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  • 99
    Thermo Fisher igg4
    Recognition of NH and SMT antigens in patients from a L. donovani endemic area in Bangladesh. ( a ) Total <t>IgG</t> against L. donovani SLA, NH and SMT are shown. Antibodies against the antigens were measured by ELISA in sera (used at 1:400 dilution) in VL individuals ( n =25), asymptomatic individuals ( n =32) and non-endemic controls ( n =24). ( b ) PMBCs were recalled with 10 μg ml −1 L. donovani SLA, NH or SMT and analyzed for CD4 T cells production of indicated cytokines by flow cytometry. ( a and b ) Black bars indicate median OD for each group. Statistical significance indicated is vs the non-endemic normal group; statistic was calculated by Kolmogorov–Smirnov t -test. **** P
    Igg4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 553 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore biotin labelled antihuman igg1 igg2 igg3 igg4
    Recognition of NH and SMT antigens in patients from a L. donovani endemic area in Bangladesh. ( a ) Total <t>IgG</t> against L. donovani SLA, NH and SMT are shown. Antibodies against the antigens were measured by ELISA in sera (used at 1:400 dilution) in VL individuals ( n =25), asymptomatic individuals ( n =32) and non-endemic controls ( n =24). ( b ) PMBCs were recalled with 10 μg ml −1 L. donovani SLA, NH or SMT and analyzed for CD4 T cells production of indicated cytokines by flow cytometry. ( a and b ) Black bars indicate median OD for each group. Statistical significance indicated is vs the non-endemic normal group; statistic was calculated by Kolmogorov–Smirnov t -test. **** P
    Biotin Labelled Antihuman Igg1 Igg2 Igg3 Igg4, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Nordic-Mubio igg3
    The number of anti-CarP antibody isotypes and <t>IgG</t> subclasses differs between RA patients and is level dependent. Anti-carbamylated protein ( anti-CarP ) antibody isotypes and immunoglobulin G ( IgG ) subclasses were measured by ELISA in 373 rheumatoid arthritis ( RA ) patients and 196 healthy controls ( HC ). Pie charts show the percentage of RA patients and HC negative or positive for one, two, or three anti-CarP antibody isotypes ( a ) and negative or positive for one, two, three, or four anti-CarP IgG subclasses ( b ). An increase in level of anti-CarP antibody IgG associates with an increase in the number of anti-CarP antibody isotypes ( c ) and IgG subclasses ( d ) in RA patients. Red lines depict means ( e ). Heat maps show the presence of anti-CarP antibody isotypes and IgG subclasses in RA and HC, ranked according to anti-CarP antibody IgG levels. Green and red mark positive and negative sera, respectively. AU arbitrary units
    Igg3, supplied by Nordic-Mubio, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    SouthernBiotech isotype igg1 igg2 igg3 or igg4
    The number of anti-CarP antibody isotypes and <t>IgG</t> subclasses differs between RA patients and is level dependent. Anti-carbamylated protein ( anti-CarP ) antibody isotypes and immunoglobulin G ( IgG ) subclasses were measured by ELISA in 373 rheumatoid arthritis ( RA ) patients and 196 healthy controls ( HC ). Pie charts show the percentage of RA patients and HC negative or positive for one, two, or three anti-CarP antibody isotypes ( a ) and negative or positive for one, two, three, or four anti-CarP IgG subclasses ( b ). An increase in level of anti-CarP antibody IgG associates with an increase in the number of anti-CarP antibody isotypes ( c ) and IgG subclasses ( d ) in RA patients. Red lines depict means ( e ). Heat maps show the presence of anti-CarP antibody isotypes and IgG subclasses in RA and HC, ranked according to anti-CarP antibody IgG levels. Green and red mark positive and negative sera, respectively. AU arbitrary units
    Isotype Igg1 Igg2 Igg3 Or Igg4, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Merck & Co subclasses igg1 igg4
    Serological response to AAV-2 in Irish blood donors. (a) AAV-2-specific total <t>IgG</t> in human donor plasma ( n =45). Specific IgG was determined by indirect AAV-2 antigen ELISA, normalized by comparison with standard positive and negative samples. The negative cut-off (dashed line) was defined at 2 sd above the concentration of a known seronegative standard. (b–e) AAV-2-specific IgG subclasses present in IgG-positive samples ( n =41). <t>IgG1(b),</t> <t>IgG2</t> (c), <t>IgG3</t> (d) and <t>IgG4</t> (e) determined by AAV-2-specific direct antigen ELISA for triplicate samples and quantified by comparison with known reference sera.
    Subclasses Igg1 Igg4, supplied by Merck & Co, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LakePharma 3c1 igg1 igg3
    Antibodies that target the NANP epitope of CSP can promote C1q-fixation. Antibodies from malaria-exposed adults living in PNG ( n = 30) ( a ) and Kenya ( n = 30) ( b ) were tested for IgG, IgM, and C1q-fixation to (NANP) 15 peptide by ELISA, and correlated with C1q fixation to CSP (Spearman’s correlation coefficient, rho). Results were standardized to arbitrary units (AU) based on malaria-naïve negative controls from Melbourne (seropositivity defined as AU > 1, shown as dotted lines) and mean of duplicates were graphed. c Mouse anti-CSP MAbs <t>2H8-IgG1/IgG2a</t> and <t>3C1-IgG1/IgG3</t> were tested for CSP-IgG, NANP-IgG, and C1q-fixation to CSP by ELISA. Results were corrected for background reactivity using no-IgG controls, and the mean and range of duplicates were graphed. AU arbitrary units, CSP circumsporozoite protein, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, IgM immunoglobulin M, MAb monoclonal antibody, OD optical density, PNG Papua New Guinea
    3c1 Igg1 Igg3, supplied by LakePharma, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore igg1 igg4
    Examples of serum-dependent cross-reactivity of <t>IgG</t> subclass detection on control beads. The control beads were incubated with different patient sera, washed, and then detected with subclass-specific secondary antibodies ((a): patient serum 1; (b): patient serum 2; (c): patient serum 3).
    Igg1 Igg4, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cellular Technology Ltd human igg1 igg2 igg3 igg4 four color fluorospot
    Viral reactivity among thymic <t>IgG-secreting</t> plasma cells
    Human Igg1 Igg2 Igg3 Igg4 Four Color Fluorospot, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher mouse anti human igg1 igg2 igg3 igg4 antibodies
    Viral reactivity among thymic <t>IgG-secreting</t> plasma cells
    Mouse Anti Human Igg1 Igg2 Igg3 Igg4 Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher igg1 igg3 fc
    PA and PG binding assessment of engineered antibodies by linear-gradient chromatography. (a) Overlay of HiTrap® MabSelect™ SuRe™ PA chromatograms (RT). An <t>IgG3-like</t> antibody (IgG 1133) based on the VH3 subclass (blue) still bound the MabSelect™ SuRe™ resin even though elution occurred at a mild pH (~pH 4.2). Adding the Fab substitution G65S (green) or N82aS (red) completely abolished binding (Kabat numbering). (b) Overlay of HiTrap® PG HP chromatograms (RT). An <t>IgG1</t> antibody carrying the M428G/N434A substitutions in its Fc region (blue) still bound PG, while the same antibody with the added Fab substitution T209G (green) or K213V (red) was found in the flow through (Eu numbering).
    Igg1 Igg3 Fc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Siemens Healthineers n latex igg3 igg4 assay
    PA and PG binding assessment of engineered antibodies by linear-gradient chromatography. (a) Overlay of HiTrap® MabSelect™ SuRe™ PA chromatograms (RT). An <t>IgG3-like</t> antibody (IgG 1133) based on the VH3 subclass (blue) still bound the MabSelect™ SuRe™ resin even though elution occurred at a mild pH (~pH 4.2). Adding the Fab substitution G65S (green) or N82aS (red) completely abolished binding (Kabat numbering). (b) Overlay of HiTrap® PG HP chromatograms (RT). An <t>IgG1</t> antibody carrying the M428G/N434A substitutions in its Fc region (blue) still bound PG, while the same antibody with the added Fab substitution T209G (green) or K213V (red) was found in the flow through (Eu numbering).
    N Latex Igg3 Igg4 Assay, supplied by Siemens Healthineers, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Boehringer Ingelheim anti igg1 igg4 antibodies
    PA and PG binding assessment of engineered antibodies by linear-gradient chromatography. (a) Overlay of HiTrap® MabSelect™ SuRe™ PA chromatograms (RT). An <t>IgG3-like</t> antibody (IgG 1133) based on the VH3 subclass (blue) still bound the MabSelect™ SuRe™ resin even though elution occurred at a mild pH (~pH 4.2). Adding the Fab substitution G65S (green) or N82aS (red) completely abolished binding (Kabat numbering). (b) Overlay of HiTrap® PG HP chromatograms (RT). An <t>IgG1</t> antibody carrying the M428G/N434A substitutions in its Fc region (blue) still bound PG, while the same antibody with the added Fab substitution T209G (green) or K213V (red) was found in the flow through (Eu numbering).
    Anti Igg1 Igg4 Antibodies, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Sanquin igg4 antibody depletion igg1 anti igg4 antibody
    <t>IgG</t> 4 /IgE ratios to peanut and peanut allergens in children with PA (n = 65) and PS children (n = 27). Values are presented as medians and interquartile ranges. P values refer to the comparison between children with PA and PS children using the Mann-Whitney U test.
    Igg4 Antibody Depletion Igg1 Anti Igg4 Antibody, supplied by Sanquin, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore igg4 kappa
    <t>IgG</t> 4 /IgE ratios to peanut and peanut allergens in children with PA (n = 65) and PS children (n = 27). Values are presented as medians and interquartile ranges. P values refer to the comparison between children with PA and PS children using the Mann-Whitney U test.
    Igg4 Kappa, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher anti igg
    Antibody production after stimulation with toll-like receptor (TLR) ligands. (A) Spontaneous and induced <t>IgM</t> responses by B cell subsets. The B cell subsets P1–P4 defined as shown in Figure 2 were FACS-sorted, cultured at 0.5 × 10 6 cells/ml and stimulated with resiquimod in the absence or presence of interleukin-2 (IL-2) and B-cell activating factor (BAFF) for 5 days. IgM production in the supernatants was quantified by ELISA. (B) TLR-induced IgM production induced of full PBMCs, of plasmacytoid dendritic cell (pDC)-depleted PBMCs (C) , and of PBMCs reconstituted with pDC following sorting (D) . (E–G) FMDV-specific <t>IgG-antibody</t> levels detected in the same setup as in (B–D) . (B–G) Cells were cultured for 7 days. Six biological replicates of two independent experiments are shown. Statistical significance was calculated using ANOVA followed by Tukey’s test (**** P
    Anti Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 446 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher control igg
    ZNF282 interacts with E2F1 and functions as an E2F1 co-activator (A)Endogenous interaction between ZNF282 and E2F1. TE10 cell lysates were immunoprecipitated with anti-E2F1 antibody or control normal <t>IgG,</t> followed by immunoblot with the indicated antibodies. (B) <t>293T</t> cells were transfected with pSG5.HA-E2F1 and pFLAG.CMV4-ZNF282. Cell lysates were immunoprecipitated with anti-E2F1 antibody or control normal IgG. Immunoprecipitated E2F1 and coimmunoprecipitated ZNF282 were detected byanti-FLAG antibody and anti-E2F1 antibody, respectively. (C) In vitro translated HA-tagged ZNF282 was incubated with recombinant GST-E2F1. Bound proteins were analyzed by immunoblot with anti-HA antibody. (D) Coactivator activity of ZNF282 for E2F1. 293T cells were transfected with pSG5.HA-E2F1 (20 ng) and 5xE2F-TA-LUC reporter (200 ng) in combination with various amounts (200, 400, 600 ng) of pSG5.HA- ZNF282. Cell extracts were assayed for luciferase activity. Results shown are mean and SD of triplicate points. (E and F) ZNF282 is required for the transcriptional activity of E2F1. 293T cells were transfected with pSG5.HA-E2F1, 5xE2F-TA-LUC, and 40 pmol of ZNF282 siRNA#5, #9 or NS (non-specific) siRNA duplex. 72 h after transfection, cells extracts were harvested for immunoblot analysis (E) and luciferase assays (F). (G-I) Requirement of ZNF282 for the expression of a subset of E2F1 target genes and recruitment of ZNF282 to selective E2F1 target genes. (G, H, and I) TE10 cells were transfected with 40 pmol of ZNF282 siRNA#5or NS siRNA duplex. 72 h after transfection, protein extracts and totalRNA were prepared. Protein levels were monitored by immunoblot using the indicated antibodies (G). Total RNA was examined by real-time quantitative RT–PCR (qRT–PCR) analysis with primers specific for the indicated mRNAs (H and I). Results shown were normalized to b-actin mRNAlevels and are means ± standard deviation (n=3). (J) ChIP assay. Crosslinked, sheared chromatin from TE10 cells was immunoprecipitated with the indicated antibodies. Quantitative PCR (qPCR) analyses were performed using primers specific for the indicated promoters. The results are shown as percentage of input and are means ± standard deviation (n=3).
    Control Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    BioLegend isotype controls igg1 igg4
    ZNF282 interacts with E2F1 and functions as an E2F1 co-activator (A)Endogenous interaction between ZNF282 and E2F1. TE10 cell lysates were immunoprecipitated with anti-E2F1 antibody or control normal <t>IgG,</t> followed by immunoblot with the indicated antibodies. (B) <t>293T</t> cells were transfected with pSG5.HA-E2F1 and pFLAG.CMV4-ZNF282. Cell lysates were immunoprecipitated with anti-E2F1 antibody or control normal IgG. Immunoprecipitated E2F1 and coimmunoprecipitated ZNF282 were detected byanti-FLAG antibody and anti-E2F1 antibody, respectively. (C) In vitro translated HA-tagged ZNF282 was incubated with recombinant GST-E2F1. Bound proteins were analyzed by immunoblot with anti-HA antibody. (D) Coactivator activity of ZNF282 for E2F1. 293T cells were transfected with pSG5.HA-E2F1 (20 ng) and 5xE2F-TA-LUC reporter (200 ng) in combination with various amounts (200, 400, 600 ng) of pSG5.HA- ZNF282. Cell extracts were assayed for luciferase activity. Results shown are mean and SD of triplicate points. (E and F) ZNF282 is required for the transcriptional activity of E2F1. 293T cells were transfected with pSG5.HA-E2F1, 5xE2F-TA-LUC, and 40 pmol of ZNF282 siRNA#5, #9 or NS (non-specific) siRNA duplex. 72 h after transfection, cells extracts were harvested for immunoblot analysis (E) and luciferase assays (F). (G-I) Requirement of ZNF282 for the expression of a subset of E2F1 target genes and recruitment of ZNF282 to selective E2F1 target genes. (G, H, and I) TE10 cells were transfected with 40 pmol of ZNF282 siRNA#5or NS siRNA duplex. 72 h after transfection, protein extracts and totalRNA were prepared. Protein levels were monitored by immunoblot using the indicated antibodies (G). Total RNA was examined by real-time quantitative RT–PCR (qRT–PCR) analysis with primers specific for the indicated mRNAs (H and I). Results shown were normalized to b-actin mRNAlevels and are means ± standard deviation (n=3). (J) ChIP assay. Crosslinked, sheared chromatin from TE10 cells was immunoprecipitated with the indicated antibodies. Quantitative PCR (qPCR) analyses were performed using primers specific for the indicated promoters. The results are shown as percentage of input and are means ± standard deviation (n=3).
    Isotype Controls Igg1 Igg4, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher mouse igg
    Aurora B associates with <t>p53</t> at various phases of the cell cycle. ( A ) Lysates of U2OS cells were IP with either anti-Aurora B (AB2254; Abcam) or anti-p53 antibodies (AB-2) or preimmune <t>IgG</t> (negative control) followed by immunoblotting (IB) with antibodies as labeled to show co-IP of Aurora B and p53. ( B ) GST pull-down assay was performed with combinations of in vitro translated Aurora B, GST, and GST-tagged p53 as labeled. Aurora B that was bound to GST-p53 was detected by IB. Coomassie staining of GST and GST-p53 inputs are in C . In vitro translated Aurora B was detected by immunoblot ( Lower ). ( C ) As presented in B , results of in vitro GST pull-down assay of immunopurified Flag-Aurora B with GST or GST-tagged p53 deletion constructs are shown. The asterisks indicate the stained bands of GST, GST-p53, and GST-p53 deletion mutants. ( D ) Hct116 cells were synchronized to S phase by double thymidine block. Cell samples at labeled time points after release of thymidine block were stained with PI and analyzed by FACS for DNA content. DNA content histograms are shown for the time points as labeled. ( E ) Lysates of synchronized Hct116 cells from D were analyzed by immunoblot with indicated antibodies. Aurora B–p53 interaction at various phases of the cell cycle (as labeled above) was detected by IP with anti-Aurora B antibody followed by IB for p53 and Aurora B (IP:AurB and IB:AurB).
    Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Recognition of NH and SMT antigens in patients from a L. donovani endemic area in Bangladesh. ( a ) Total IgG against L. donovani SLA, NH and SMT are shown. Antibodies against the antigens were measured by ELISA in sera (used at 1:400 dilution) in VL individuals ( n =25), asymptomatic individuals ( n =32) and non-endemic controls ( n =24). ( b ) PMBCs were recalled with 10 μg ml −1 L. donovani SLA, NH or SMT and analyzed for CD4 T cells production of indicated cytokines by flow cytometry. ( a and b ) Black bars indicate median OD for each group. Statistical significance indicated is vs the non-endemic normal group; statistic was calculated by Kolmogorov–Smirnov t -test. **** P

    Journal: Clinical & Translational Immunology

    Article Title: From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE

    doi: 10.1038/cti.2015.6

    Figure Lengend Snippet: Recognition of NH and SMT antigens in patients from a L. donovani endemic area in Bangladesh. ( a ) Total IgG against L. donovani SLA, NH and SMT are shown. Antibodies against the antigens were measured by ELISA in sera (used at 1:400 dilution) in VL individuals ( n =25), asymptomatic individuals ( n =32) and non-endemic controls ( n =24). ( b ) PMBCs were recalled with 10 μg ml −1 L. donovani SLA, NH or SMT and analyzed for CD4 T cells production of indicated cytokines by flow cytometry. ( a and b ) Black bars indicate median OD for each group. Statistical significance indicated is vs the non-endemic normal group; statistic was calculated by Kolmogorov–Smirnov t -test. **** P

    Article Snippet: After incubation, peroxidase-labeled anti-human IgG, IgG1, IgG2, IgG3, IgG4 and IgE (Life Technologies, Carlsbad, CA, USA) in serum diluent was added.

    Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    LEISH-F3 formulated with GLA-SE is immunogenic in humans. Thirty-six healthy adult subjects were immunized on days 0, 28 and 56 with 20 μg LEISH-F3+2 μg GLA-SE ( n =12), 20 μg LEISH-F3+5 μg GLA-SE ( n =12) and 20μg LEISH-F3 protein alone ( n =12). The immunogenicity of the vaccine was evaluated by assessing antibody and T-cell responses at days 0, 35, 63, 84 and 168 and days 0, 7, 35, 63, 84 and 168, respectively. ( a and b ) ELISAs for titers of LEISH-F3-specific antibodies (total and IgG subclasses and total IgE) in volunteer serum were conducted for the indicated time points. ( c ) Quantitative T-cell responses to LEISH-F3 was measured by IL-2, IL-5, IL-10, IFN-γ and TNF cytokine production in whole blood Luminex assay (WBA). Nine subjects were excluded from the per-protocol population immunology summaries resulting in evaluable groups such as: 2 μg GLA-SE ( n =8), 5 μg GLA-SE ( n =10) and LEISH-F3 alone ( n =9). P -value for comparison was performed for various treatment groups: between 2 and 5 μg GLA-SE vaccine groups and between vaccine (2 and 5 μg GLA-SE vaccine groups combined) and 20 μg LEISH-F3 alone. P -values were considered significant at the 0.05 significance level. * P -values significant at 0.05 significance level when compared between vaccine (2 and 5 μg GLA-SE vaccine groups combined) and 20 μg LEISH-F3 alone. ** P -values significant at 0.05 significance level when compared between vaccine (2 and 5 μg GLA-SE vaccine groups separately).

    Journal: Clinical & Translational Immunology

    Article Title: From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE

    doi: 10.1038/cti.2015.6

    Figure Lengend Snippet: LEISH-F3 formulated with GLA-SE is immunogenic in humans. Thirty-six healthy adult subjects were immunized on days 0, 28 and 56 with 20 μg LEISH-F3+2 μg GLA-SE ( n =12), 20 μg LEISH-F3+5 μg GLA-SE ( n =12) and 20μg LEISH-F3 protein alone ( n =12). The immunogenicity of the vaccine was evaluated by assessing antibody and T-cell responses at days 0, 35, 63, 84 and 168 and days 0, 7, 35, 63, 84 and 168, respectively. ( a and b ) ELISAs for titers of LEISH-F3-specific antibodies (total and IgG subclasses and total IgE) in volunteer serum were conducted for the indicated time points. ( c ) Quantitative T-cell responses to LEISH-F3 was measured by IL-2, IL-5, IL-10, IFN-γ and TNF cytokine production in whole blood Luminex assay (WBA). Nine subjects were excluded from the per-protocol population immunology summaries resulting in evaluable groups such as: 2 μg GLA-SE ( n =8), 5 μg GLA-SE ( n =10) and LEISH-F3 alone ( n =9). P -value for comparison was performed for various treatment groups: between 2 and 5 μg GLA-SE vaccine groups and between vaccine (2 and 5 μg GLA-SE vaccine groups combined) and 20 μg LEISH-F3 alone. P -values were considered significant at the 0.05 significance level. * P -values significant at 0.05 significance level when compared between vaccine (2 and 5 μg GLA-SE vaccine groups combined) and 20 μg LEISH-F3 alone. ** P -values significant at 0.05 significance level when compared between vaccine (2 and 5 μg GLA-SE vaccine groups separately).

    Article Snippet: After incubation, peroxidase-labeled anti-human IgG, IgG1, IgG2, IgG3, IgG4 and IgE (Life Technologies, Carlsbad, CA, USA) in serum diluent was added.

    Techniques: Luminex

    Analysis of neutralization sensitivity of Env variants taken from donor 45 at three time points to autologous serum IgG (left panels) and autologous CD4bs MAbs VRC01, VRC03, VRC06, and VRC06b (right panels). The horizontal bars indicate the group geometric

    Journal: Journal of Virology

    Article Title: Selection Pressure on HIV-1 Envelope by Broadly Neutralizing Antibodies to the Conserved CD4-Binding Site

    doi: 10.1128/JVI.07139-11

    Figure Lengend Snippet: Analysis of neutralization sensitivity of Env variants taken from donor 45 at three time points to autologous serum IgG (left panels) and autologous CD4bs MAbs VRC01, VRC03, VRC06, and VRC06b (right panels). The horizontal bars indicate the group geometric

    Article Snippet: The concentrations of purified serum IgG or IgG MAbs were determined using NanoDrop (Thermo Scientific) with 1.40 as the extinction coefficient.

    Techniques: Neutralization

    Comparative analysis of different serodiagnostic assays from serum samples obtained from PKDL patients, Healthy controls (EC and NEC) and disease control groups (Leprosy and Vitiligo). (A) Comparative evaluation of PEG CIC at 450 nm obtained from PEG precipitation of PKDL patients’ sera; cut-off 0.274. (B) Comparative evaluation of ELISA reactivity of anti-leishmanial antibody (IgG) in serum against Leishmania antigen; cut-off 0.350. (C) Comparative evaluation of ELISA reactivity of anti-leishmanial antibody (IgM) in serum against Leishmania antigen; cut-off 0.191. (D) Comparative evaluation of Glyco CIC assay based on CICs isolated from sera of PKDL patients; cut-off 0.091. The study population in all cases comprised of panel of PKDL patients (PKDL; n = 90), Endemic Healthy controls (n = 19), Non-Endemic Healthy controls (n = 34) and other disease (OD; n = 83) including Leprosy (n = 37) and Vitiligo (n = 46). Each sample was tested in triplicates and mean was taken. Each dot represent mean of single sample and the black dotted horizontal lines represent cut-off values. The cut-off value of the assays was established as means + 3 SD of 136 controls. Significance was determined by unpaired Student's t-test at 95% confidence intervals and p-values

    Journal: PLoS ONE

    Article Title: Glycoproteins in circulating immune complexes are biomarkers of patients with Indian PKDL: A study from endemic districts of West Bengal, India

    doi: 10.1371/journal.pone.0192302

    Figure Lengend Snippet: Comparative analysis of different serodiagnostic assays from serum samples obtained from PKDL patients, Healthy controls (EC and NEC) and disease control groups (Leprosy and Vitiligo). (A) Comparative evaluation of PEG CIC at 450 nm obtained from PEG precipitation of PKDL patients’ sera; cut-off 0.274. (B) Comparative evaluation of ELISA reactivity of anti-leishmanial antibody (IgG) in serum against Leishmania antigen; cut-off 0.350. (C) Comparative evaluation of ELISA reactivity of anti-leishmanial antibody (IgM) in serum against Leishmania antigen; cut-off 0.191. (D) Comparative evaluation of Glyco CIC assay based on CICs isolated from sera of PKDL patients; cut-off 0.091. The study population in all cases comprised of panel of PKDL patients (PKDL; n = 90), Endemic Healthy controls (n = 19), Non-Endemic Healthy controls (n = 34) and other disease (OD; n = 83) including Leprosy (n = 37) and Vitiligo (n = 46). Each sample was tested in triplicates and mean was taken. Each dot represent mean of single sample and the black dotted horizontal lines represent cut-off values. The cut-off value of the assays was established as means + 3 SD of 136 controls. Significance was determined by unpaired Student's t-test at 95% confidence intervals and p-values

    Article Snippet: The wells were then incubated for 1hr at RT with HRP-conjugated anti-human monoclonal IgG (1:15,000) (Sigma-Aldrich, Cat # :A0170), monoclonal IgG1 (1:1000) (Thermo Fisher Scientific, Cat#: A-10648, USA), monoclonal IgG2 (1:1000) (Thermo Fisher Scientific, Cat#:MH1772, USA), monoclonal IgG3 (1:1000) (Thermo Fisher Scientific, Cat#:05–3620,USA), monoclonal IgG4 (1:1000) (Thermo Fisher Scientific, Cat#:A-10654,USA), and polyclonal IgM (1:10,000) (Sigma-Aldrich, Cat#:A6907), respectively, to measure the levels of IgM, IgG and IgG subclasses of anti-leishmanial antibodies with Tetramethylbenzidine (TMB).

    Techniques: Enzyme-linked Immunosorbent Assay, Isolation

    Suppression of granulocyte activation and degranulation is IgG-independent in EN but IgG-dependent in Mf+. Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone (dark bars) or in presence of 5 μg/ml of IgG negative fractions (n = 8) (light bars) from EN (A), Mf+ (B), Mf- (C), CP (D) and NEC (E) or the corresponding IgG positive fractions (grey bars). After that, cells were stained for CD63 and HLADR expression. Activated granulocytes were characterized as CD63+/HLADR- cells. Bars represent means ± SEM of the percentage of activated granulocyte CD63 + /HLADR - cells. The release of histamine after 30 min (F), neutrophil elastase (G) and eosinophil cationic protein (H) after 18 hours in culture supernatants was assessed. Graphs are representative of 3 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes

    doi: 10.1371/journal.pntd.0005777

    Figure Lengend Snippet: Suppression of granulocyte activation and degranulation is IgG-independent in EN but IgG-dependent in Mf+. Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone (dark bars) or in presence of 5 μg/ml of IgG negative fractions (n = 8) (light bars) from EN (A), Mf+ (B), Mf- (C), CP (D) and NEC (E) or the corresponding IgG positive fractions (grey bars). After that, cells were stained for CD63 and HLADR expression. Activated granulocytes were characterized as CD63+/HLADR- cells. Bars represent means ± SEM of the percentage of activated granulocyte CD63 + /HLADR - cells. The release of histamine after 30 min (F), neutrophil elastase (G) and eosinophil cationic protein (H) after 18 hours in culture supernatants was assessed. Graphs are representative of 3 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p

    Article Snippet: Assessment of immunoglobulin isotypes expression in plasma and IgG/IgG4 positive and negative fractions To analyze the isotype composition in IgA, IgG/IgG4 positive and negative fractions and in the plasma of EN and LF patients, ProcartaPlex Human Antibody Isotyping Panels (eBioscience, Vienna, Austria) were used according to manufacturer’s instructions.

    Techniques: Activation Assay, Isolation, Staining, Expressing

    Anti-inflammatory IgG4 antibodies modulate granulocyte functions via FcγRI and FcγRII. Granulocytes from healthy blood donors were purified and stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml), and Brugia antigen extracts (10 μg/ml) and incubated with either medium or anti-FcγRI (2μg/ml), FcγRII (1 μg/ml) or FcγRIII (4 μg/ml) antibodies alone or in combinations. Thereafter granulocytes were incubated with 2.5 μg/ml of affinity purified IgG4 for 18 hours. The cells were then stained with DAPI (blue) and the presence of IgG4 was detected with anti-IgG Alexa fluor 488 antibody (green). A representative experiment out of 6 is shown: A-G are representative fluorescence microscopy images of granulocytes incubated with culture medium (A) or in the presence of blocking antibodies against FcγRI (B), FcγRII (C), FcγRIII (D) or a combination of antibodies against FcγRI and II (E), FcγRI and III (F), or FcγRII and III (G). Bars represent mean fluorescence intensities ± SEM (H) or the percentages of CD63+/HLADR- activated granulocytes (I) in 6 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test; *: p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes

    doi: 10.1371/journal.pntd.0005777

    Figure Lengend Snippet: Anti-inflammatory IgG4 antibodies modulate granulocyte functions via FcγRI and FcγRII. Granulocytes from healthy blood donors were purified and stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml), and Brugia antigen extracts (10 μg/ml) and incubated with either medium or anti-FcγRI (2μg/ml), FcγRII (1 μg/ml) or FcγRIII (4 μg/ml) antibodies alone or in combinations. Thereafter granulocytes were incubated with 2.5 μg/ml of affinity purified IgG4 for 18 hours. The cells were then stained with DAPI (blue) and the presence of IgG4 was detected with anti-IgG Alexa fluor 488 antibody (green). A representative experiment out of 6 is shown: A-G are representative fluorescence microscopy images of granulocytes incubated with culture medium (A) or in the presence of blocking antibodies against FcγRI (B), FcγRII (C), FcγRIII (D) or a combination of antibodies against FcγRI and II (E), FcγRI and III (F), or FcγRII and III (G). Bars represent mean fluorescence intensities ± SEM (H) or the percentages of CD63+/HLADR- activated granulocytes (I) in 6 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test; *: p

    Article Snippet: Assessment of immunoglobulin isotypes expression in plasma and IgG/IgG4 positive and negative fractions To analyze the isotype composition in IgA, IgG/IgG4 positive and negative fractions and in the plasma of EN and LF patients, ProcartaPlex Human Antibody Isotyping Panels (eBioscience, Vienna, Austria) were used according to manufacturer’s instructions.

    Techniques: Purification, Incubation, Affinity Purification, Staining, Fluorescence, Microscopy, Blocking Assay

    Preferential expression of IgG4 in Mf+. Plasma samples from EN (n = 14) and LF infected Mf+ (n = 14), Mf-(n = 14) and CP (n = 14) patients were diluted and analyzed for the expression of IgG1 (A), IgG2 (B), IgG3 (C), IgG4 (D), IgE (E), IgA (F) and IgM (G) using Luminex-based immunoassay. Bars depict the plasmatic antibody expressions as mean ± SEM. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes

    doi: 10.1371/journal.pntd.0005777

    Figure Lengend Snippet: Preferential expression of IgG4 in Mf+. Plasma samples from EN (n = 14) and LF infected Mf+ (n = 14), Mf-(n = 14) and CP (n = 14) patients were diluted and analyzed for the expression of IgG1 (A), IgG2 (B), IgG3 (C), IgG4 (D), IgE (E), IgA (F) and IgM (G) using Luminex-based immunoassay. Bars depict the plasmatic antibody expressions as mean ± SEM. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p

    Article Snippet: Assessment of immunoglobulin isotypes expression in plasma and IgG/IgG4 positive and negative fractions To analyze the isotype composition in IgA, IgG/IgG4 positive and negative fractions and in the plasma of EN and LF patients, ProcartaPlex Human Antibody Isotyping Panels (eBioscience, Vienna, Austria) were used according to manufacturer’s instructions.

    Techniques: Expressing, Infection, Luminex

    IgA expression correlated with the suppressive capacity in the IgG-negative fractions of EN. Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone or in the presence of 5 μg/ml of IgG negative fractions of EN. Cells were stained with anti-CD63 and HLADR antibodies. Activated granulocytes were characterized as CD63+/HLADR- cells and the percentage of inhibition was calculated using the control without plasma as 100% of activation (0% of inhibition). The percentage of inhibition was then correlated with the expression of IgE, IgM and IgA determined by Luminex assay. Each dot represents the plot of the expression of IgE (A), IgM (B) or IgA (C) to the corresponding value of the inhibition. Thereafter, the percentage of activated granulocytes (CD63+/HLADR- cells) was determined after 18 hours of incubation (D) in the presence of IgE/IL-3 alone (dark bar) or in combination with either bulk plasma from EN (EN), IgA negative fractions (EN) IgA- or IgA positive fractions (EN) IgA+. Bars represent means ± SEM of the percentage of activated granulocyte CD63 + /HLADR - cells. Statistical comparison was based on Spearman’s rank correlation (A-C) or Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test (D). The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes

    doi: 10.1371/journal.pntd.0005777

    Figure Lengend Snippet: IgA expression correlated with the suppressive capacity in the IgG-negative fractions of EN. Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone or in the presence of 5 μg/ml of IgG negative fractions of EN. Cells were stained with anti-CD63 and HLADR antibodies. Activated granulocytes were characterized as CD63+/HLADR- cells and the percentage of inhibition was calculated using the control without plasma as 100% of activation (0% of inhibition). The percentage of inhibition was then correlated with the expression of IgE, IgM and IgA determined by Luminex assay. Each dot represents the plot of the expression of IgE (A), IgM (B) or IgA (C) to the corresponding value of the inhibition. Thereafter, the percentage of activated granulocytes (CD63+/HLADR- cells) was determined after 18 hours of incubation (D) in the presence of IgE/IL-3 alone (dark bar) or in combination with either bulk plasma from EN (EN), IgA negative fractions (EN) IgA- or IgA positive fractions (EN) IgA+. Bars represent means ± SEM of the percentage of activated granulocyte CD63 + /HLADR - cells. Statistical comparison was based on Spearman’s rank correlation (A-C) or Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test (D). The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p

    Article Snippet: Assessment of immunoglobulin isotypes expression in plasma and IgG/IgG4 positive and negative fractions To analyze the isotype composition in IgA, IgG/IgG4 positive and negative fractions and in the plasma of EN and LF patients, ProcartaPlex Human Antibody Isotyping Panels (eBioscience, Vienna, Austria) were used according to manufacturer’s instructions.

    Techniques: Expressing, Isolation, Staining, Inhibition, Activation Assay, Luminex, Incubation

    IgG4 antibodies from Mf+ patients present a higher affinity for granulocytes compared to IgG4 from EN and Mf-. Purified granulocytes from healthy blood donors were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml), and Brugia antigen extracts (10 μg/ml) and cultured in presence of 2.5 μg/ml of IgG4 molecules from EN (A), Mf+ (B), Mf- (C) and CP (D) for 18 hours. The cells were then stained with DAPI (blue) and IgG4 binding on activated cells was revealed with Alexa fluor 488 labelled antibody (green). Original magnification x100. The median green fluorescence intensities are illustrated in E. A representative experiment of 3 is shown. Thereafter, the relative affinity of IgG4 from EN, Mf+, Mf- and CP with Brugia antigen was analyzed by ELISA (F). Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-value refers to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test; *: p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes

    doi: 10.1371/journal.pntd.0005777

    Figure Lengend Snippet: IgG4 antibodies from Mf+ patients present a higher affinity for granulocytes compared to IgG4 from EN and Mf-. Purified granulocytes from healthy blood donors were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml), and Brugia antigen extracts (10 μg/ml) and cultured in presence of 2.5 μg/ml of IgG4 molecules from EN (A), Mf+ (B), Mf- (C) and CP (D) for 18 hours. The cells were then stained with DAPI (blue) and IgG4 binding on activated cells was revealed with Alexa fluor 488 labelled antibody (green). Original magnification x100. The median green fluorescence intensities are illustrated in E. A representative experiment of 3 is shown. Thereafter, the relative affinity of IgG4 from EN, Mf+, Mf- and CP with Brugia antigen was analyzed by ELISA (F). Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-value refers to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test; *: p

    Article Snippet: Assessment of immunoglobulin isotypes expression in plasma and IgG/IgG4 positive and negative fractions To analyze the isotype composition in IgA, IgG/IgG4 positive and negative fractions and in the plasma of EN and LF patients, ProcartaPlex Human Antibody Isotyping Panels (eBioscience, Vienna, Austria) were used according to manufacturer’s instructions.

    Techniques: Purification, Cell Culture, Staining, Binding Assay, Fluorescence, Enzyme-linked Immunosorbent Assay

    Depletion of IgG4 abrogates the suppressive capacity of IgG positive fractions from plasma of LF infected individuals. Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone (dark bars) or in presence of 2.5 μg/ml of IgG4 negative fractions obtained from IgG enriched fractions (n = 8) (light bars) from EN (A), Mf+ (B), Mf- (C), CP (D) or the corresponding IgG4 positive fractions (n = 8) (grey bars) and increasing concentration (1.25 μg/ml, 2.5 μg/ml and 5 μg/ml) of IgG4 positive fractions from different groups (E). Cells were stained with anti-CD63 and HLADR antibodies. Activated granulocytes were characterized as CD63+/HLADR- cells. The release of histamine (F), neutrophil elastase (G) and eosinophil cationic protein (H) in supernatants from cultures with IgG4 positive fractions was assessed after 18 hours. Bars represent means ± SEM. Graphs are representative of 3 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test;*: p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes

    doi: 10.1371/journal.pntd.0005777

    Figure Lengend Snippet: Depletion of IgG4 abrogates the suppressive capacity of IgG positive fractions from plasma of LF infected individuals. Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone (dark bars) or in presence of 2.5 μg/ml of IgG4 negative fractions obtained from IgG enriched fractions (n = 8) (light bars) from EN (A), Mf+ (B), Mf- (C), CP (D) or the corresponding IgG4 positive fractions (n = 8) (grey bars) and increasing concentration (1.25 μg/ml, 2.5 μg/ml and 5 μg/ml) of IgG4 positive fractions from different groups (E). Cells were stained with anti-CD63 and HLADR antibodies. Activated granulocytes were characterized as CD63+/HLADR- cells. The release of histamine (F), neutrophil elastase (G) and eosinophil cationic protein (H) in supernatants from cultures with IgG4 positive fractions was assessed after 18 hours. Bars represent means ± SEM. Graphs are representative of 3 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test;*: p

    Article Snippet: Assessment of immunoglobulin isotypes expression in plasma and IgG/IgG4 positive and negative fractions To analyze the isotype composition in IgA, IgG/IgG4 positive and negative fractions and in the plasma of EN and LF patients, ProcartaPlex Human Antibody Isotyping Panels (eBioscience, Vienna, Austria) were used according to manufacturer’s instructions.

    Techniques: Infection, Isolation, Concentration Assay, Staining

    Induction of tumor-protective IgE requires γδ TCR + IEL (a) FACS analysis of humoral immunity in the skin draining LNs of FVB wild-type and Tcrd -/- mice exposed topically to DMBA on the dorsal side of the ears twice, 3 days apart, and analyzed 4 or 7 days after last exposure (wild-type naïve (n=10), wild-type 4 days (n=8), wild-type 7 days (n=10), Tcrd -/- 4 days (n=5), Tcrd -/- 7 days (n=10)). Graphs show number of total LN cells, B220 + CD95 + GL7 + GC B cells and IgG1 + or IgE + FSC hi CD95 + CD138 + PCs. (b) ELISA of IgE in serum of wild-type and Tcrd -/- mice topically exposed to DMBA on shaved back skin and bled before and 4 days after exposure (n=10/group). (c-d) FACS analysis and enumeration of (c) LN and skin γδ T cells and (d) humoral immunity in the skin draining LNs of Tcrd -/- →wild-type and wild-type→ Tcrd -/- chimeric mice relative to chimeric control mice following exposure to DMBA and analysis at day 7 as in (a) (n=5/group). (e) FACS analysis of IgE + PCs in the skin draining LNs of FVB wild-type (n=6) and Vg5Vd1 -/- (n=8) mice exposed to DMBA and analysed as in (a). Statistics in (a-d) by one-way ANOVA multiple comparison and (e) two-tailed Student’s t-test for unpaired data; *p

    Journal: Nature immunology

    Article Title: Epithelial damage and tissue γδ T cells promote a unique tumor-protective IgE response

    doi: 10.1038/s41590-018-0161-8

    Figure Lengend Snippet: Induction of tumor-protective IgE requires γδ TCR + IEL (a) FACS analysis of humoral immunity in the skin draining LNs of FVB wild-type and Tcrd -/- mice exposed topically to DMBA on the dorsal side of the ears twice, 3 days apart, and analyzed 4 or 7 days after last exposure (wild-type naïve (n=10), wild-type 4 days (n=8), wild-type 7 days (n=10), Tcrd -/- 4 days (n=5), Tcrd -/- 7 days (n=10)). Graphs show number of total LN cells, B220 + CD95 + GL7 + GC B cells and IgG1 + or IgE + FSC hi CD95 + CD138 + PCs. (b) ELISA of IgE in serum of wild-type and Tcrd -/- mice topically exposed to DMBA on shaved back skin and bled before and 4 days after exposure (n=10/group). (c-d) FACS analysis and enumeration of (c) LN and skin γδ T cells and (d) humoral immunity in the skin draining LNs of Tcrd -/- →wild-type and wild-type→ Tcrd -/- chimeric mice relative to chimeric control mice following exposure to DMBA and analysis at day 7 as in (a) (n=5/group). (e) FACS analysis of IgE + PCs in the skin draining LNs of FVB wild-type (n=6) and Vg5Vd1 -/- (n=8) mice exposed to DMBA and analysed as in (a). Statistics in (a-d) by one-way ANOVA multiple comparison and (e) two-tailed Student’s t-test for unpaired data; *p

    Article Snippet: For IgG1 and IgG2a antibodies, NUNC Immune Maxisorp 96-well plates (Thermo Scientific) were coated with 5 μg/ml goat anti-mouse IgH+L (Southern Biotech) in borate buffered saline at 37°C for 3hr.

    Techniques: FACS, Mouse Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Carcinogen-induced humoral immunity depends on αβ T cell-derived IL-4 (a) FACS analysis of humoral immunity in the skin draining LNs of FVB wild-type (n=6), Il4 -/- (n=5) and Tcrb -/- (n=5) mice exposed topically to DMBA on the dorsal side of the ears twice, 3 days apart, and analysed 7 days after the last exposure relative naïve wild-type controls (n=6). Graphs show number of total LN cells, B220 + CD95 + GL7 + GC B cells and IgG1 + or IgE + FSC hi CD95 + CD138 + PCs presented as mean ± SEM. Representative flow plots show IgG1 + and IgE + PCs in wild-type, Il4 -/- and Tcrb -/- mice. (b) FACS analysis of humoral immunity in the skin draining LNs of Tcrb -/- mice reconstituted with wild-type CD4 + αβ T cells (n=3) or Il4 -/- CD4 + αβ T cells (n=4) 1 day prior to topical DMBA exposure as described in (a). Statistics in (a-b) by two-tailed Student’s t-test for unpaired data; *p

    Journal: Nature immunology

    Article Title: Epithelial damage and tissue γδ T cells promote a unique tumor-protective IgE response

    doi: 10.1038/s41590-018-0161-8

    Figure Lengend Snippet: Carcinogen-induced humoral immunity depends on αβ T cell-derived IL-4 (a) FACS analysis of humoral immunity in the skin draining LNs of FVB wild-type (n=6), Il4 -/- (n=5) and Tcrb -/- (n=5) mice exposed topically to DMBA on the dorsal side of the ears twice, 3 days apart, and analysed 7 days after the last exposure relative naïve wild-type controls (n=6). Graphs show number of total LN cells, B220 + CD95 + GL7 + GC B cells and IgG1 + or IgE + FSC hi CD95 + CD138 + PCs presented as mean ± SEM. Representative flow plots show IgG1 + and IgE + PCs in wild-type, Il4 -/- and Tcrb -/- mice. (b) FACS analysis of humoral immunity in the skin draining LNs of Tcrb -/- mice reconstituted with wild-type CD4 + αβ T cells (n=3) or Il4 -/- CD4 + αβ T cells (n=4) 1 day prior to topical DMBA exposure as described in (a). Statistics in (a-b) by two-tailed Student’s t-test for unpaired data; *p

    Article Snippet: For IgG1 and IgG2a antibodies, NUNC Immune Maxisorp 96-well plates (Thermo Scientific) were coated with 5 μg/ml goat anti-mouse IgH+L (Southern Biotech) in borate buffered saline at 37°C for 3hr.

    Techniques: Derivative Assay, FACS, Mouse Assay, Flow Cytometry, Two Tailed Test

    γδ T cells promote a distinct IgE repertoire in response to carcinogen (a-j) High-throughput sequencing and heavy-chain repertoire analysis of IgG1 and IgE in sorted B220 + CD95 + GL7 + GC B cells and FSC hi CD95 hi CD138 + PCs from skin draining LNs of wild-type and Tcrd -/- mice 7 days after the last of two topical exposures to DMBA (n=6/group). (a-b) Average clone size. (c) Average clonal diversity of IgG1 and IgE PC repertoires in wild-type and Tcrd -/- mice (‘General diversity index ( q D)’ displayed as a measure of ‘clonal frequencies (q)’; p

    Journal: Nature immunology

    Article Title: Epithelial damage and tissue γδ T cells promote a unique tumor-protective IgE response

    doi: 10.1038/s41590-018-0161-8

    Figure Lengend Snippet: γδ T cells promote a distinct IgE repertoire in response to carcinogen (a-j) High-throughput sequencing and heavy-chain repertoire analysis of IgG1 and IgE in sorted B220 + CD95 + GL7 + GC B cells and FSC hi CD95 hi CD138 + PCs from skin draining LNs of wild-type and Tcrd -/- mice 7 days after the last of two topical exposures to DMBA (n=6/group). (a-b) Average clone size. (c) Average clonal diversity of IgG1 and IgE PC repertoires in wild-type and Tcrd -/- mice (‘General diversity index ( q D)’ displayed as a measure of ‘clonal frequencies (q)’; p

    Article Snippet: For IgG1 and IgG2a antibodies, NUNC Immune Maxisorp 96-well plates (Thermo Scientific) were coated with 5 μg/ml goat anti-mouse IgH+L (Southern Biotech) in borate buffered saline at 37°C for 3hr.

    Techniques: Next-Generation Sequencing, Mouse Assay

    K10-dependent endothelial apoptosis. A . HUVEC were exposed for 24 hours to 10% dilutions of sera from healthy subjects (Ctrl) or RNP+ patients with or without Raynaud’s (RP+ or -) and anti-K10 IgG antibodies (ELISA > 2 S.D. above healthy subject mean to be anti-K10+), loaded with CellEvent Caspase 3/7 Green Detection Reagent (10μM/ml) and assayed for green absorbance in 24 hours in duplicate wells. Anti-K10+ sera induced high levels of caspase activity (an indicator of apoptosis) from patients with or without RP. ** t test p

    Journal: Arthritis & rheumatology (Hoboken, N.J.)

    Article Title: An Autoimmune Basis for Raynaud’s Phenomenon: Murine Model and Human Disease

    doi: 10.1002/art.40505

    Figure Lengend Snippet: K10-dependent endothelial apoptosis. A . HUVEC were exposed for 24 hours to 10% dilutions of sera from healthy subjects (Ctrl) or RNP+ patients with or without Raynaud’s (RP+ or -) and anti-K10 IgG antibodies (ELISA > 2 S.D. above healthy subject mean to be anti-K10+), loaded with CellEvent Caspase 3/7 Green Detection Reagent (10μM/ml) and assayed for green absorbance in 24 hours in duplicate wells. Anti-K10+ sera induced high levels of caspase activity (an indicator of apoptosis) from patients with or without RP. ** t test p

    Article Snippet: Anti-K10 monoclonal antibodies included Clone LH2, a murine IgG1 monoclonal raised against human K10 with reported reactivity against both human and mouse cytokeratin 10 (LifeSpan BioSciences, Seattle); Clone VIK-10, a murine monoclonal IgG1 with anti-K10 specificity raised against a human epidermal cytoskeletal extract (Thermo Fisher Scientific, Waltham MA); and clone AE20, a murine monoclonal IgG specific for human K10 (Genway Biotech, San Diego).

    Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay

    The anti-Aβ MABT mAb binds with high affinity to different Aβ peptides and has antiaggregation properties. A , An in vivo efficacy study administering mMABT to hAPP (V717I) mice showed a reduction in Aβ plaque load (left panel) and improved memory performance (right panel). Results show means (±95% CI). B , C , An Aβ ELISA was used to compare the binding to human and murine Aβ1–42 and human Aβ1–40, for mMABT ( B ) and MABT ( C ). D , MABT was also tested for binding to different Aβ1–42 assembly states, showing equal binding for oligomers, monomers, and fibers. E , MABT binds Aβ plaques present in brain sections from transgenic hAPP (V717I) mice (top panels) and human AD temporal neocortical sections (bottom panels). F , In vitro functionality was shown by the ability of MABT to impede Aβ1–42 aggregation (left panel), and to disassemble preformed Aβ1–42 aggregates (right panel) in a ThT-based assay, with an Aβ1–42 to MABT molar ratio of 10:1. An anti-Aβ IgG mAb with a N-terminal epitope was used as control. Results show the mean (±SD) of three independent experiments. * p

    Journal: The Journal of Neuroscience

    Article Title: An Effector-Reduced Anti-β-Amyloid (Aβ) Antibody with Unique Aβ Binding Properties Promotes Neuroprotection and Glial Engulfment of Aβ

    doi: 10.1523/JNEUROSCI.4742-11.2012

    Figure Lengend Snippet: The anti-Aβ MABT mAb binds with high affinity to different Aβ peptides and has antiaggregation properties. A , An in vivo efficacy study administering mMABT to hAPP (V717I) mice showed a reduction in Aβ plaque load (left panel) and improved memory performance (right panel). Results show means (±95% CI). B , C , An Aβ ELISA was used to compare the binding to human and murine Aβ1–42 and human Aβ1–40, for mMABT ( B ) and MABT ( C ). D , MABT was also tested for binding to different Aβ1–42 assembly states, showing equal binding for oligomers, monomers, and fibers. E , MABT binds Aβ plaques present in brain sections from transgenic hAPP (V717I) mice (top panels) and human AD temporal neocortical sections (bottom panels). F , In vitro functionality was shown by the ability of MABT to impede Aβ1–42 aggregation (left panel), and to disassemble preformed Aβ1–42 aggregates (right panel) in a ThT-based assay, with an Aβ1–42 to MABT molar ratio of 10:1. An anti-Aβ IgG mAb with a N-terminal epitope was used as control. Results show the mean (±SD) of three independent experiments. * p

    Article Snippet: Serial dilutions of anti-Aβ MABT, MABT-IgG1, MABT-IgG1-D265A, or IgG1 control mAbs were prepared in the assay buffer containing 10% Blocker Casein in PBS (Pierce).

    Techniques: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Transgenic Assay, In Vitro, ThT Assay

    Aβ1–42 oligomers are taken up via an FcR-mediated mechanism into microglia upon MABT treatment. A , Confocal imaging was used to show that Aβ1–42 oligomers complexed to MABT are taken up into microglia. Aβ1–42 oligomers are shown in red, and blue is DAPI. An apical-to-distal slice was obtained from a three-dimensional image rendered from Z -stacks. Confocal imaging was done on mixed cortical cells labeled for Aβ (red) and DAPI (blue) as described in Materials and Methods. B , C , Microglia were identified and scanned for the maximum fluorescence intensity through a series of confocal stacks representing intracellular locations ( B ), and the total area of fluorescent signal above a minimum threshold was quantified ( C ). Each mark on the graph represents the total area of Aβ staining within a single cell. A minimum of 20 cells was analyzed for each treatment condition. Data were compared using one-way ANOVA followed by Tukey's post hoc multiple comparison. Means (±SEM) are shown. D , Microglia (Iba1 + ) was verified as the cell type taking up Aβ1–42 complexed to MABT. Red is Iba1, green is HyLite Fluor 488-labeled Aβ1–42, and blue is DAPI. E , Differential binding to FcγRIIIa-V158 was verified in a binding assay. F , An anti-Aβ mAb requires effector function via FcγR binding for full protective effect against Aβ1–42 oligomer toxicity. Mixed cortical cells from P1 rats were treated with Aβ1–42 oligomers, with or without MABT, MABT-IgG1-D265A, MABT-IgG1, or an IgG1 control mAb. The graph shows the mean (±SEM) percentage increase in cell survival compared with Aβ1–42 oligomer treated cells, from five independent experiments. Statistical analysis was done using one-way ANOVA followed by Tukey's post hoc multiple comparison.

    Journal: The Journal of Neuroscience

    Article Title: An Effector-Reduced Anti-β-Amyloid (Aβ) Antibody with Unique Aβ Binding Properties Promotes Neuroprotection and Glial Engulfment of Aβ

    doi: 10.1523/JNEUROSCI.4742-11.2012

    Figure Lengend Snippet: Aβ1–42 oligomers are taken up via an FcR-mediated mechanism into microglia upon MABT treatment. A , Confocal imaging was used to show that Aβ1–42 oligomers complexed to MABT are taken up into microglia. Aβ1–42 oligomers are shown in red, and blue is DAPI. An apical-to-distal slice was obtained from a three-dimensional image rendered from Z -stacks. Confocal imaging was done on mixed cortical cells labeled for Aβ (red) and DAPI (blue) as described in Materials and Methods. B , C , Microglia were identified and scanned for the maximum fluorescence intensity through a series of confocal stacks representing intracellular locations ( B ), and the total area of fluorescent signal above a minimum threshold was quantified ( C ). Each mark on the graph represents the total area of Aβ staining within a single cell. A minimum of 20 cells was analyzed for each treatment condition. Data were compared using one-way ANOVA followed by Tukey's post hoc multiple comparison. Means (±SEM) are shown. D , Microglia (Iba1 + ) was verified as the cell type taking up Aβ1–42 complexed to MABT. Red is Iba1, green is HyLite Fluor 488-labeled Aβ1–42, and blue is DAPI. E , Differential binding to FcγRIIIa-V158 was verified in a binding assay. F , An anti-Aβ mAb requires effector function via FcγR binding for full protective effect against Aβ1–42 oligomer toxicity. Mixed cortical cells from P1 rats were treated with Aβ1–42 oligomers, with or without MABT, MABT-IgG1-D265A, MABT-IgG1, or an IgG1 control mAb. The graph shows the mean (±SEM) percentage increase in cell survival compared with Aβ1–42 oligomer treated cells, from five independent experiments. Statistical analysis was done using one-way ANOVA followed by Tukey's post hoc multiple comparison.

    Article Snippet: Serial dilutions of anti-Aβ MABT, MABT-IgG1, MABT-IgG1-D265A, or IgG1 control mAbs were prepared in the assay buffer containing 10% Blocker Casein in PBS (Pierce).

    Techniques: Imaging, Labeling, Fluorescence, Staining, Binding Assay

    The number of anti-CarP antibody isotypes and IgG subclasses differs between RA patients and is level dependent. Anti-carbamylated protein ( anti-CarP ) antibody isotypes and immunoglobulin G ( IgG ) subclasses were measured by ELISA in 373 rheumatoid arthritis ( RA ) patients and 196 healthy controls ( HC ). Pie charts show the percentage of RA patients and HC negative or positive for one, two, or three anti-CarP antibody isotypes ( a ) and negative or positive for one, two, three, or four anti-CarP IgG subclasses ( b ). An increase in level of anti-CarP antibody IgG associates with an increase in the number of anti-CarP antibody isotypes ( c ) and IgG subclasses ( d ) in RA patients. Red lines depict means ( e ). Heat maps show the presence of anti-CarP antibody isotypes and IgG subclasses in RA and HC, ranked according to anti-CarP antibody IgG levels. Green and red mark positive and negative sera, respectively. AU arbitrary units

    Journal: Arthritis Research & Therapy

    Article Title: The isotype and IgG subclass distribution of anti-carbamylated protein antibodies in rheumatoid arthritis patients

    doi: 10.1186/s13075-017-1392-z

    Figure Lengend Snippet: The number of anti-CarP antibody isotypes and IgG subclasses differs between RA patients and is level dependent. Anti-carbamylated protein ( anti-CarP ) antibody isotypes and immunoglobulin G ( IgG ) subclasses were measured by ELISA in 373 rheumatoid arthritis ( RA ) patients and 196 healthy controls ( HC ). Pie charts show the percentage of RA patients and HC negative or positive for one, two, or three anti-CarP antibody isotypes ( a ) and negative or positive for one, two, three, or four anti-CarP IgG subclasses ( b ). An increase in level of anti-CarP antibody IgG associates with an increase in the number of anti-CarP antibody isotypes ( c ) and IgG subclasses ( d ) in RA patients. Red lines depict means ( e ). Heat maps show the presence of anti-CarP antibody isotypes and IgG subclasses in RA and HC, ranked according to anti-CarP antibody IgG levels. Green and red mark positive and negative sera, respectively. AU arbitrary units

    Article Snippet: Bound human IgG2 or IgG3 was detected using MAH-IgG2 (Nordic MUbio, Clone HP6014) or MAH-IgG3 (Nordic MUbio, Clone HP6080) continued with HRP-conjugated GAM-Ig (DAKO, P0447) and rabbit-anti-goat (RAG)-Ig (DAKO, P0449).

    Techniques: Enzyme-linked Immunosorbent Assay

    Anti-CarP IgG1 is associated with more severe radiological progression. The extent and rate of joint destruction was analysed in all rheumatoid arthritis ( RA ) patients or separately for anti-citrullinated protein antibody ( ACPA )-negative and ACPA-positive RA and within the ACPA-negative RA patients also separately for rheumatoid factor (RF) negative and positive. The severity of joint damage is depicted as median Sharp-van der Heijde score ( SHS ) on the y axis and the follow-up years on the x axis for anti-carbamylated protein ( anti-CarP ) antibody immunoglobulin ( Ig )G1-positive and -negative patients in all RA patients analysed ( a ) and for ACPA- and RF-negative RA patients ( b ). β and p values are derived from the analysis model as described in the Methods and Results sections

    Journal: Arthritis Research & Therapy

    Article Title: The isotype and IgG subclass distribution of anti-carbamylated protein antibodies in rheumatoid arthritis patients

    doi: 10.1186/s13075-017-1392-z

    Figure Lengend Snippet: Anti-CarP IgG1 is associated with more severe radiological progression. The extent and rate of joint destruction was analysed in all rheumatoid arthritis ( RA ) patients or separately for anti-citrullinated protein antibody ( ACPA )-negative and ACPA-positive RA and within the ACPA-negative RA patients also separately for rheumatoid factor (RF) negative and positive. The severity of joint damage is depicted as median Sharp-van der Heijde score ( SHS ) on the y axis and the follow-up years on the x axis for anti-carbamylated protein ( anti-CarP ) antibody immunoglobulin ( Ig )G1-positive and -negative patients in all RA patients analysed ( a ) and for ACPA- and RF-negative RA patients ( b ). β and p values are derived from the analysis model as described in the Methods and Results sections

    Article Snippet: Bound human IgG2 or IgG3 was detected using MAH-IgG2 (Nordic MUbio, Clone HP6014) or MAH-IgG3 (Nordic MUbio, Clone HP6080) continued with HRP-conjugated GAM-Ig (DAKO, P0447) and rabbit-anti-goat (RAG)-Ig (DAKO, P0449).

    Techniques: Derivative Assay

    Distribution of anti-CarP and ACPA isotypes and IgG subclasses. a Percentage positivity of anti-citrullinated protein antibody ( ACPA ; black bars ) or anti-carbamylated protein ( anti-CarP ) antibody ( grey bars ) immunoglobulin ( Ig )M, IgA, and IgG subclasses in ACPA and anti-CarP antibody IgG double-positive RA patients ( n = 114). b Percentage single- or double-positive for ACPA ( grey ) and anti-CarP antibody ( light grey ) isotypes and IgG subclasses in IgG double-positive RA patients. Circles are not to scale. Number of anti-CarP antibody and ACPA isotypes ( c ) and IgG subclasses ( d ) in anti-CarP antibody and ACPA IgG double-positive RA patients and at least positive for one IgG subclass ( n = 90)

    Journal: Arthritis Research & Therapy

    Article Title: The isotype and IgG subclass distribution of anti-carbamylated protein antibodies in rheumatoid arthritis patients

    doi: 10.1186/s13075-017-1392-z

    Figure Lengend Snippet: Distribution of anti-CarP and ACPA isotypes and IgG subclasses. a Percentage positivity of anti-citrullinated protein antibody ( ACPA ; black bars ) or anti-carbamylated protein ( anti-CarP ) antibody ( grey bars ) immunoglobulin ( Ig )M, IgA, and IgG subclasses in ACPA and anti-CarP antibody IgG double-positive RA patients ( n = 114). b Percentage single- or double-positive for ACPA ( grey ) and anti-CarP antibody ( light grey ) isotypes and IgG subclasses in IgG double-positive RA patients. Circles are not to scale. Number of anti-CarP antibody and ACPA isotypes ( c ) and IgG subclasses ( d ) in anti-CarP antibody and ACPA IgG double-positive RA patients and at least positive for one IgG subclass ( n = 90)

    Article Snippet: Bound human IgG2 or IgG3 was detected using MAH-IgG2 (Nordic MUbio, Clone HP6014) or MAH-IgG3 (Nordic MUbio, Clone HP6080) continued with HRP-conjugated GAM-Ig (DAKO, P0447) and rabbit-anti-goat (RAG)-Ig (DAKO, P0449).

    Techniques:

    Anti-CarP antibody isotypes and IgG subclasses are present in RA sera. ELISAs were performed to detect anti-carbamylated protein ( anti-CarP ) antibody isotypes ( a ) and immunoglobulin G ( IgG ) subclasses ( b ) in sera of 196 healthy controls ( HC ) and 373 rheumatoid arthritis ( RA ) patients. The mean ( red line ) plus two times the standard deviation in HC was established as the cut-off for the anti-CarP antibody isotypes. The 97 th percentile in HC was used as the cut-off for the IgG subclasses. The dotted line represents the cut-off. The specific anti-CarP reactivity is depicted in arbitrary units ( AU ) per millilitre. The number of samples tested and the percentage positivity is shown below the graphs. c Percentage positivity of anti-CarP antibody isotypes and IgG subclasses in all RA patients ( grey bars , n = 373), anti-citrullinated protein antibody ( ACPA ) IgG-positive RA patients ( dark grey bars , n = 217), ACPA IgG-negative RA patients ( black bars , n = 156), and HC ( light grey bars , n = 196)

    Journal: Arthritis Research & Therapy

    Article Title: The isotype and IgG subclass distribution of anti-carbamylated protein antibodies in rheumatoid arthritis patients

    doi: 10.1186/s13075-017-1392-z

    Figure Lengend Snippet: Anti-CarP antibody isotypes and IgG subclasses are present in RA sera. ELISAs were performed to detect anti-carbamylated protein ( anti-CarP ) antibody isotypes ( a ) and immunoglobulin G ( IgG ) subclasses ( b ) in sera of 196 healthy controls ( HC ) and 373 rheumatoid arthritis ( RA ) patients. The mean ( red line ) plus two times the standard deviation in HC was established as the cut-off for the anti-CarP antibody isotypes. The 97 th percentile in HC was used as the cut-off for the IgG subclasses. The dotted line represents the cut-off. The specific anti-CarP reactivity is depicted in arbitrary units ( AU ) per millilitre. The number of samples tested and the percentage positivity is shown below the graphs. c Percentage positivity of anti-CarP antibody isotypes and IgG subclasses in all RA patients ( grey bars , n = 373), anti-citrullinated protein antibody ( ACPA ) IgG-positive RA patients ( dark grey bars , n = 217), ACPA IgG-negative RA patients ( black bars , n = 156), and HC ( light grey bars , n = 196)

    Article Snippet: Bound human IgG2 or IgG3 was detected using MAH-IgG2 (Nordic MUbio, Clone HP6014) or MAH-IgG3 (Nordic MUbio, Clone HP6080) continued with HRP-conjugated GAM-Ig (DAKO, P0447) and rabbit-anti-goat (RAG)-Ig (DAKO, P0449).

    Techniques: Standard Deviation

    Serological response to AAV-2 in Irish blood donors. (a) AAV-2-specific total IgG in human donor plasma ( n =45). Specific IgG was determined by indirect AAV-2 antigen ELISA, normalized by comparison with standard positive and negative samples. The negative cut-off (dashed line) was defined at 2 sd above the concentration of a known seronegative standard. (b–e) AAV-2-specific IgG subclasses present in IgG-positive samples ( n =41). IgG1(b), IgG2 (c), IgG3 (d) and IgG4 (e) determined by AAV-2-specific direct antigen ELISA for triplicate samples and quantified by comparison with known reference sera.

    Journal: The Journal of General Virology

    Article Title: Adeno-associated virus serotype 2 induces cell-mediated immune responses directed against multiple epitopes of the capsid protein VP1

    doi: 10.1099/vir.0.014175-0

    Figure Lengend Snippet: Serological response to AAV-2 in Irish blood donors. (a) AAV-2-specific total IgG in human donor plasma ( n =45). Specific IgG was determined by indirect AAV-2 antigen ELISA, normalized by comparison with standard positive and negative samples. The negative cut-off (dashed line) was defined at 2 sd above the concentration of a known seronegative standard. (b–e) AAV-2-specific IgG subclasses present in IgG-positive samples ( n =41). IgG1(b), IgG2 (c), IgG3 (d) and IgG4 (e) determined by AAV-2-specific direct antigen ELISA for triplicate samples and quantified by comparison with known reference sera.

    Article Snippet: Plates were probed for human IgG using anti-human IgG subclass-biotin antibodies for subclasses IgG1–IgG4 (Merck).

    Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

    Antibodies that target the NANP epitope of CSP can promote C1q-fixation. Antibodies from malaria-exposed adults living in PNG ( n = 30) ( a ) and Kenya ( n = 30) ( b ) were tested for IgG, IgM, and C1q-fixation to (NANP) 15 peptide by ELISA, and correlated with C1q fixation to CSP (Spearman’s correlation coefficient, rho). Results were standardized to arbitrary units (AU) based on malaria-naïve negative controls from Melbourne (seropositivity defined as AU > 1, shown as dotted lines) and mean of duplicates were graphed. c Mouse anti-CSP MAbs 2H8-IgG1/IgG2a and 3C1-IgG1/IgG3 were tested for CSP-IgG, NANP-IgG, and C1q-fixation to CSP by ELISA. Results were corrected for background reactivity using no-IgG controls, and the mean and range of duplicates were graphed. AU arbitrary units, CSP circumsporozoite protein, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, IgM immunoglobulin M, MAb monoclonal antibody, OD optical density, PNG Papua New Guinea

    Journal: BMC Medicine

    Article Title: Human antibodies activate complement against Plasmodium falciparum sporozoites, and are associated with protection against malaria in children

    doi: 10.1186/s12916-018-1054-2

    Figure Lengend Snippet: Antibodies that target the NANP epitope of CSP can promote C1q-fixation. Antibodies from malaria-exposed adults living in PNG ( n = 30) ( a ) and Kenya ( n = 30) ( b ) were tested for IgG, IgM, and C1q-fixation to (NANP) 15 peptide by ELISA, and correlated with C1q fixation to CSP (Spearman’s correlation coefficient, rho). Results were standardized to arbitrary units (AU) based on malaria-naïve negative controls from Melbourne (seropositivity defined as AU > 1, shown as dotted lines) and mean of duplicates were graphed. c Mouse anti-CSP MAbs 2H8-IgG1/IgG2a and 3C1-IgG1/IgG3 were tested for CSP-IgG, NANP-IgG, and C1q-fixation to CSP by ELISA. Results were corrected for background reactivity using no-IgG controls, and the mean and range of duplicates were graphed. AU arbitrary units, CSP circumsporozoite protein, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, IgM immunoglobulin M, MAb monoclonal antibody, OD optical density, PNG Papua New Guinea

    Article Snippet: We were provided with subclass switched mouse anti-NANP monoclonal antibodies (MAbs) 2H8-IgG1/IgG2a and 3C1-IgG1/IgG3 (LakePharma, Belmont, USA) by PATH’s MVI [ ].

    Techniques: Peptide ELISA, Enzyme-linked Immunosorbent Assay

    Acquisition of C1q-fixing antibodies and total IgG to CSP and MSP2 antigens in malaria-exposed Kenyan children and adults. Kenyan children and adults ( N = 75) were categorized into groups based on the median ages 0.5, 1, 2, 5, and 35 years ( n = 11, n = 14, n = 18, n = 16, and n = 16, respectively). Samples were tested for C1q-fixation and total IgG to CSP ( a ) and MSP2 ( b ) by ELISA. Results were standardized to arbitrary units (AU) based on malaria-naive negative controls from Melbourne (seropositivity defined as AU > 1, shown as dotted lines), and the mean of duplicates were graphed along with the group median, interquartile range, and percentage of positive samples. Reactivity between two groups and more than two groups were compared using the Mann–Whitney U test and Kruskal–Wallis test, respectively. AU arbitrary units, CSP circumsporozoite protein, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, MSP2 Merozoite surface protein 2

    Journal: BMC Medicine

    Article Title: Human antibodies activate complement against Plasmodium falciparum sporozoites, and are associated with protection against malaria in children

    doi: 10.1186/s12916-018-1054-2

    Figure Lengend Snippet: Acquisition of C1q-fixing antibodies and total IgG to CSP and MSP2 antigens in malaria-exposed Kenyan children and adults. Kenyan children and adults ( N = 75) were categorized into groups based on the median ages 0.5, 1, 2, 5, and 35 years ( n = 11, n = 14, n = 18, n = 16, and n = 16, respectively). Samples were tested for C1q-fixation and total IgG to CSP ( a ) and MSP2 ( b ) by ELISA. Results were standardized to arbitrary units (AU) based on malaria-naive negative controls from Melbourne (seropositivity defined as AU > 1, shown as dotted lines), and the mean of duplicates were graphed along with the group median, interquartile range, and percentage of positive samples. Reactivity between two groups and more than two groups were compared using the Mann–Whitney U test and Kruskal–Wallis test, respectively. AU arbitrary units, CSP circumsporozoite protein, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, MSP2 Merozoite surface protein 2

    Article Snippet: We were provided with subclass switched mouse anti-NANP monoclonal antibodies (MAbs) 2H8-IgG1/IgG2a and 3C1-IgG1/IgG3 (LakePharma, Belmont, USA) by PATH’s MVI [ ].

    Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Naturally acquired human anti-CSP antibodies are predominately IgG1, IgG3, and IgM, and can promote complement fixation to CSP. Antibodies from malaria-exposed adults ( n = 30 in each group) living in PNG ( a , c-e ) and Kenyan ( b ) were tested for IgG/IgM and complement-fixation to CSP by ELISA. Results were standardized to arbitrary units (AU) based on malaria-naïve negative controls from Melbourne (seropositivity defined as AU > 1, shown as dotted lines), and mean and range of duplicates were graphed (mean only for scatter plots). a,b IgG subclasses and IgM reactivity to CSP. The median, interquartile range, and percentage of positive samples are shown. c Correlations between C1q-fixation and C4/C3/C3/C5b-C9-fixation to CSP (Spearman’s correlation coefficient, rho). d Examples of C1q and C3-fixation to CSP by individual serum samples (V15, V32, V33, V45, and V46 from PNG donors, n = 5). e C3-fixation to CSP by individual samples (V6, V7, V32, V37, and V43 from PNG donors, n = 5) in the presence of normal human serum (NHS) and serum depleted of C1q (C1q dep.) (Wilcoxon matched-pairs signed rank test). Non-standardized data are shown, as only one Melbourne control (Melb.) was tested. AU arbitrary units, CSP circumsporozoite protein, dep. depleted, ELISA enzyme-linked immunosorbent assay, Melb. Melbourne, NHS normal human serum, PNG Papua New Guinea

    Journal: BMC Medicine

    Article Title: Human antibodies activate complement against Plasmodium falciparum sporozoites, and are associated with protection against malaria in children

    doi: 10.1186/s12916-018-1054-2

    Figure Lengend Snippet: Naturally acquired human anti-CSP antibodies are predominately IgG1, IgG3, and IgM, and can promote complement fixation to CSP. Antibodies from malaria-exposed adults ( n = 30 in each group) living in PNG ( a , c-e ) and Kenyan ( b ) were tested for IgG/IgM and complement-fixation to CSP by ELISA. Results were standardized to arbitrary units (AU) based on malaria-naïve negative controls from Melbourne (seropositivity defined as AU > 1, shown as dotted lines), and mean and range of duplicates were graphed (mean only for scatter plots). a,b IgG subclasses and IgM reactivity to CSP. The median, interquartile range, and percentage of positive samples are shown. c Correlations between C1q-fixation and C4/C3/C3/C5b-C9-fixation to CSP (Spearman’s correlation coefficient, rho). d Examples of C1q and C3-fixation to CSP by individual serum samples (V15, V32, V33, V45, and V46 from PNG donors, n = 5). e C3-fixation to CSP by individual samples (V6, V7, V32, V37, and V43 from PNG donors, n = 5) in the presence of normal human serum (NHS) and serum depleted of C1q (C1q dep.) (Wilcoxon matched-pairs signed rank test). Non-standardized data are shown, as only one Melbourne control (Melb.) was tested. AU arbitrary units, CSP circumsporozoite protein, dep. depleted, ELISA enzyme-linked immunosorbent assay, Melb. Melbourne, NHS normal human serum, PNG Papua New Guinea

    Article Snippet: We were provided with subclass switched mouse anti-NANP monoclonal antibodies (MAbs) 2H8-IgG1/IgG2a and 3C1-IgG1/IgG3 (LakePharma, Belmont, USA) by PATH’s MVI [ ].

    Techniques: Enzyme-linked Immunosorbent Assay

    Vaccine-induced rabbit anti-CSP IgG can fix human complement proteins to CSP. Purified IgG from rabbit serum before (pre-imm.) and after (a-CSP) CSP immunization was tested for IgG and complement-fixation to CSP by ELISA. Results were corrected for background reactivity using no-IgG controls, and the mean and range of the duplicates were graphed. a IgG reactivity to CSP and (NANP) 15 peptide (pre-immune IgG shown with the open symbol was tested at 10 μg/ml). Results from two independent experiments are shown. b C1q and C3-fixation to CSP tested in the presence (+) and absence (−) of complement to confirm specificity for complement fixation. a-CSP after CSP immunization, CSP circumsporozoite protein, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, pre-imm. before CSP immunization

    Journal: BMC Medicine

    Article Title: Human antibodies activate complement against Plasmodium falciparum sporozoites, and are associated with protection against malaria in children

    doi: 10.1186/s12916-018-1054-2

    Figure Lengend Snippet: Vaccine-induced rabbit anti-CSP IgG can fix human complement proteins to CSP. Purified IgG from rabbit serum before (pre-imm.) and after (a-CSP) CSP immunization was tested for IgG and complement-fixation to CSP by ELISA. Results were corrected for background reactivity using no-IgG controls, and the mean and range of the duplicates were graphed. a IgG reactivity to CSP and (NANP) 15 peptide (pre-immune IgG shown with the open symbol was tested at 10 μg/ml). Results from two independent experiments are shown. b C1q and C3-fixation to CSP tested in the presence (+) and absence (−) of complement to confirm specificity for complement fixation. a-CSP after CSP immunization, CSP circumsporozoite protein, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, pre-imm. before CSP immunization

    Article Snippet: We were provided with subclass switched mouse anti-NANP monoclonal antibodies (MAbs) 2H8-IgG1/IgG2a and 3C1-IgG1/IgG3 (LakePharma, Belmont, USA) by PATH’s MVI [ ].

    Techniques: Purification, Enzyme-linked Immunosorbent Assay

    Antibodies promote complement-fixation on P. falciparum sporozoites, which enhances antibody-mediated traversal inhibition and can lead to sporozoite death. a Antibody samples from malaria-exposed (PNG and Kenyan) and malaria-naïve (Melbourne) individuals were tested for the ability to fix human C1q and C3 to 3D7 P. falciparum sporozoites by ELISA and Western blot. ELISA samples (top panel) were tested in duplicate, and the mean and range were graphed (C1q-fixation data is from two independent experiments). Western blot sporozoites (bottom panel) were incubated with human antibody samples and normal human serum (NHS, active complement), and then washed and processed for Western blotting under reduced conditions. Any complement proteins that had deposited on the sporozoite surface were detected using C1q- and C3-specific antibodies, and the sporozoite surface antigen, CSP, was used as a loading control. b,c In vitro traversal inhibition of freshly dissected sporozoites incubated with HC-04 cells, in the presence of NHS and heat-inactivated human serum (HIS). Each condition was tested in duplicate, and the mean and range were graphed. b Traversal inhibition of NF54 sporozoites treated with rabbit anti-CSP IgG (compared to rabbit pre-immune IgG). c Traversal inhibition of NF54 and NF166.C8 sporozoites treated with malaria-exposed Kenyan pool (compared to malaria-naïve Melbourne pool). Results from two independent experiments are shown. d Percentage of dead 3D7 sporozoites (PI+ cells) treated with rabbit anti-CSP IgG and pre-immune IgG, in the presence of NHS or C5-depleted serum (C5dep.). The mean and range of two independent experiments are graphed. a-CSP after CSP immunization, C5dep. C5-depleted serum, CSP circumsporozoite protein, ELISA enzyme-linked immunosorbent assay, HIS heat-inactivated human serum, IgG immunoglobulin G, Melb. Melbourne, NHS normal human serum, OD optical density, PI propidium iodide, PNG Papua New Guinea, pre-imm. before CSP immunization

    Journal: BMC Medicine

    Article Title: Human antibodies activate complement against Plasmodium falciparum sporozoites, and are associated with protection against malaria in children

    doi: 10.1186/s12916-018-1054-2

    Figure Lengend Snippet: Antibodies promote complement-fixation on P. falciparum sporozoites, which enhances antibody-mediated traversal inhibition and can lead to sporozoite death. a Antibody samples from malaria-exposed (PNG and Kenyan) and malaria-naïve (Melbourne) individuals were tested for the ability to fix human C1q and C3 to 3D7 P. falciparum sporozoites by ELISA and Western blot. ELISA samples (top panel) were tested in duplicate, and the mean and range were graphed (C1q-fixation data is from two independent experiments). Western blot sporozoites (bottom panel) were incubated with human antibody samples and normal human serum (NHS, active complement), and then washed and processed for Western blotting under reduced conditions. Any complement proteins that had deposited on the sporozoite surface were detected using C1q- and C3-specific antibodies, and the sporozoite surface antigen, CSP, was used as a loading control. b,c In vitro traversal inhibition of freshly dissected sporozoites incubated with HC-04 cells, in the presence of NHS and heat-inactivated human serum (HIS). Each condition was tested in duplicate, and the mean and range were graphed. b Traversal inhibition of NF54 sporozoites treated with rabbit anti-CSP IgG (compared to rabbit pre-immune IgG). c Traversal inhibition of NF54 and NF166.C8 sporozoites treated with malaria-exposed Kenyan pool (compared to malaria-naïve Melbourne pool). Results from two independent experiments are shown. d Percentage of dead 3D7 sporozoites (PI+ cells) treated with rabbit anti-CSP IgG and pre-immune IgG, in the presence of NHS or C5-depleted serum (C5dep.). The mean and range of two independent experiments are graphed. a-CSP after CSP immunization, C5dep. C5-depleted serum, CSP circumsporozoite protein, ELISA enzyme-linked immunosorbent assay, HIS heat-inactivated human serum, IgG immunoglobulin G, Melb. Melbourne, NHS normal human serum, OD optical density, PI propidium iodide, PNG Papua New Guinea, pre-imm. before CSP immunization

    Article Snippet: We were provided with subclass switched mouse anti-NANP monoclonal antibodies (MAbs) 2H8-IgG1/IgG2a and 3C1-IgG1/IgG3 (LakePharma, Belmont, USA) by PATH’s MVI [ ].

    Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, In Vitro

    Examples of serum-dependent cross-reactivity of IgG subclass detection on control beads. The control beads were incubated with different patient sera, washed, and then detected with subclass-specific secondary antibodies ((a): patient serum 1; (b): patient serum 2; (c): patient serum 3).

    Journal: Journal of Immunology Research

    Article Title: Renal Transplant Patients Biopsied for Cause and Tested for C4d, DSA, and IgG Subclasses and C1q: Which Humoral Markers Improve Diagnosis and Outcomes?

    doi: 10.1155/2017/1652931

    Figure Lengend Snippet: Examples of serum-dependent cross-reactivity of IgG subclass detection on control beads. The control beads were incubated with different patient sera, washed, and then detected with subclass-specific secondary antibodies ((a): patient serum 1; (b): patient serum 2; (c): patient serum 3).

    Article Snippet: Positive control beads were produced by Acuimmune (Chatsworth, CA) using IgG1–IgG4 and IgM purified from myeloma plasma (Sigma-Aldrich, Saint Louis, MO) and coupled to Microplex Microspheres (Luminex, Austin, TX).

    Techniques: Incubation

    PE-conjugated secondary antibodies for IgG subclass detection. The control beads were incubated with negative control serum, washed, and then detected with subclass-specific antibodies ((a): IgG1-PE; (b): IgG2-PE; (c): IgG3-PE; (d): IgG4-PE).

    Journal: Journal of Immunology Research

    Article Title: Renal Transplant Patients Biopsied for Cause and Tested for C4d, DSA, and IgG Subclasses and C1q: Which Humoral Markers Improve Diagnosis and Outcomes?

    doi: 10.1155/2017/1652931

    Figure Lengend Snippet: PE-conjugated secondary antibodies for IgG subclass detection. The control beads were incubated with negative control serum, washed, and then detected with subclass-specific antibodies ((a): IgG1-PE; (b): IgG2-PE; (c): IgG3-PE; (d): IgG4-PE).

    Article Snippet: Positive control beads were produced by Acuimmune (Chatsworth, CA) using IgG1–IgG4 and IgM purified from myeloma plasma (Sigma-Aldrich, Saint Louis, MO) and coupled to Microplex Microspheres (Luminex, Austin, TX).

    Techniques: Incubation, Negative Control

    Evaluation of endpoint titers of anti-PfCelTOS total IgG, IgG2a, and IgG2b antibodies. For titration, 1:200 to 1:409,600 dilutions of mouse sera from different vaccine groups (groups 1 to 5) were analyzed using an ELISA. The bars show the last dilution

    Journal: Infection and Immunity

    Article Title: Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) of Plasmodium falciparum in BALB/c Mice

    doi: 10.1128/IAI.00911-18

    Figure Lengend Snippet: Evaluation of endpoint titers of anti-PfCelTOS total IgG, IgG2a, and IgG2b antibodies. For titration, 1:200 to 1:409,600 dilutions of mouse sera from different vaccine groups (groups 1 to 5) were analyzed using an ELISA. The bars show the last dilution

    Article Snippet: In order to evaluate the IgG subclass responses to PfCelTOS, the test was performed as described above, and instead of goat anti-mouse IgG-HRP, 100 μl of either goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (diluted 1:1,000 in PBS-T buffer) (Sigma-Aldrich) as a secondary antibody was added to each well and incubated at RT for 1 h.

    Techniques: Titration, Enzyme-linked Immunosorbent Assay

    Anti-PfCelTOS IgG antibody responses and persistence of IgG responses.

    Journal: Infection and Immunity

    Article Title: Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) of Plasmodium falciparum in BALB/c Mice

    doi: 10.1128/IAI.00911-18

    Figure Lengend Snippet: Anti-PfCelTOS IgG antibody responses and persistence of IgG responses.

    Article Snippet: In order to evaluate the IgG subclass responses to PfCelTOS, the test was performed as described above, and instead of goat anti-mouse IgG-HRP, 100 μl of either goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (diluted 1:1,000 in PBS-T buffer) (Sigma-Aldrich) as a secondary antibody was added to each well and incubated at RT for 1 h.

    Techniques:

    Avidity analyses of anti-PfCelTOS IgG, IgG2a, and IgG2b antibodies by an ELISA. The avidity index (AI) was calculated as the portion of the OD value of urea-treated serum samples compared to that of untreated samples multiplied by 100. AI values of

    Journal: Infection and Immunity

    Article Title: Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) of Plasmodium falciparum in BALB/c Mice

    doi: 10.1128/IAI.00911-18

    Figure Lengend Snippet: Avidity analyses of anti-PfCelTOS IgG, IgG2a, and IgG2b antibodies by an ELISA. The avidity index (AI) was calculated as the portion of the OD value of urea-treated serum samples compared to that of untreated samples multiplied by 100. AI values of

    Article Snippet: In order to evaluate the IgG subclass responses to PfCelTOS, the test was performed as described above, and instead of goat anti-mouse IgG-HRP, 100 μl of either goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (diluted 1:1,000 in PBS-T buffer) (Sigma-Aldrich) as a secondary antibody was added to each well and incubated at RT for 1 h.

    Techniques: Enzyme-linked Immunosorbent Assay

    Assessment of anti-PfCelTOS IgG subclass profiles and longevity. Preimmune sera ( n  = 30) were used as negative controls to determine the ELISA cutoffs, which were calculated as the mean OD 490 plus 3 SD. The cutoffs for total IgG (TIgG),

    Journal: Infection and Immunity

    Article Title: Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) of Plasmodium falciparum in BALB/c Mice

    doi: 10.1128/IAI.00911-18

    Figure Lengend Snippet: Assessment of anti-PfCelTOS IgG subclass profiles and longevity. Preimmune sera ( n  = 30) were used as negative controls to determine the ELISA cutoffs, which were calculated as the mean OD 490 plus 3 SD. The cutoffs for total IgG (TIgG),

    Article Snippet: In order to evaluate the IgG subclass responses to PfCelTOS, the test was performed as described above, and instead of goat anti-mouse IgG-HRP, 100 μl of either goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (diluted 1:1,000 in PBS-T buffer) (Sigma-Aldrich) as a secondary antibody was added to each well and incubated at RT for 1 h.

    Techniques: Enzyme-linked Immunosorbent Assay

    Anti-PfCelTOS IgG antibody levels in vaccine mouse sera at different time points by an ELISA. Groups of 6- to 8-week-old female BALB/c mice were immunized subcutaneously with recombinant PfCelTOS alone or formulated with different adjuvants (MPL, CpG,

    Journal: Infection and Immunity

    Article Title: Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) of Plasmodium falciparum in BALB/c Mice

    doi: 10.1128/IAI.00911-18

    Figure Lengend Snippet: Anti-PfCelTOS IgG antibody levels in vaccine mouse sera at different time points by an ELISA. Groups of 6- to 8-week-old female BALB/c mice were immunized subcutaneously with recombinant PfCelTOS alone or formulated with different adjuvants (MPL, CpG,

    Article Snippet: In order to evaluate the IgG subclass responses to PfCelTOS, the test was performed as described above, and instead of goat anti-mouse IgG-HRP, 100 μl of either goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (diluted 1:1,000 in PBS-T buffer) (Sigma-Aldrich) as a secondary antibody was added to each well and incubated at RT for 1 h.

    Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay, Recombinant

    Evaluation of Th1/Th2 ratios. Th1 (anti-PfCelTOS IgG2a and IgG2b antibodies and cytokines IFN-γ and TNF-α) and Th2 (anti-PfCelTOS IgG1 and the cytokine IL-10) responses were analyzed in different vaccine groups on days 38 and 180 after

    Journal: Infection and Immunity

    Article Title: Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) of Plasmodium falciparum in BALB/c Mice

    doi: 10.1128/IAI.00911-18

    Figure Lengend Snippet: Evaluation of Th1/Th2 ratios. Th1 (anti-PfCelTOS IgG2a and IgG2b antibodies and cytokines IFN-γ and TNF-α) and Th2 (anti-PfCelTOS IgG1 and the cytokine IL-10) responses were analyzed in different vaccine groups on days 38 and 180 after

    Article Snippet: In order to evaluate the IgG subclass responses to PfCelTOS, the test was performed as described above, and instead of goat anti-mouse IgG-HRP, 100 μl of either goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (diluted 1:1,000 in PBS-T buffer) (Sigma-Aldrich) as a secondary antibody was added to each well and incubated at RT for 1 h.

    Techniques:

    Viral reactivity among thymic IgG-secreting plasma cells

    Journal: Science immunology

    Article Title: The human thymus perivascular space is a functional niche for viral-specific plasma cells

    doi: 10.1126/sciimmunol.aah4447

    Figure Lengend Snippet: Viral reactivity among thymic IgG-secreting plasma cells

    Article Snippet: Enumeration of IgG1, IgG2, IgG3, and IgG4 secreting cells was done using Human IgG1/IgG2/IgG3/IgG4 Four color Fluorospot (Cellular Technology).

    Techniques:

    PA and PG binding assessment of engineered antibodies by linear-gradient chromatography. (a) Overlay of HiTrap® MabSelect™ SuRe™ PA chromatograms (RT). An IgG3-like antibody (IgG 1133) based on the VH3 subclass (blue) still bound the MabSelect™ SuRe™ resin even though elution occurred at a mild pH (~pH 4.2). Adding the Fab substitution G65S (green) or N82aS (red) completely abolished binding (Kabat numbering). (b) Overlay of HiTrap® PG HP chromatograms (RT). An IgG1 antibody carrying the M428G/N434A substitutions in its Fc region (blue) still bound PG, while the same antibody with the added Fab substitution T209G (green) or K213V (red) was found in the flow through (Eu numbering).

    Journal: mAbs

    Article Title: Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

    doi: 10.1080/19420862.2019.1660564

    Figure Lengend Snippet: PA and PG binding assessment of engineered antibodies by linear-gradient chromatography. (a) Overlay of HiTrap® MabSelect™ SuRe™ PA chromatograms (RT). An IgG3-like antibody (IgG 1133) based on the VH3 subclass (blue) still bound the MabSelect™ SuRe™ resin even though elution occurred at a mild pH (~pH 4.2). Adding the Fab substitution G65S (green) or N82aS (red) completely abolished binding (Kabat numbering). (b) Overlay of HiTrap® PG HP chromatograms (RT). An IgG1 antibody carrying the M428G/N434A substitutions in its Fc region (blue) still bound PG, while the same antibody with the added Fab substitution T209G (green) or K213V (red) was found in the flow through (Eu numbering).

    Article Snippet: Both Fabs utilized the same VH subfamily (VH1-69) and only varied by their CDRs. cDNA coding sequences of IgG1 Hc, kappa Lc, and the mixed IgG1/IgG3 Fc were gene synthetized by Geneart AG (ThermoFisher Scientific).

    Techniques: Binding Assay, Chromatography, Flow Cytometry

    Single-step avidity purification of a full-length heterodimeric common light chain IgG. (a) Schematic of single-step PG avidity purification. The Fab #1 dG homodimer did not significantly bind PG, the heterodimeric IgG was eluted with glycine buffer pH 3.2, and Fab #2 IgG1 antibody was stripped from the column with glycine buffer pH 2.5. (b) HiTrap® PG HP chromatogram at 4 °C, pH measurements of the main peak fractions are indicated. (c) Total ion chromatogram obtained by RP-HPLC-MS of the main peak (1) fraction with baseline-separation between homo- and heterodimers. Fractional abundances were obtained by integrating the extracted ion chromatograms of homo- and heterodimer species (inset). (G-) denotes a nonfunctional PG binding site.

    Journal: mAbs

    Article Title: Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

    doi: 10.1080/19420862.2019.1660564

    Figure Lengend Snippet: Single-step avidity purification of a full-length heterodimeric common light chain IgG. (a) Schematic of single-step PG avidity purification. The Fab #1 dG homodimer did not significantly bind PG, the heterodimeric IgG was eluted with glycine buffer pH 3.2, and Fab #2 IgG1 antibody was stripped from the column with glycine buffer pH 2.5. (b) HiTrap® PG HP chromatogram at 4 °C, pH measurements of the main peak fractions are indicated. (c) Total ion chromatogram obtained by RP-HPLC-MS of the main peak (1) fraction with baseline-separation between homo- and heterodimers. Fractional abundances were obtained by integrating the extracted ion chromatograms of homo- and heterodimer species (inset). (G-) denotes a nonfunctional PG binding site.

    Article Snippet: Both Fabs utilized the same VH subfamily (VH1-69) and only varied by their CDRs. cDNA coding sequences of IgG1 Hc, kappa Lc, and the mixed IgG1/IgG3 Fc were gene synthetized by Geneart AG (ThermoFisher Scientific).

    Techniques: Purification, High Performance Liquid Chromatography, Mass Spectrometry, Binding Assay

    Single-step avidity purification of heterodimeric IgGs. (a) Schematic of single-step PA avidity purification. The IgG dA homodimer did not bind PA, the heterodimeric IgG was eluted with NaAc pH 4.2 (4 °C) or 4.1 (RT), and the scFv-Fc homodimer was stripped from the column with glycine buffer pH 3 (RT or 4 °C). (b) HiTrap® MabSelect SuRe™ chromatogram at 4 °C, pH measurements of the main peak fractions are indicated. (c) Total ion chromatogram obtained by RP-HPLC-MS of the main peak (1) fraction with baseline-separation between homo- and heterodimers. Fractional abundances were obtained by integrating the extracted ion chromatograms of homo- and heterodimer species (inset). (d) Schematic of single-step PG avidity purification. The scFv dG homodimer did not significantly bind PG, the heterodimeric IgG was eluted with glycine buffer pH 3.2 (RT or 4 °C), and the IgG1 antibody was stripped from the column with glycine buffer pH 2.5 (RT or 4 °C). (e) HiTrap® PG HP chromatogram at 4 °C, pH measurements of the main peak fractions are indicated. (f) RP-HPLC-MS analysis of the main peak (1) fraction as in (c). (A-) denotes a nonfunctional PA binding site. (G-) denotes a nonfunctional PG binding site.

    Journal: mAbs

    Article Title: Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

    doi: 10.1080/19420862.2019.1660564

    Figure Lengend Snippet: Single-step avidity purification of heterodimeric IgGs. (a) Schematic of single-step PA avidity purification. The IgG dA homodimer did not bind PA, the heterodimeric IgG was eluted with NaAc pH 4.2 (4 °C) or 4.1 (RT), and the scFv-Fc homodimer was stripped from the column with glycine buffer pH 3 (RT or 4 °C). (b) HiTrap® MabSelect SuRe™ chromatogram at 4 °C, pH measurements of the main peak fractions are indicated. (c) Total ion chromatogram obtained by RP-HPLC-MS of the main peak (1) fraction with baseline-separation between homo- and heterodimers. Fractional abundances were obtained by integrating the extracted ion chromatograms of homo- and heterodimer species (inset). (d) Schematic of single-step PG avidity purification. The scFv dG homodimer did not significantly bind PG, the heterodimeric IgG was eluted with glycine buffer pH 3.2 (RT or 4 °C), and the IgG1 antibody was stripped from the column with glycine buffer pH 2.5 (RT or 4 °C). (e) HiTrap® PG HP chromatogram at 4 °C, pH measurements of the main peak fractions are indicated. (f) RP-HPLC-MS analysis of the main peak (1) fraction as in (c). (A-) denotes a nonfunctional PA binding site. (G-) denotes a nonfunctional PG binding site.

    Article Snippet: Both Fabs utilized the same VH subfamily (VH1-69) and only varied by their CDRs. cDNA coding sequences of IgG1 Hc, kappa Lc, and the mixed IgG1/IgG3 Fc were gene synthetized by Geneart AG (ThermoFisher Scientific).

    Techniques: Purification, High Performance Liquid Chromatography, Mass Spectrometry, Binding Assay

    PK profiles of engineered antibodies and heterodimeric IgGs in Sprague-Dawley rats. Blood concentrations over time are shown. Antibodies and heterodimers were administrated at 10 mg/kg in a single intravenous bolus injection. Data points represent the mean concentration ± SD. (a) PK profile comparison of engineered antibodies: trastuzumab dA (IgG dA) and trastuzumab dG (IgG dG) vs. trastuzumab analog (IgG1). (b) PK profile comparison of the different heterodimeric IgGs. The trastuzumab analog profile (IgG1) generated from the same in vivo experiments is shown, heterodimers as indicated. (A-) denotes a nonfunctional PA binding site. (G-) denotes a nonfunctional PG binding site. (*) denotes the presence of the L234A and L235A substitutions in both BEAT Fc chains (Eu numbering).

    Journal: mAbs

    Article Title: Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

    doi: 10.1080/19420862.2019.1660564

    Figure Lengend Snippet: PK profiles of engineered antibodies and heterodimeric IgGs in Sprague-Dawley rats. Blood concentrations over time are shown. Antibodies and heterodimers were administrated at 10 mg/kg in a single intravenous bolus injection. Data points represent the mean concentration ± SD. (a) PK profile comparison of engineered antibodies: trastuzumab dA (IgG dA) and trastuzumab dG (IgG dG) vs. trastuzumab analog (IgG1). (b) PK profile comparison of the different heterodimeric IgGs. The trastuzumab analog profile (IgG1) generated from the same in vivo experiments is shown, heterodimers as indicated. (A-) denotes a nonfunctional PA binding site. (G-) denotes a nonfunctional PG binding site. (*) denotes the presence of the L234A and L235A substitutions in both BEAT Fc chains (Eu numbering).

    Article Snippet: Both Fabs utilized the same VH subfamily (VH1-69) and only varied by their CDRs. cDNA coding sequences of IgG1 Hc, kappa Lc, and the mixed IgG1/IgG3 Fc were gene synthetized by Geneart AG (ThermoFisher Scientific).

    Techniques: Injection, Concentration Assay, Generated, In Vivo, Binding Assay

    IgG 4 /IgE ratios to peanut and peanut allergens in children with PA (n = 65) and PS children (n = 27). Values are presented as medians and interquartile ranges. P values refer to the comparison between children with PA and PS children using the Mann-Whitney U test.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens

    doi: 10.1016/j.jaci.2015.01.012

    Figure Lengend Snippet: IgG 4 /IgE ratios to peanut and peanut allergens in children with PA (n = 65) and PS children (n = 27). Values are presented as medians and interquartile ranges. P values refer to the comparison between children with PA and PS children using the Mann-Whitney U test.

    Article Snippet: IgG4 antibody depletion IgG1 anti-IgG4 antibody (clone MH164-4; Sanquin, Amsterdam, The Netherlands) was coupled to cyanogen bromide–activated Sepharose (GE Healthcare, Hertfordshire, United Kingdom) during an overnight incubation at 4°C.

    Techniques: MANN-WHITNEY

    Inhibition of peanut-induced activation of mast cells sensitized with plasma from a patient with PA in the presence of mock- or IgG 4 -depleted plasma samples from peanut-sensitized tolerant patients (median inhibition, 75% vs 30%, respectively; P = .007, n = 12). % Inhibition = (% CD63 + cells sensitized with plasma from a patient with PA − % CD63 + cells sensitized with plasma from a patient with PA in the presence of test plasma)/% CD63 + cells sensitized with plasma from a patient with PA. The P value refers to the comparison between IgG 4 - and mock-depleted paired samples by using the Wilcoxon signed-rank test. ** P

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens

    doi: 10.1016/j.jaci.2015.01.012

    Figure Lengend Snippet: Inhibition of peanut-induced activation of mast cells sensitized with plasma from a patient with PA in the presence of mock- or IgG 4 -depleted plasma samples from peanut-sensitized tolerant patients (median inhibition, 75% vs 30%, respectively; P = .007, n = 12). % Inhibition = (% CD63 + cells sensitized with plasma from a patient with PA − % CD63 + cells sensitized with plasma from a patient with PA in the presence of test plasma)/% CD63 + cells sensitized with plasma from a patient with PA. The P value refers to the comparison between IgG 4 - and mock-depleted paired samples by using the Wilcoxon signed-rank test. ** P

    Article Snippet: IgG4 antibody depletion IgG1 anti-IgG4 antibody (clone MH164-4; Sanquin, Amsterdam, The Netherlands) was coupled to cyanogen bromide–activated Sepharose (GE Healthcare, Hertfordshire, United Kingdom) during an overnight incubation at 4°C.

    Techniques: Inhibition, Activation Assay

    Antibody production after stimulation with toll-like receptor (TLR) ligands. (A) Spontaneous and induced IgM responses by B cell subsets. The B cell subsets P1–P4 defined as shown in Figure 2 were FACS-sorted, cultured at 0.5 × 10 6 cells/ml and stimulated with resiquimod in the absence or presence of interleukin-2 (IL-2) and B-cell activating factor (BAFF) for 5 days. IgM production in the supernatants was quantified by ELISA. (B) TLR-induced IgM production induced of full PBMCs, of plasmacytoid dendritic cell (pDC)-depleted PBMCs (C) , and of PBMCs reconstituted with pDC following sorting (D) . (E–G) FMDV-specific IgG-antibody levels detected in the same setup as in (B–D) . (B–G) Cells were cultured for 7 days. Six biological replicates of two independent experiments are shown. Statistical significance was calculated using ANOVA followed by Tukey’s test (**** P

    Journal: Frontiers in Immunology

    Article Title: Porcine B Cell Subset Responses to Toll-like Receptor Ligands

    doi: 10.3389/fimmu.2017.01044

    Figure Lengend Snippet: Antibody production after stimulation with toll-like receptor (TLR) ligands. (A) Spontaneous and induced IgM responses by B cell subsets. The B cell subsets P1–P4 defined as shown in Figure 2 were FACS-sorted, cultured at 0.5 × 10 6 cells/ml and stimulated with resiquimod in the absence or presence of interleukin-2 (IL-2) and B-cell activating factor (BAFF) for 5 days. IgM production in the supernatants was quantified by ELISA. (B) TLR-induced IgM production induced of full PBMCs, of plasmacytoid dendritic cell (pDC)-depleted PBMCs (C) , and of PBMCs reconstituted with pDC following sorting (D) . (E–G) FMDV-specific IgG-antibody levels detected in the same setup as in (B–D) . (B–G) Cells were cultured for 7 days. Six biological replicates of two independent experiments are shown. Statistical significance was calculated using ANOVA followed by Tukey’s test (**** P

    Article Snippet: Monoclonal antibodies used included CD4 (clone 74-12-4; ATCC), CD14 (clone MIL2; Biorad, USA), CD16 (clone G7, Biorad), CD21 (clone BB6-11C9.6; Southern Biotech, USA), CD40 (clone G28.5, ATCC, USA), CD79a-APC (clone HM57, BioLegend, USA), CD80/86 (detection through human cytotoxic T-lymphocyte associated antigen-mouse immunoglobulin fusion protein; Enzo Biochem, USA), CD115 (clone ROS8G11-1; kindly provided by Prof. David Hume, Roslin Institute, University of Edinburgh, UK), CD172a (clone 74-22-15A, hybridoma kindly provided by Prof. Armin Saalmüller, Veterinary University, Vienna, Austria), CD172a-PE (Southern Biotech, USA), anti-IgM (clone PIG45A, ATCC), anti-IgG (23.7.1b, Thermofisher Scientific, Switzerland), anti-CD25 (clone K231.3B3, Biorad), and CADM1 (clone 3E1, MBL International, USA).

    Techniques: FACS, Cell Culture, Enzyme-linked Immunosorbent Assay

    B cell subsets in peripheral blood of pigs. (A) Gating strategies for identifying B cell subsets based on expression of IgG, IgM, CD21, and CD11R1. After doublet exclusion, population 1 (P1) was defined as CD21 + IgM + , P2 as IgG + , P3 as CD21 − IgM + CD11R1 + , and P4 as CD21 − IgM + CD11R1 − . (B) Histogram overlays of FSC, IgM, CD11c, CD80/86, CD14, phosphor-PLC-γ2, CD115, and CD172a for the subsets P1–P4 identified in (A) . A representative animal out of three is shown.

    Journal: Frontiers in Immunology

    Article Title: Porcine B Cell Subset Responses to Toll-like Receptor Ligands

    doi: 10.3389/fimmu.2017.01044

    Figure Lengend Snippet: B cell subsets in peripheral blood of pigs. (A) Gating strategies for identifying B cell subsets based on expression of IgG, IgM, CD21, and CD11R1. After doublet exclusion, population 1 (P1) was defined as CD21 + IgM + , P2 as IgG + , P3 as CD21 − IgM + CD11R1 + , and P4 as CD21 − IgM + CD11R1 − . (B) Histogram overlays of FSC, IgM, CD11c, CD80/86, CD14, phosphor-PLC-γ2, CD115, and CD172a for the subsets P1–P4 identified in (A) . A representative animal out of three is shown.

    Article Snippet: Monoclonal antibodies used included CD4 (clone 74-12-4; ATCC), CD14 (clone MIL2; Biorad, USA), CD16 (clone G7, Biorad), CD21 (clone BB6-11C9.6; Southern Biotech, USA), CD40 (clone G28.5, ATCC, USA), CD79a-APC (clone HM57, BioLegend, USA), CD80/86 (detection through human cytotoxic T-lymphocyte associated antigen-mouse immunoglobulin fusion protein; Enzo Biochem, USA), CD115 (clone ROS8G11-1; kindly provided by Prof. David Hume, Roslin Institute, University of Edinburgh, UK), CD172a (clone 74-22-15A, hybridoma kindly provided by Prof. Armin Saalmüller, Veterinary University, Vienna, Austria), CD172a-PE (Southern Biotech, USA), anti-IgM (clone PIG45A, ATCC), anti-IgG (23.7.1b, Thermofisher Scientific, Switzerland), anti-CD25 (clone K231.3B3, Biorad), and CADM1 (clone 3E1, MBL International, USA).

    Techniques: Expressing, Planar Chromatography

    Proliferative responses of B cell subsets after stimulation with toll-like receptor (TLR) ligands (A) Gating strategy defining CD21 + IgM + , CD21 + IgM + , and IgG + B cells, exemplified for unstimulated and Pam3Cys-SK4 stimulated peripheral blood mononuclear cells (PBMCs) after 5 days of culture. (B) CellTrace Violet staining of the three B-cell subsets defined in (A) using multicolor flow cytometry. PBMCs were cultured for 5d. A representative animal out of three is shown. (C) Percentage of proliferating B cell subsets, defined as shown in (A,B) , after stimulation of PBMCs with the indicated TLR ligands. Triplicates cultures from three different animals are shown. (D) Proliferation of FACS-sorted B cell subsets after stimulation for 3 days was determined using 3 H-thymidine incorporation. P1 was defined and sorted as CD21 + IgM + subset, P2 as IgG + subset, P3 as CD21 − IgM + CD11R1 + subset, and P4 as CD21 − IgM + CD11R1 − subset (for gate definition see Figure 2 ). (C,D) Mean values are indicated by horizontal bars. Error bars show SDs. Statistical significance was calculated using ANOVA followed by Tukey’s test.

    Journal: Frontiers in Immunology

    Article Title: Porcine B Cell Subset Responses to Toll-like Receptor Ligands

    doi: 10.3389/fimmu.2017.01044

    Figure Lengend Snippet: Proliferative responses of B cell subsets after stimulation with toll-like receptor (TLR) ligands (A) Gating strategy defining CD21 + IgM + , CD21 + IgM + , and IgG + B cells, exemplified for unstimulated and Pam3Cys-SK4 stimulated peripheral blood mononuclear cells (PBMCs) after 5 days of culture. (B) CellTrace Violet staining of the three B-cell subsets defined in (A) using multicolor flow cytometry. PBMCs were cultured for 5d. A representative animal out of three is shown. (C) Percentage of proliferating B cell subsets, defined as shown in (A,B) , after stimulation of PBMCs with the indicated TLR ligands. Triplicates cultures from three different animals are shown. (D) Proliferation of FACS-sorted B cell subsets after stimulation for 3 days was determined using 3 H-thymidine incorporation. P1 was defined and sorted as CD21 + IgM + subset, P2 as IgG + subset, P3 as CD21 − IgM + CD11R1 + subset, and P4 as CD21 − IgM + CD11R1 − subset (for gate definition see Figure 2 ). (C,D) Mean values are indicated by horizontal bars. Error bars show SDs. Statistical significance was calculated using ANOVA followed by Tukey’s test.

    Article Snippet: Monoclonal antibodies used included CD4 (clone 74-12-4; ATCC), CD14 (clone MIL2; Biorad, USA), CD16 (clone G7, Biorad), CD21 (clone BB6-11C9.6; Southern Biotech, USA), CD40 (clone G28.5, ATCC, USA), CD79a-APC (clone HM57, BioLegend, USA), CD80/86 (detection through human cytotoxic T-lymphocyte associated antigen-mouse immunoglobulin fusion protein; Enzo Biochem, USA), CD115 (clone ROS8G11-1; kindly provided by Prof. David Hume, Roslin Institute, University of Edinburgh, UK), CD172a (clone 74-22-15A, hybridoma kindly provided by Prof. Armin Saalmüller, Veterinary University, Vienna, Austria), CD172a-PE (Southern Biotech, USA), anti-IgM (clone PIG45A, ATCC), anti-IgG (23.7.1b, Thermofisher Scientific, Switzerland), anti-CD25 (clone K231.3B3, Biorad), and CADM1 (clone 3E1, MBL International, USA).

    Techniques: Staining, Flow Cytometry, Cytometry, Cell Culture, FACS

    ZNF282 interacts with E2F1 and functions as an E2F1 co-activator (A)Endogenous interaction between ZNF282 and E2F1. TE10 cell lysates were immunoprecipitated with anti-E2F1 antibody or control normal IgG, followed by immunoblot with the indicated antibodies. (B) 293T cells were transfected with pSG5.HA-E2F1 and pFLAG.CMV4-ZNF282. Cell lysates were immunoprecipitated with anti-E2F1 antibody or control normal IgG. Immunoprecipitated E2F1 and coimmunoprecipitated ZNF282 were detected byanti-FLAG antibody and anti-E2F1 antibody, respectively. (C) In vitro translated HA-tagged ZNF282 was incubated with recombinant GST-E2F1. Bound proteins were analyzed by immunoblot with anti-HA antibody. (D) Coactivator activity of ZNF282 for E2F1. 293T cells were transfected with pSG5.HA-E2F1 (20 ng) and 5xE2F-TA-LUC reporter (200 ng) in combination with various amounts (200, 400, 600 ng) of pSG5.HA- ZNF282. Cell extracts were assayed for luciferase activity. Results shown are mean and SD of triplicate points. (E and F) ZNF282 is required for the transcriptional activity of E2F1. 293T cells were transfected with pSG5.HA-E2F1, 5xE2F-TA-LUC, and 40 pmol of ZNF282 siRNA#5, #9 or NS (non-specific) siRNA duplex. 72 h after transfection, cells extracts were harvested for immunoblot analysis (E) and luciferase assays (F). (G-I) Requirement of ZNF282 for the expression of a subset of E2F1 target genes and recruitment of ZNF282 to selective E2F1 target genes. (G, H, and I) TE10 cells were transfected with 40 pmol of ZNF282 siRNA#5or NS siRNA duplex. 72 h after transfection, protein extracts and totalRNA were prepared. Protein levels were monitored by immunoblot using the indicated antibodies (G). Total RNA was examined by real-time quantitative RT–PCR (qRT–PCR) analysis with primers specific for the indicated mRNAs (H and I). Results shown were normalized to b-actin mRNAlevels and are means ± standard deviation (n=3). (J) ChIP assay. Crosslinked, sheared chromatin from TE10 cells was immunoprecipitated with the indicated antibodies. Quantitative PCR (qPCR) analyses were performed using primers specific for the indicated promoters. The results are shown as percentage of input and are means ± standard deviation (n=3).

    Journal: Oncotarget

    Article Title: ZNF282 (Zinc finger protein 282), a novel E2F1 co-activator, promotes esophageal squamous cell carcinoma

    doi:

    Figure Lengend Snippet: ZNF282 interacts with E2F1 and functions as an E2F1 co-activator (A)Endogenous interaction between ZNF282 and E2F1. TE10 cell lysates were immunoprecipitated with anti-E2F1 antibody or control normal IgG, followed by immunoblot with the indicated antibodies. (B) 293T cells were transfected with pSG5.HA-E2F1 and pFLAG.CMV4-ZNF282. Cell lysates were immunoprecipitated with anti-E2F1 antibody or control normal IgG. Immunoprecipitated E2F1 and coimmunoprecipitated ZNF282 were detected byanti-FLAG antibody and anti-E2F1 antibody, respectively. (C) In vitro translated HA-tagged ZNF282 was incubated with recombinant GST-E2F1. Bound proteins were analyzed by immunoblot with anti-HA antibody. (D) Coactivator activity of ZNF282 for E2F1. 293T cells were transfected with pSG5.HA-E2F1 (20 ng) and 5xE2F-TA-LUC reporter (200 ng) in combination with various amounts (200, 400, 600 ng) of pSG5.HA- ZNF282. Cell extracts were assayed for luciferase activity. Results shown are mean and SD of triplicate points. (E and F) ZNF282 is required for the transcriptional activity of E2F1. 293T cells were transfected with pSG5.HA-E2F1, 5xE2F-TA-LUC, and 40 pmol of ZNF282 siRNA#5, #9 or NS (non-specific) siRNA duplex. 72 h after transfection, cells extracts were harvested for immunoblot analysis (E) and luciferase assays (F). (G-I) Requirement of ZNF282 for the expression of a subset of E2F1 target genes and recruitment of ZNF282 to selective E2F1 target genes. (G, H, and I) TE10 cells were transfected with 40 pmol of ZNF282 siRNA#5or NS siRNA duplex. 72 h after transfection, protein extracts and totalRNA were prepared. Protein levels were monitored by immunoblot using the indicated antibodies (G). Total RNA was examined by real-time quantitative RT–PCR (qRT–PCR) analysis with primers specific for the indicated mRNAs (H and I). Results shown were normalized to b-actin mRNAlevels and are means ± standard deviation (n=3). (J) ChIP assay. Crosslinked, sheared chromatin from TE10 cells was immunoprecipitated with the indicated antibodies. Quantitative PCR (qPCR) analyses were performed using primers specific for the indicated promoters. The results are shown as percentage of input and are means ± standard deviation (n=3).

    Article Snippet: For coimmunoprecipitation (CoIP) assays, 293T or TE10 cell extracts were immunoprecipitated by specific antibodies or control IgG and protein G Dynabeads (Invitrogen) as indicated in figure legends.

    Techniques: Immunoprecipitation, Transfection, In Vitro, Incubation, Recombinant, Activity Assay, Luciferase, Expressing, Quantitative RT-PCR, Standard Deviation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Aurora B associates with p53 at various phases of the cell cycle. ( A ) Lysates of U2OS cells were IP with either anti-Aurora B (AB2254; Abcam) or anti-p53 antibodies (AB-2) or preimmune IgG (negative control) followed by immunoblotting (IB) with antibodies as labeled to show co-IP of Aurora B and p53. ( B ) GST pull-down assay was performed with combinations of in vitro translated Aurora B, GST, and GST-tagged p53 as labeled. Aurora B that was bound to GST-p53 was detected by IB. Coomassie staining of GST and GST-p53 inputs are in C . In vitro translated Aurora B was detected by immunoblot ( Lower ). ( C ) As presented in B , results of in vitro GST pull-down assay of immunopurified Flag-Aurora B with GST or GST-tagged p53 deletion constructs are shown. The asterisks indicate the stained bands of GST, GST-p53, and GST-p53 deletion mutants. ( D ) Hct116 cells were synchronized to S phase by double thymidine block. Cell samples at labeled time points after release of thymidine block were stained with PI and analyzed by FACS for DNA content. DNA content histograms are shown for the time points as labeled. ( E ) Lysates of synchronized Hct116 cells from D were analyzed by immunoblot with indicated antibodies. Aurora B–p53 interaction at various phases of the cell cycle (as labeled above) was detected by IP with anti-Aurora B antibody followed by IB for p53 and Aurora B (IP:AurB and IB:AurB).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Aurora B kinase phosphorylates and instigates degradation of p53

    doi: 10.1073/pnas.1110287109

    Figure Lengend Snippet: Aurora B associates with p53 at various phases of the cell cycle. ( A ) Lysates of U2OS cells were IP with either anti-Aurora B (AB2254; Abcam) or anti-p53 antibodies (AB-2) or preimmune IgG (negative control) followed by immunoblotting (IB) with antibodies as labeled to show co-IP of Aurora B and p53. ( B ) GST pull-down assay was performed with combinations of in vitro translated Aurora B, GST, and GST-tagged p53 as labeled. Aurora B that was bound to GST-p53 was detected by IB. Coomassie staining of GST and GST-p53 inputs are in C . In vitro translated Aurora B was detected by immunoblot ( Lower ). ( C ) As presented in B , results of in vitro GST pull-down assay of immunopurified Flag-Aurora B with GST or GST-tagged p53 deletion constructs are shown. The asterisks indicate the stained bands of GST, GST-p53, and GST-p53 deletion mutants. ( D ) Hct116 cells were synchronized to S phase by double thymidine block. Cell samples at labeled time points after release of thymidine block were stained with PI and analyzed by FACS for DNA content. DNA content histograms are shown for the time points as labeled. ( E ) Lysates of synchronized Hct116 cells from D were analyzed by immunoblot with indicated antibodies. Aurora B–p53 interaction at various phases of the cell cycle (as labeled above) was detected by IP with anti-Aurora B antibody followed by IB for p53 and Aurora B (IP:AurB and IB:AurB).

    Article Snippet: The following antibodies were used in this study: 14-3-3σ (1433S01; RDI), Actin (A2066; Sigma), Annexin V-FITC (556419; BD Biosciences), Aurora B (Ab2254; Abcam), Bad ( ; BD Biosciences), Bax ( ; BD Transduction), Cyclin A (SC751; Santa Cruz), Cyclin B1 (SC245; Santa Cruz), Cyclin D (MS-2110; Neomarkers), Cyclin E (SC-247; Santa Cruz), Flag (A804-200; Sigma), GFP (SC-9996; Santa Cruz), HA (12CA5; Roche), His (SC-803; Santa Cruz), HH3 (p-S10, 05–817; Upstate), MDM2 (S3813; Santa Cruz), Mouse IgG (488; Alexa; ; Molecular Probes), Mouse IgG (568; Alexa; ; Molecular Probes), p53 for IP (AB-1, PAB1801; Oncogene Science), p53 for IF (SC-6243; Santa Cruz), p53 (610183; BD Biosciences), p53 (p-S315, 2528S0; Cell Signaling), P21 (610233; Transduction Labs), PUMA (SC-28226; Santa Cruz), Rabbit IgG (488; Alexa; A1103; Molecular Probes), rabbit IgG (568; Alexa; A11011; Molecular Probes), and Survivin (2808; Cell Signaling).

    Techniques: Negative Control, Labeling, Co-Immunoprecipitation Assay, Pull Down Assay, In Vitro, Staining, Construct, Blocking Assay, FACS