( a ) IgE, ( b ) IgG, and ( c ) IgG1 and IgG2a at 24 h after final topical treatment of NC/Nga mice with Dp alone or Dp + nSP30 in water, as analyzed by ELISA. Data are given as means ± SEMs ( n = 5 or 6). * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Induction of systemic immune responses by Dp + nSP30 agglomerates in PBS. a–c Plasma levels of Dp-specific ( a ) IgE, ( b ) IgG, and ( c ) IgG1 and IgG2a as analyzed by ELISA at 24 h after final topical treatment of NC/Nga mice with Dp alone or Dp + nSP30 in PBS. (D) Numbers of Dp-specific IFN-γ- and IL-4-producing splenocytes after re-stimulation with 100 μg mL −1 Dp, as determined by using ELISPOT assays specific for each cytokine. Data are given as means ± SEMs ( n = 5–12). ** P
    Figure Legend Snippet: Induction of systemic immune responses by Dp + nSP30 agglomerates in PBS. a–c Plasma levels of Dp-specific ( a ) IgE, ( b ) IgG, and ( c ) IgG1 and IgG2a as analyzed by ELISA at 24 h after final topical treatment of NC/Nga mice with Dp alone or Dp + nSP30 in PBS. (D) Numbers of Dp-specific IFN-γ- and IL-4-producing splenocytes after re-stimulation with 100 μg mL −1 Dp, as determined by using ELISPOT assays specific for each cytokine. Data are given as means ± SEMs ( n = 5–12). ** P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Enzyme-linked Immunospot

    60) Product Images from "Low-Dose Methotrexate Prevents Primary and Secondary Humoral Immune Responses and Induces Immune Tolerance to a Recombinant Immunotoxin"

    Article Title: Low-Dose Methotrexate Prevents Primary and Secondary Humoral Immune Responses and Induces Immune Tolerance to a Recombinant Immunotoxin

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1701430

    Anti-LMB-100 antibody class switching is not affected by MTX treatment Female Balb/c mice were treated with 20 μg LMB-100 i.v. twice a week for 6 weeks. One group received 1 mg/kg MTX i.p. 0, 24, and 48 hours after LMB-100 for the first 3 weeks. A) ELISA plates were coated with LMB-100 and 1 μl of plasma was diluted 1:100 and then by 4 down the plate. Anti-LMB-100 antibodies in plasma samples from week 6 were detected with anti-isotype secondary antibodies (n=4). Circle: LMB-100 treatment. Square: LMB-100+MTX. B) Anti-LMB-100 IgM antibodies were detected in plasma samples from mice treated with 40 μg LMB-100 i.v. and 1 mg/kg MTX (n=8). Titer is reported as dilution factor. Data points are mean, Error bars are SEM. Assays were repeated once.
    Figure Legend Snippet: Anti-LMB-100 antibody class switching is not affected by MTX treatment Female Balb/c mice were treated with 20 μg LMB-100 i.v. twice a week for 6 weeks. One group received 1 mg/kg MTX i.p. 0, 24, and 48 hours after LMB-100 for the first 3 weeks. A) ELISA plates were coated with LMB-100 and 1 μl of plasma was diluted 1:100 and then by 4 down the plate. Anti-LMB-100 antibodies in plasma samples from week 6 were detected with anti-isotype secondary antibodies (n=4). Circle: LMB-100 treatment. Square: LMB-100+MTX. B) Anti-LMB-100 IgM antibodies were detected in plasma samples from mice treated with 40 μg LMB-100 i.v. and 1 mg/kg MTX (n=8). Titer is reported as dilution factor. Data points are mean, Error bars are SEM. Assays were repeated once.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    MTX prevents the immune memory response A) Mice were immunized with three doses of 50 μg LMB-100 i.v. in weeks 1 and 3 and challenged in week 11 with either LMB-100 (50 μg i.v.), LMB-100 + MTX (1 mg/kg i.p), MTX, or PBS. MTX was given 0, 24, and 48 hours after LMB-100. Plasma were collected and analyzed for anti-LMB-100 antibodies by ELISA (n=11). B) Week 12 titers. Titer is reported as dilution factor. Data points are mean, error bars are SEM.
    Figure Legend Snippet: MTX prevents the immune memory response A) Mice were immunized with three doses of 50 μg LMB-100 i.v. in weeks 1 and 3 and challenged in week 11 with either LMB-100 (50 μg i.v.), LMB-100 + MTX (1 mg/kg i.p), MTX, or PBS. MTX was given 0, 24, and 48 hours after LMB-100. Plasma were collected and analyzed for anti-LMB-100 antibodies by ELISA (n=11). B) Week 12 titers. Titer is reported as dilution factor. Data points are mean, error bars are SEM.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    MTX-induced tolerance is antigen-specific Mice were immunized with two doses of 40 μg LMB-100 i.v. in weeks 1 through 3 or in combination with 1 mg/kg MTX i.p given 0, 24, and 48 hours after each LMB-100 dose. In weeks 4–6 mice were challenged with 40 μg LMB-100 i.v. + 40 μg OVA i.v. Plasma samples were collected and analyzed for anti-LMB-100 (A) and anti-OVA (B) antibodies by ELISA (n=14). Titer is reported as dilution factor. Data points are mean, Error bars are SEM. Data compiled from two separate experiments.
    Figure Legend Snippet: MTX-induced tolerance is antigen-specific Mice were immunized with two doses of 40 μg LMB-100 i.v. in weeks 1 through 3 or in combination with 1 mg/kg MTX i.p given 0, 24, and 48 hours after each LMB-100 dose. In weeks 4–6 mice were challenged with 40 μg LMB-100 i.v. + 40 μg OVA i.v. Plasma samples were collected and analyzed for anti-LMB-100 (A) and anti-OVA (B) antibodies by ELISA (n=14). Titer is reported as dilution factor. Data points are mean, Error bars are SEM. Data compiled from two separate experiments.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Adoptive transfer of whole splenocytes from tolerized mice confers LMB-100 tolerance to naïve recipients A) Female Balb/c mice were immunized with three doses of 40 μg or 50 μg LMB-100 i.v. in weeks 1 and 3, with 1 mg/kg MTX given i.p. 0, 24, and 48 hours after each LMB-100 dose. Blood samples were collected in week 4 prior to sacrifice, and anti-LMB-100 antibodies were detected by ELISA (n=8–12). B) In week 4 donor mice were sacrificed, splenocytes were isolated, and 10×10 6 to 25×10 6 splenocytes were injected i.v. into naïve recipient mice. Recipient mice were challenged with three doses of LMB-100 in weeks 5 and 7. Plasma was collected in week 8 and analyzed for anti-LMB-100 antibodies by ELISA (n=8–34). Data shown is a compilation of two separate experiments, table below indicates treatment of donor splenocytes. Titer is reported as dilution factor. Bars show mean, Error bars are SEM.
    Figure Legend Snippet: Adoptive transfer of whole splenocytes from tolerized mice confers LMB-100 tolerance to naïve recipients A) Female Balb/c mice were immunized with three doses of 40 μg or 50 μg LMB-100 i.v. in weeks 1 and 3, with 1 mg/kg MTX given i.p. 0, 24, and 48 hours after each LMB-100 dose. Blood samples were collected in week 4 prior to sacrifice, and anti-LMB-100 antibodies were detected by ELISA (n=8–12). B) In week 4 donor mice were sacrificed, splenocytes were isolated, and 10×10 6 to 25×10 6 splenocytes were injected i.v. into naïve recipient mice. Recipient mice were challenged with three doses of LMB-100 in weeks 5 and 7. Plasma was collected in week 8 and analyzed for anti-LMB-100 antibodies by ELISA (n=8–34). Data shown is a compilation of two separate experiments, table below indicates treatment of donor splenocytes. Titer is reported as dilution factor. Bars show mean, Error bars are SEM.

    Techniques Used: Adoptive Transfer Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation, Injection

    MTX prevents the formation of ADAs A) Female Balb/c mice were immunized with 40 μg LMB-100 i.p. twice per week (days 1 and 3) together with 1 mg/kg MTX i.p., given at 0, 24, and 48 hours after LMB-100 for a total of 5 doses per week. MTX was given for 1, 2, 3, or 6 weeks. Plasma was collected and analyzed for anti-LMB-100 antibodies by ELISA (n=8). B) Mice were immunized with 20 μg LMB-100 i.v. twice weekly. MTX was given by i.p. injection at the doses indicated for 5 days per week (n=4). Blood samples were collected in week 6 and analyzed for anti-LMB-100 antibodies by ELISA. Titer is reported as dilution factor. Data points are mean, error bars are SEM. Each experiment was repeated at least once.
    Figure Legend Snippet: MTX prevents the formation of ADAs A) Female Balb/c mice were immunized with 40 μg LMB-100 i.p. twice per week (days 1 and 3) together with 1 mg/kg MTX i.p., given at 0, 24, and 48 hours after LMB-100 for a total of 5 doses per week. MTX was given for 1, 2, 3, or 6 weeks. Plasma was collected and analyzed for anti-LMB-100 antibodies by ELISA (n=8). B) Mice were immunized with 20 μg LMB-100 i.v. twice weekly. MTX was given by i.p. injection at the doses indicated for 5 days per week (n=4). Blood samples were collected in week 6 and analyzed for anti-LMB-100 antibodies by ELISA. Titer is reported as dilution factor. Data points are mean, error bars are SEM. Each experiment was repeated at least once.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

    Related Articles

    Clone Assay:

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions
    Article Snippet: Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific). .. For the GFP standard curve, GST-GFP was cloned by standard methods, expressed in DL21 cells, purified with glutathione resin (GE Healthcare), and quantified with a BCA protein assay (Thermo Fisher Scientific) versus BSA standard curves.

    Binding Assay:

    Article Title: Macrophage migration inhibitory factor mediates peripheral nerve injury-induced hypersensitivity by curbing dopaminergic descending inhibition
    Article Snippet: Ninety-six well ELISA plates were coated with rabbit anti-mouse MIF (Invitrogen, Carlsbad, CA, USA). .. Unspecific binding was blocked with PBS containing 1% BSA/5% sucrose/ 0.05% NaHN3 .

    Article Title: Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice against Cryptococcus neoformans Infection ▿
    Article Snippet: .. To characterize antigen binding, recombinant IgGs were added to ELISA plates (Immulon 1B; Thermo Labsystems) coated with 10 μg/ml GXM purified from culture of C. neoformans serotype D strain 24067, obtained from the American Type Culture Collection (ATCC) as described previously ( ). .. Bound antibody was detected by using goat anti-human IgG(κ) L chain conjugated to alkaline phosphatase (AP; Sigma-Aldrich).

    Concentration Assay:

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL
    Article Snippet: Protein quantification The concentration of the chimeras was quantified in cell culture using specific sandwich ELISA assays. .. The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Serial Dilution:

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions
    Article Snippet: Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific). .. The GFP standard curve was created by serial dilution of the pure GST-GFP in WT embryo lysate (prepared in the same way as the lysates of embryos expressing GFP-tagged proteins) and applied in neighboring wells of the ELISA plate.

    Western Blot:

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions
    Article Snippet: Paragraph title: ELISAs and Western blots ... Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific).

    Blocking Assay:

    Article Title: Significant role for IRF3 in both T cell and APC effector functions during T cell responses
    Article Snippet: ELISA plates were coated with antibodies to mouse IFN-γ or IL-17 (Ebiosciences, Sand Diego, CA). .. After blocking and washing, supernatants or serial dilutions of recombinant IFN-γ or IL-17 (Ebiosciences) were added.

    Purification:

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions
    Article Snippet: Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific). .. For the GFP standard curve, GST-GFP was cloned by standard methods, expressed in DL21 cells, purified with glutathione resin (GE Healthcare), and quantified with a BCA protein assay (Thermo Fisher Scientific) versus BSA standard curves.

    Article Title: Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice against Cryptococcus neoformans Infection ▿
    Article Snippet: .. To characterize antigen binding, recombinant IgGs were added to ELISA plates (Immulon 1B; Thermo Labsystems) coated with 10 μg/ml GXM purified from culture of C. neoformans serotype D strain 24067, obtained from the American Type Culture Collection (ATCC) as described previously ( ). .. Bound antibody was detected by using goat anti-human IgG(κ) L chain conjugated to alkaline phosphatase (AP; Sigma-Aldrich).

    Enzyme-linked Immunosorbent Assay:

    Article Title: IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity
    Article Snippet: .. Total IgG auto-Ab titers were measured in ELISA plates coated with salmon sperm dsDNA (Invitrogen), histone (Sigma-Aldrich), or nucleosome (histone plated on a layer of dsDNA coating) and detected with biotinylated anti-κ Abs (Invitrogen). .. IgG subtype–specific auto-Ab titers were detected by biotinylated IgG1 , biotinylated IgG2b , and alkaline phosphatase IgG2c Abs (SouthernBiotech).

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions
    Article Snippet: .. Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific). .. For the GFP standard curve, GST-GFP was cloned by standard methods, expressed in DL21 cells, purified with glutathione resin (GE Healthcare), and quantified with a BCA protein assay (Thermo Fisher Scientific) versus BSA standard curves.

    Article Title: Macrophage migration inhibitory factor mediates peripheral nerve injury-induced hypersensitivity by curbing dopaminergic descending inhibition
    Article Snippet: .. Ninety-six well ELISA plates were coated with rabbit anti-mouse MIF (Invitrogen, Carlsbad, CA, USA). .. Unspecific binding was blocked with PBS containing 1% BSA/5% sucrose/ 0.05% NaHN3 .

    Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity
    Article Snippet: .. Extracted DNA was then coated onto ELISA plates and detected by AP-site specific reagent, Aldehyde-Reactive Probe (ARP) (Invitrogen), followed by detection with HRP-conjugated streptavidin and TMB. ..

    Article Title: Takinib, a selective TAK1 inhibitor, broadens the therapeutic efficacy of TNFα inhibition for cancer and autoimmune disease
    Article Snippet: .. 24h after treatment, supernatant was added to ELISA plates (IL-6, IL-8, TNF, Ready-SET-Go! from Thermo). ..

    Article Title: In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120
    Article Snippet: .. Briefly, gp120 proteins (1 μg/ml) or peptides (1 μg/ml) were adsorbed onto ELISA plates (Immunoblot 2HB or Microfluor 2 Black; Thermo, Milford, MA) overnight at 4°C. ..

    Article Title: Genetic and pharmacological validation of TAK1 inhibition in macrophages as a therapeutic strategy to effectively inhibit TNF secretion
    Article Snippet: .. 24 h after treatment, supernatant was added to ELISA plates (IL-6, IL-8, TNF, IL-1B, Ready- SET-Go! from Thermo). ..

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL
    Article Snippet: .. The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6). ..

    Article Title: Significant role for IRF3 in both T cell and APC effector functions during T cell responses
    Article Snippet: .. ELISA plates were coated with antibodies to mouse IFN-γ or IL-17 (Ebiosciences, Sand Diego, CA). .. After blocking and washing, supernatants or serial dilutions of recombinant IFN-γ or IL-17 (Ebiosciences) were added.

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA
    Article Snippet: .. To detect the presence of LPS from M15 cells in the wells of ELISA plates coated with rUS9-FhLAP, or mAb US9 plus rUS9-FhLAP, the plates were incubated with 100 μL/well of a goat anti-lipid A LPS polyclonal antibody (ThermoFisher Scientific, Rockford, USA) diluted 1/100 in PBS-T-SM and incubated for 30 min at RT with shaking at 750 rpm. .. The plates were then washed five times with PBS-T, and bound IgG antibodies were detected with HRP-conjugated mouse anti-sheep/goat IgG monoclonal antibodies (Sigma–Aldrich, 1/30,000 in PBS-T-SM).

    Article Title: Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice against Cryptococcus neoformans Infection ▿
    Article Snippet: .. To characterize antigen binding, recombinant IgGs were added to ELISA plates (Immulon 1B; Thermo Labsystems) coated with 10 μg/ml GXM purified from culture of C. neoformans serotype D strain 24067, obtained from the American Type Culture Collection (ATCC) as described previously ( ). .. Bound antibody was detected by using goat anti-human IgG(κ) L chain conjugated to alkaline phosphatase (AP; Sigma-Aldrich).

    Sandwich ELISA:

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL
    Article Snippet: Protein quantification The concentration of the chimeras was quantified in cell culture using specific sandwich ELISA assays. .. The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Incubation:

    Article Title: Macrophage migration inhibitory factor mediates peripheral nerve injury-induced hypersensitivity by curbing dopaminergic descending inhibition
    Article Snippet: Ninety-six well ELISA plates were coated with rabbit anti-mouse MIF (Invitrogen, Carlsbad, CA, USA). .. After washing three times, recombinant mouse MIF test samples and standard sera were added to the wells and incubated for 2 h. Biotinylated polyclonal rabbit anti-mouse MIF was used as the detection antibody, and streptavidin-HRP was used as the second-step reagent.

    Article Title: Takinib, a selective TAK1 inhibitor, broadens the therapeutic efficacy of TNFα inhibition for cancer and autoimmune disease
    Article Snippet: After overnight incubation, cells were serum-starved with 1%CMRL medium, 1% Glutamax, 1%Pen/Strep for 4h, after which treatment with inhibitors and TNF stimulation occurred. .. 24h after treatment, supernatant was added to ELISA plates (IL-6, IL-8, TNF, Ready-SET-Go! from Thermo).

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL
    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6). .. After 2-h incubation with 100 µl/well of the chimeras to be measured, the plate was washed and incubated 1 h with biotinylated anti-human FasL mAb 10F2 at 0.1 µg/well in 100 µL diluted in PBS with 1% BSA.

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA
    Article Snippet: .. To detect the presence of LPS from M15 cells in the wells of ELISA plates coated with rUS9-FhLAP, or mAb US9 plus rUS9-FhLAP, the plates were incubated with 100 μL/well of a goat anti-lipid A LPS polyclonal antibody (ThermoFisher Scientific, Rockford, USA) diluted 1/100 in PBS-T-SM and incubated for 30 min at RT with shaking at 750 rpm. .. The plates were then washed five times with PBS-T, and bound IgG antibodies were detected with HRP-conjugated mouse anti-sheep/goat IgG monoclonal antibodies (Sigma–Aldrich, 1/30,000 in PBS-T-SM).

    Article Title: Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice against Cryptococcus neoformans Infection ▿
    Article Snippet: To characterize antigen binding, recombinant IgGs were added to ELISA plates (Immulon 1B; Thermo Labsystems) coated with 10 μg/ml GXM purified from culture of C. neoformans serotype D strain 24067, obtained from the American Type Culture Collection (ATCC) as described previously ( ). .. To estimate relative affinity in the fluid phase, a fixed amount of each recombinant IgG was first incubated for 1 h at 37°C with different concentrations of a peptide mimic of GXM (P206) recognized with high affinity by 3E5 ( ).

    Cell Culture:

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL
    Article Snippet: Protein quantification The concentration of the chimeras was quantified in cell culture using specific sandwich ELISA assays. .. The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Microscopy:

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions
    Article Snippet: ELISAs and Western blots For ELISAs, late cellularization DE-cad∷GFP and Arm∷GFP embryos were selected by hand under a dissecting microscope and placed on ice (10 embryos collected each). .. Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific).

    Expressing:

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions
    Article Snippet: Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific). .. The GFP standard curve was created by serial dilution of the pure GST-GFP in WT embryo lysate (prepared in the same way as the lysates of embryos expressing GFP-tagged proteins) and applied in neighboring wells of the ELISA plate.

    BIA-KA:

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions
    Article Snippet: Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific). .. For the GFP standard curve, GST-GFP was cloned by standard methods, expressed in DL21 cells, purified with glutathione resin (GE Healthcare), and quantified with a BCA protein assay (Thermo Fisher Scientific) versus BSA standard curves.

    Lysis:

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions
    Article Snippet: Embryos were dechorionated and transferred to a prechilled mini homogenizer and lysed with 100 µl NP-40 lysis buffer (150 mM sodium chloride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml PMSF, 1.0% NP-40, and 50 mM Tris, pH 8.0). .. Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific).

    Recombinant:

    Article Title: Macrophage migration inhibitory factor mediates peripheral nerve injury-induced hypersensitivity by curbing dopaminergic descending inhibition
    Article Snippet: Ninety-six well ELISA plates were coated with rabbit anti-mouse MIF (Invitrogen, Carlsbad, CA, USA). .. After washing three times, recombinant mouse MIF test samples and standard sera were added to the wells and incubated for 2 h. Biotinylated polyclonal rabbit anti-mouse MIF was used as the detection antibody, and streptavidin-HRP was used as the second-step reagent.

    Article Title: Significant role for IRF3 in both T cell and APC effector functions during T cell responses
    Article Snippet: ELISA plates were coated with antibodies to mouse IFN-γ or IL-17 (Ebiosciences, Sand Diego, CA). .. After blocking and washing, supernatants or serial dilutions of recombinant IFN-γ or IL-17 (Ebiosciences) were added.

    Article Title: Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice against Cryptococcus neoformans Infection ▿
    Article Snippet: .. To characterize antigen binding, recombinant IgGs were added to ELISA plates (Immulon 1B; Thermo Labsystems) coated with 10 μg/ml GXM purified from culture of C. neoformans serotype D strain 24067, obtained from the American Type Culture Collection (ATCC) as described previously ( ). .. Bound antibody was detected by using goat anti-human IgG(κ) L chain conjugated to alkaline phosphatase (AP; Sigma-Aldrich).

    Plasmid Preparation:

    Article Title: IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity
    Article Snippet: Total IgG auto-Ab titers were measured in ELISA plates coated with salmon sperm dsDNA (Invitrogen), histone (Sigma-Aldrich), or nucleosome (histone plated on a layer of dsDNA coating) and detected with biotinylated anti-κ Abs (Invitrogen). .. Total IgG auto-Ab titers were measured in ELISA plates coated with salmon sperm dsDNA (Invitrogen), histone (Sigma-Aldrich), or nucleosome (histone plated on a layer of dsDNA coating) and detected with biotinylated anti-κ Abs (Invitrogen).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher elisa plates
    1:7:7 stoichiometry of Baz, DE-cad, and Arm at SAJs. (A–D) Mapping protein distributions. (A, top) X-y planes are shown with single-cell sides boxed. (middle) Montages of single-cell sides shown from apical surface to 12 µm basal (0.3-µm z steps). SAJ is shown in a bracket. Dots indicate five SAJs and are shown at 3-µm intervals. (bottom) SAJ parameters are shown (50 sides each). (B) SAJ RFI profiles are shown of DE-cad∷GFP ( n = 288), Arm∷GFP ( n = 229), and Baz∷GFP ( n = 188). (C) IM RFI profiles ( n = 50 each) are shown. (D) Maps of SAJ and IM RFIs are shown over cell cortex. (E–G) Embryo protein counts are shown. (E) Purified <t>GST-GFP</t> is shown (12% SDS-PAGE; coomassie). (F) Example <t>ELISA</t> assay is shown with GST-GFP standard curve, and Baz∷GFP late cellularization embryos are shown. (G) Graph of the example standard curve is shown (done in duplicate), and the example Baz sample is indicated in red. (H) Levels of GFP-tagged proteins versus endogenous proteins in WT embryos (3–7-h embryo lysates; 6% SDS-PAGE). Blots were probed with DE-cad, Arm, Baz, and β-tubulin antibodies (non–GFP-tagged portion of DE-cad∷GFP detected; Oda and Tsukita, 1999 ). (I) Protein counts are shown. (J) Single-plane images are shown with the same coverslips and settings. Late cellularization is shown. RFIs were measured at the 10 brightest SAJs and equal IM areas. Normalized means ± SD are shown for five embryos each. WB, Western blot.
    Elisa Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/elisa plates/product/Thermo Fisher
    Average 99 stars, based on 430 article reviews
    Price from $9.99 to $1999.99
    elisa plates - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    95
    Thermo Fisher streptavidin coated elisa plates
    Three different mimotope peptides mimic different domains of the the P berghei GAPDH molecule. (A) The anti-P39 antibody specifically recognizes PbGAPDH in Western blots of P berghei sporozoite lysates. Ponceau stain and the anti-actin antibody were used as loading controls. “S”: lysates of sporozoites purified from infected A stephensi salivary glands; “M”: mock lysates obtained from uninfected A stephensi salivary glands. Anti-CSP antibody served as a positive control for the sporozoite lysate. A polyclonal anti-PbGAPDH antibody (red arrow) identifies a sporozoite GAPDH band with identical mobility to the anti-P39 band. (B) Antibodies against each of the mimotope peptides (P39, P61, and P52) recognize bands with identical mobility as PbGAPDH (red arrow), but not the pET tag protein (blue arrow). Each panel shows two lanes, the left containing the pET tag protein alone and the right the tagged recombinant PbGAPDH protein. The antibodies used to probe the blots are indicated at the bottom of each panel. The anti-tag and anti-KLH antibodies served as a positive and negative controls, respectively. The anti-mouse GAPDH antibody was used as a positive control for identification of the GAPDH protein. The position of the recombinant protein and tag proteins is indicated by arrows to the right. All data are representative of two independent experiments. (C) Each anti-peptide antibody specifically recognizes its own peptide. The biotinylated peptide indicated at the top of each panel was bound to the wells of <t>streptavidin-coated</t> <t>ELISA</t> plates and tested for binding by the antibodies denoted at the bottom of each panel. No evidence of cross-reaction was detected. All data are representative of two independent assays.
    Streptavidin Coated Elisa Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin coated elisa plates/product/Thermo Fisher
    Average 95 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    streptavidin coated elisa plates - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

    Image Search Results


    1:7:7 stoichiometry of Baz, DE-cad, and Arm at SAJs. (A–D) Mapping protein distributions. (A, top) X-y planes are shown with single-cell sides boxed. (middle) Montages of single-cell sides shown from apical surface to 12 µm basal (0.3-µm z steps). SAJ is shown in a bracket. Dots indicate five SAJs and are shown at 3-µm intervals. (bottom) SAJ parameters are shown (50 sides each). (B) SAJ RFI profiles are shown of DE-cad∷GFP ( n = 288), Arm∷GFP ( n = 229), and Baz∷GFP ( n = 188). (C) IM RFI profiles ( n = 50 each) are shown. (D) Maps of SAJ and IM RFIs are shown over cell cortex. (E–G) Embryo protein counts are shown. (E) Purified GST-GFP is shown (12% SDS-PAGE; coomassie). (F) Example ELISA assay is shown with GST-GFP standard curve, and Baz∷GFP late cellularization embryos are shown. (G) Graph of the example standard curve is shown (done in duplicate), and the example Baz sample is indicated in red. (H) Levels of GFP-tagged proteins versus endogenous proteins in WT embryos (3–7-h embryo lysates; 6% SDS-PAGE). Blots were probed with DE-cad, Arm, Baz, and β-tubulin antibodies (non–GFP-tagged portion of DE-cad∷GFP detected; Oda and Tsukita, 1999 ). (I) Protein counts are shown. (J) Single-plane images are shown with the same coverslips and settings. Late cellularization is shown. RFIs were measured at the 10 brightest SAJs and equal IM areas. Normalized means ± SD are shown for five embryos each. WB, Western blot.

    Journal: The Journal of Cell Biology

    Article Title: Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions

    doi: 10.1083/jcb.200812146

    Figure Lengend Snippet: 1:7:7 stoichiometry of Baz, DE-cad, and Arm at SAJs. (A–D) Mapping protein distributions. (A, top) X-y planes are shown with single-cell sides boxed. (middle) Montages of single-cell sides shown from apical surface to 12 µm basal (0.3-µm z steps). SAJ is shown in a bracket. Dots indicate five SAJs and are shown at 3-µm intervals. (bottom) SAJ parameters are shown (50 sides each). (B) SAJ RFI profiles are shown of DE-cad∷GFP ( n = 288), Arm∷GFP ( n = 229), and Baz∷GFP ( n = 188). (C) IM RFI profiles ( n = 50 each) are shown. (D) Maps of SAJ and IM RFIs are shown over cell cortex. (E–G) Embryo protein counts are shown. (E) Purified GST-GFP is shown (12% SDS-PAGE; coomassie). (F) Example ELISA assay is shown with GST-GFP standard curve, and Baz∷GFP late cellularization embryos are shown. (G) Graph of the example standard curve is shown (done in duplicate), and the example Baz sample is indicated in red. (H) Levels of GFP-tagged proteins versus endogenous proteins in WT embryos (3–7-h embryo lysates; 6% SDS-PAGE). Blots were probed with DE-cad, Arm, Baz, and β-tubulin antibodies (non–GFP-tagged portion of DE-cad∷GFP detected; Oda and Tsukita, 1999 ). (I) Protein counts are shown. (J) Single-plane images are shown with the same coverslips and settings. Late cellularization is shown. RFIs were measured at the 10 brightest SAJs and equal IM areas. Normalized means ± SD are shown for five embryos each. WB, Western blot.

    Article Snippet: Then, the full lysate volumes were loaded into ELISA plates coated with goat anti-GFP antibody (Thermo Fisher Scientific).

    Techniques: Purification, SDS Page, Enzyme-linked Immunosorbent Assay, Western Blot

    Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Derivative Assay, Construct, Cotransfection, FLAG-tag, Transfection, Plasmid Preparation, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Incubation, MTT Assay, Viability Assay

    Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Produced, Plasmid Preparation, Immunoprecipitation, SDS Page, Transfection, Incubation, Recombinant

    Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Construct, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Produced, SDS Page

    Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Expressing, Staining, Transfection, Plasmid Preparation, MTT Assay, Viability Assay, Incubation, Produced, Enzyme-linked Immunosorbent Assay, Blocking Assay

    Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Activity Assay, Derivative Assay, Cotransfection, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Construct, MTT Assay, Transfection

    Least squares linear regression analysis between OD values obtained in indirect ELISA testing sera from non-infected cattle in response to rUS9-FhLAP (rUS9-FhLAP response; X-axis) and LPS from E . coli serotype O55:B5 (LPS response; Y-axis). Each point represents the paired OD values obtained for each serum. Blue lines represent the 95% upper and lower confidence levels.

    Journal: PLoS ONE

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

    doi: 10.1371/journal.pone.0211035

    Figure Lengend Snippet: Least squares linear regression analysis between OD values obtained in indirect ELISA testing sera from non-infected cattle in response to rUS9-FhLAP (rUS9-FhLAP response; X-axis) and LPS from E . coli serotype O55:B5 (LPS response; Y-axis). Each point represents the paired OD values obtained for each serum. Blue lines represent the 95% upper and lower confidence levels.

    Article Snippet: To detect the presence of LPS from M15 cells in the wells of ELISA plates coated with rUS9-FhLAP, or mAb US9 plus rUS9-FhLAP, the plates were incubated with 100 μL/well of a goat anti-lipid A LPS polyclonal antibody (ThermoFisher Scientific, Rockford, USA) diluted 1/100 in PBS-T-SM and incubated for 30 min at RT with shaking at 750 rpm.

    Techniques: Indirect ELISA, Infection

    Analysis of soluble E . coli proteins binding to Nickel Affinity Gel columns. (A) SDS-PAGE analysis of M15 E . coli soluble proteins present in the starting sample (lane 1), in the non-retained fraction (lane 2), and in the eluted fraction (lane 3, concentrated). The relative mobility of MW standards is shown on the left. (B) Least squares linear regression analysis comparing OD values obtained in indirect ELISA for sera from non-infected cattle (n = 35) using as target IMAC-purified proteins from non-transformed (M15 response; X-axis) or transformed (rUS9-FhLAP response; Y-axis) M15 E . coli cells. Each point represents the paired OD values obtained for each serum in the indirect ELISAs. Blue lines represent the 95% upper and lower confidence levels.

    Journal: PLoS ONE

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

    doi: 10.1371/journal.pone.0211035

    Figure Lengend Snippet: Analysis of soluble E . coli proteins binding to Nickel Affinity Gel columns. (A) SDS-PAGE analysis of M15 E . coli soluble proteins present in the starting sample (lane 1), in the non-retained fraction (lane 2), and in the eluted fraction (lane 3, concentrated). The relative mobility of MW standards is shown on the left. (B) Least squares linear regression analysis comparing OD values obtained in indirect ELISA for sera from non-infected cattle (n = 35) using as target IMAC-purified proteins from non-transformed (M15 response; X-axis) or transformed (rUS9-FhLAP response; Y-axis) M15 E . coli cells. Each point represents the paired OD values obtained for each serum in the indirect ELISAs. Blue lines represent the 95% upper and lower confidence levels.

    Article Snippet: To detect the presence of LPS from M15 cells in the wells of ELISA plates coated with rUS9-FhLAP, or mAb US9 plus rUS9-FhLAP, the plates were incubated with 100 μL/well of a goat anti-lipid A LPS polyclonal antibody (ThermoFisher Scientific, Rockford, USA) diluted 1/100 in PBS-T-SM and incubated for 30 min at RT with shaking at 750 rpm.

    Techniques: Binding Assay, SDS Page, Indirect ELISA, Infection, Purification, Transformation Assay

    ELISA OD values obtained testing sera from Fasciola –free (open circles) and Fasciola –infected (closed circles) cattle. Each point in the figure represents the mean OD value (two replicates) obtained for one individual serum sample. IND = sera tested in indirect ELISA with rUS9-FhLAP antigen as target. US9 = sera tested in capture ELISA with mAb US9 coupled to ELISA plates and rUS9-FhLAP as target antigen. MM3 = sera tested in capture ELISA with mAb MM3 (MM3-ELISA; reference test) and Fasciola ESAs as target antigen. Horizontal lines represent the cut-off values (red dashed line: cut-off 1; blue line: cut-off 2). Cut-off 1 and cut-off 2 values for each ELISA were as follows: OD = 1.174 and 0.963 (IND); OD = 0.111 and 0.084 (US9); OD = 0.075 and 0.059 (MM3).

    Journal: PLoS ONE

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

    doi: 10.1371/journal.pone.0211035

    Figure Lengend Snippet: ELISA OD values obtained testing sera from Fasciola –free (open circles) and Fasciola –infected (closed circles) cattle. Each point in the figure represents the mean OD value (two replicates) obtained for one individual serum sample. IND = sera tested in indirect ELISA with rUS9-FhLAP antigen as target. US9 = sera tested in capture ELISA with mAb US9 coupled to ELISA plates and rUS9-FhLAP as target antigen. MM3 = sera tested in capture ELISA with mAb MM3 (MM3-ELISA; reference test) and Fasciola ESAs as target antigen. Horizontal lines represent the cut-off values (red dashed line: cut-off 1; blue line: cut-off 2). Cut-off 1 and cut-off 2 values for each ELISA were as follows: OD = 1.174 and 0.963 (IND); OD = 0.111 and 0.084 (US9); OD = 0.075 and 0.059 (MM3).

    Article Snippet: To detect the presence of LPS from M15 cells in the wells of ELISA plates coated with rUS9-FhLAP, or mAb US9 plus rUS9-FhLAP, the plates were incubated with 100 μL/well of a goat anti-lipid A LPS polyclonal antibody (ThermoFisher Scientific, Rockford, USA) diluted 1/100 in PBS-T-SM and incubated for 30 min at RT with shaking at 750 rpm.

    Techniques: Enzyme-linked Immunosorbent Assay, Infection, Indirect ELISA

    Least squares linear regression analysis comparing non-inhibited and inhibited (MTFSVPIS peptide) sera from non-infected cattle (A) or sera from F . hepatica naturally-infected cattle (B). Each point represents the paired OD values obtained for each serum in indirect ELISA using rUS9-FhLAP as target antigen. Blue lines represent the 95% upper and lower confidence levels.

    Journal: PLoS ONE

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

    doi: 10.1371/journal.pone.0211035

    Figure Lengend Snippet: Least squares linear regression analysis comparing non-inhibited and inhibited (MTFSVPIS peptide) sera from non-infected cattle (A) or sera from F . hepatica naturally-infected cattle (B). Each point represents the paired OD values obtained for each serum in indirect ELISA using rUS9-FhLAP as target antigen. Blue lines represent the 95% upper and lower confidence levels.

    Article Snippet: To detect the presence of LPS from M15 cells in the wells of ELISA plates coated with rUS9-FhLAP, or mAb US9 plus rUS9-FhLAP, the plates were incubated with 100 μL/well of a goat anti-lipid A LPS polyclonal antibody (ThermoFisher Scientific, Rockford, USA) diluted 1/100 in PBS-T-SM and incubated for 30 min at RT with shaking at 750 rpm.

    Techniques: Infection, Indirect ELISA

    ELISA OD values obtained testing sera from Fasciola –free (open circles) and Fasciola –infected (closed circles) sheep. Each point in the figure represents the mean OD value (two replicates) obtained for one individual serum sample. IND = sera tested in indirect ELISA with rUS9-FhLAP antigen as target. US9 = sera tested in capture ELISA with mAb US9 coupled to ELISA plates and rUS9-FhLAP as target antigen. MM3 = sera tested in capture ELISA with mAb MM3 (MM3-ELISA; reference test). Horizontal lines represent the cut-off values (red dashed line: cut-off 1; blue line: cut-off 2). Cut-off 1 and cut-off 2 values for each ELISA were as follows: OD = 0.205 and 0.16 (IND); OD = 0.114 and 0.089 (US9); OD = 0.009 and 0.007 (MM3).

    Journal: PLoS ONE

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

    doi: 10.1371/journal.pone.0211035

    Figure Lengend Snippet: ELISA OD values obtained testing sera from Fasciola –free (open circles) and Fasciola –infected (closed circles) sheep. Each point in the figure represents the mean OD value (two replicates) obtained for one individual serum sample. IND = sera tested in indirect ELISA with rUS9-FhLAP antigen as target. US9 = sera tested in capture ELISA with mAb US9 coupled to ELISA plates and rUS9-FhLAP as target antigen. MM3 = sera tested in capture ELISA with mAb MM3 (MM3-ELISA; reference test). Horizontal lines represent the cut-off values (red dashed line: cut-off 1; blue line: cut-off 2). Cut-off 1 and cut-off 2 values for each ELISA were as follows: OD = 0.205 and 0.16 (IND); OD = 0.114 and 0.089 (US9); OD = 0.009 and 0.007 (MM3).

    Article Snippet: To detect the presence of LPS from M15 cells in the wells of ELISA plates coated with rUS9-FhLAP, or mAb US9 plus rUS9-FhLAP, the plates were incubated with 100 μL/well of a goat anti-lipid A LPS polyclonal antibody (ThermoFisher Scientific, Rockford, USA) diluted 1/100 in PBS-T-SM and incubated for 30 min at RT with shaking at 750 rpm.

    Techniques: Enzyme-linked Immunosorbent Assay, Infection, Indirect ELISA

    Least squares linear regression analysis obtained comparing non-infected (A) and Fasciola -infected (B) cattle sera OD values obtained in indirect ELISA and US9-ELISA. Each point represents the paired OD values obtained for each serum by both methods. Blue lines represent the 95% upper and lower confidence levels.

    Journal: PLoS ONE

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

    doi: 10.1371/journal.pone.0211035

    Figure Lengend Snippet: Least squares linear regression analysis obtained comparing non-infected (A) and Fasciola -infected (B) cattle sera OD values obtained in indirect ELISA and US9-ELISA. Each point represents the paired OD values obtained for each serum by both methods. Blue lines represent the 95% upper and lower confidence levels.

    Article Snippet: To detect the presence of LPS from M15 cells in the wells of ELISA plates coated with rUS9-FhLAP, or mAb US9 plus rUS9-FhLAP, the plates were incubated with 100 μL/well of a goat anti-lipid A LPS polyclonal antibody (ThermoFisher Scientific, Rockford, USA) diluted 1/100 in PBS-T-SM and incubated for 30 min at RT with shaking at 750 rpm.

    Techniques: Infection, Indirect ELISA, Enzyme-linked Immunosorbent Assay

    Analysis of the expression of the chimeric recombinant protein rUS9-FhLAP in E . coli and optimization of its use in ELISA. (A) SDS-PAGE and WB analysis of rUS9-FhLAP expression. Lane 1: MW markers. Lane 2: Coomasie blue staining of the soluble protein fraction obtained after induction with 0.5 mM IPTG. Lane 3: Coomasie blue staining of rUS9-FhLAP after purification by IMAC. Lane 4: WB analysis of rUS9-FhLAP revealed with mAb US9 as primary antibody. Lane 5: WB analysis of rUS9-FhLAP revealed with HRP-conjugated anti-polyhistidine. (B) ELISA OD values obtained testing different concentrations of rUS9-FhLAP (range: 0.7–22 μg/mL) as target antigen in capture ELISA with mAb US9. (C) ELISA OD values obtained testing different concentrations of rUS9-FhLAP (range: 0.3–11 μg/mL) as target antigen in indirect ELISA. The analysis was carried out with a serum from a Fasciola -infected sheep as primary antibody.

    Journal: PLoS ONE

    Article Title: In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

    doi: 10.1371/journal.pone.0211035

    Figure Lengend Snippet: Analysis of the expression of the chimeric recombinant protein rUS9-FhLAP in E . coli and optimization of its use in ELISA. (A) SDS-PAGE and WB analysis of rUS9-FhLAP expression. Lane 1: MW markers. Lane 2: Coomasie blue staining of the soluble protein fraction obtained after induction with 0.5 mM IPTG. Lane 3: Coomasie blue staining of rUS9-FhLAP after purification by IMAC. Lane 4: WB analysis of rUS9-FhLAP revealed with mAb US9 as primary antibody. Lane 5: WB analysis of rUS9-FhLAP revealed with HRP-conjugated anti-polyhistidine. (B) ELISA OD values obtained testing different concentrations of rUS9-FhLAP (range: 0.7–22 μg/mL) as target antigen in capture ELISA with mAb US9. (C) ELISA OD values obtained testing different concentrations of rUS9-FhLAP (range: 0.3–11 μg/mL) as target antigen in indirect ELISA. The analysis was carried out with a serum from a Fasciola -infected sheep as primary antibody.

    Article Snippet: To detect the presence of LPS from M15 cells in the wells of ELISA plates coated with rUS9-FhLAP, or mAb US9 plus rUS9-FhLAP, the plates were incubated with 100 μL/well of a goat anti-lipid A LPS polyclonal antibody (ThermoFisher Scientific, Rockford, USA) diluted 1/100 in PBS-T-SM and incubated for 30 min at RT with shaking at 750 rpm.

    Techniques: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Staining, Purification, Indirect ELISA, Infection

    Three different mimotope peptides mimic different domains of the the P berghei GAPDH molecule. (A) The anti-P39 antibody specifically recognizes PbGAPDH in Western blots of P berghei sporozoite lysates. Ponceau stain and the anti-actin antibody were used as loading controls. “S”: lysates of sporozoites purified from infected A stephensi salivary glands; “M”: mock lysates obtained from uninfected A stephensi salivary glands. Anti-CSP antibody served as a positive control for the sporozoite lysate. A polyclonal anti-PbGAPDH antibody (red arrow) identifies a sporozoite GAPDH band with identical mobility to the anti-P39 band. (B) Antibodies against each of the mimotope peptides (P39, P61, and P52) recognize bands with identical mobility as PbGAPDH (red arrow), but not the pET tag protein (blue arrow). Each panel shows two lanes, the left containing the pET tag protein alone and the right the tagged recombinant PbGAPDH protein. The antibodies used to probe the blots are indicated at the bottom of each panel. The anti-tag and anti-KLH antibodies served as a positive and negative controls, respectively. The anti-mouse GAPDH antibody was used as a positive control for identification of the GAPDH protein. The position of the recombinant protein and tag proteins is indicated by arrows to the right. All data are representative of two independent experiments. (C) Each anti-peptide antibody specifically recognizes its own peptide. The biotinylated peptide indicated at the top of each panel was bound to the wells of streptavidin-coated ELISA plates and tested for binding by the antibodies denoted at the bottom of each panel. No evidence of cross-reaction was detected. All data are representative of two independent assays.

    Journal: Life Science Alliance

    Article Title: Identification of Plasmodium GAPDH epitopes for generation of antibodies that inhibit malaria infection

    doi: 10.26508/lsa.201800111

    Figure Lengend Snippet: Three different mimotope peptides mimic different domains of the the P berghei GAPDH molecule. (A) The anti-P39 antibody specifically recognizes PbGAPDH in Western blots of P berghei sporozoite lysates. Ponceau stain and the anti-actin antibody were used as loading controls. “S”: lysates of sporozoites purified from infected A stephensi salivary glands; “M”: mock lysates obtained from uninfected A stephensi salivary glands. Anti-CSP antibody served as a positive control for the sporozoite lysate. A polyclonal anti-PbGAPDH antibody (red arrow) identifies a sporozoite GAPDH band with identical mobility to the anti-P39 band. (B) Antibodies against each of the mimotope peptides (P39, P61, and P52) recognize bands with identical mobility as PbGAPDH (red arrow), but not the pET tag protein (blue arrow). Each panel shows two lanes, the left containing the pET tag protein alone and the right the tagged recombinant PbGAPDH protein. The antibodies used to probe the blots are indicated at the bottom of each panel. The anti-tag and anti-KLH antibodies served as a positive and negative controls, respectively. The anti-mouse GAPDH antibody was used as a positive control for identification of the GAPDH protein. The position of the recombinant protein and tag proteins is indicated by arrows to the right. All data are representative of two independent experiments. (C) Each anti-peptide antibody specifically recognizes its own peptide. The biotinylated peptide indicated at the top of each panel was bound to the wells of streptavidin-coated ELISA plates and tested for binding by the antibodies denoted at the bottom of each panel. No evidence of cross-reaction was detected. All data are representative of two independent assays.

    Article Snippet: Mouse antibody was titrated 1 wk after the final boost using biotinylated peptide attached to streptavidin-coated ELISA plates (Thermo Fisher Scientific).

    Techniques: Western Blot, Staining, Purification, Infection, Positive Control, Positron Emission Tomography, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay