Structured Review

Bio-Rad peroxidase conjugated goat anti mouse igg
Characterization of chimeric mouse/human <t>IgE-DG1.</t> Chimeric mouse/human IgE-DG1 was transiently expressed in HEK293 cells, and the IgE concentrations were determined by ELISA. A. Non-reduced (NR) or reduced (R) proteins from the supernatant of transfected HEK293 cells were separated by SDS-PAGE (12%) and transferred onto nitrocellulose membrane. IgE heavy and kappa light chains were detected by peroxidase-conjugated anti-human epsilon heavy chains or by anti-human kappa light chains, respectively, prior to chemiluminescent visualization. B. The activity and specificity of chimeric IgE-DG1 and its mouse monoclonal <t>IgG</t> counterpart (mDG1) were compared by indirect ELISA. Native gliadin- or deamidated gliadin (D-GLIA 48) -coated ELISA plates were incubated with indicated concentrations of chimeric IgE-DG1 or mDG1.
Peroxidase Conjugated Goat Anti Mouse Igg, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A chimeric IgE that mimics IgE from patients allergic to acid-hydrolyzed wheat proteins is a novel tool for in vitro allergenicity assessment of functionalized glutens"

Article Title: A chimeric IgE that mimics IgE from patients allergic to acid-hydrolyzed wheat proteins is a novel tool for in vitro allergenicity assessment of functionalized glutens

Journal: PLoS ONE

doi: 10.1371/journal.pone.0187415

Characterization of chimeric mouse/human IgE-DG1. Chimeric mouse/human IgE-DG1 was transiently expressed in HEK293 cells, and the IgE concentrations were determined by ELISA. A. Non-reduced (NR) or reduced (R) proteins from the supernatant of transfected HEK293 cells were separated by SDS-PAGE (12%) and transferred onto nitrocellulose membrane. IgE heavy and kappa light chains were detected by peroxidase-conjugated anti-human epsilon heavy chains or by anti-human kappa light chains, respectively, prior to chemiluminescent visualization. B. The activity and specificity of chimeric IgE-DG1 and its mouse monoclonal IgG counterpart (mDG1) were compared by indirect ELISA. Native gliadin- or deamidated gliadin (D-GLIA 48) -coated ELISA plates were incubated with indicated concentrations of chimeric IgE-DG1 or mDG1.
Figure Legend Snippet: Characterization of chimeric mouse/human IgE-DG1. Chimeric mouse/human IgE-DG1 was transiently expressed in HEK293 cells, and the IgE concentrations were determined by ELISA. A. Non-reduced (NR) or reduced (R) proteins from the supernatant of transfected HEK293 cells were separated by SDS-PAGE (12%) and transferred onto nitrocellulose membrane. IgE heavy and kappa light chains were detected by peroxidase-conjugated anti-human epsilon heavy chains or by anti-human kappa light chains, respectively, prior to chemiluminescent visualization. B. The activity and specificity of chimeric IgE-DG1 and its mouse monoclonal IgG counterpart (mDG1) were compared by indirect ELISA. Native gliadin- or deamidated gliadin (D-GLIA 48) -coated ELISA plates were incubated with indicated concentrations of chimeric IgE-DG1 or mDG1.

Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, SDS Page, Activity Assay, Indirect ELISA, Incubation

2) Product Images from "Activation of Cell Cycle Arrest and Apoptosis by the Proto-Oncogene Pim-2"

Article Title: Activation of Cell Cycle Arrest and Apoptosis by the Proto-Oncogene Pim-2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034736

Western blot analysis of cytoplasmic(C) and nuclear(N) expression of PIM-2 in various cell lines. Nuclear or cytoplasmic proteins (50 µg) were separated on a 15% SDS-PAGE. Blots were reacted with anti PIM-2 antibodies as primary antibody and HRP conjugated anti rabbit IgG secondary antibody. The membranes were stripped twice and reacted once with anti RCC1 antibody as control for nuclear proteins and once with anti β-tubulin as control for cytoplasmic proteins.
Figure Legend Snippet: Western blot analysis of cytoplasmic(C) and nuclear(N) expression of PIM-2 in various cell lines. Nuclear or cytoplasmic proteins (50 µg) were separated on a 15% SDS-PAGE. Blots were reacted with anti PIM-2 antibodies as primary antibody and HRP conjugated anti rabbit IgG secondary antibody. The membranes were stripped twice and reacted once with anti RCC1 antibody as control for nuclear proteins and once with anti β-tubulin as control for cytoplasmic proteins.

Techniques Used: Western Blot, Expressing, SDS Page

3) Product Images from "Cross-Reactivity among Several Recombinant Calicivirus Virus-Like Particles (VLPs) with Monoclonal Antibodies Obtained from Mice Immunized Orally with One Type of VLP"

Article Title: Cross-Reactivity among Several Recombinant Calicivirus Virus-Like Particles (VLPs) with Monoclonal Antibodies Obtained from Mice Immunized Orally with One Type of VLP

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.40.7.2459-2465.2002

Western blot of polypeptides from VLPs reacting with group A MAbs. The results shown are for MAbs NV23, NV37, F8, and F120 and VLPs rSV, rNV, rSeV, rCV, rFUV, rSMA, rGV, rKAV, rNAV, rCHV, and rUEV. Proteins were boiled for 2 min and lysed with sample buffer (about 1 to 2 μg/well), and SDS-polyacrylamide gel electrophoresis (PAGE) was conducted in 10% polyacrylamide gels. The separated proteins were transferred to nitrocellulose sheets, which were incubated overnight at room temperature with the indicated MAbs (at a 1:1,000 dilution of ascites) and then incubated with HRPO-conjugated goat anti-mouse IgG, IgM, and IgA for 1 h at 37°C and treated with substrate (4-chloro-1-naphthol). Calibration kits were used for molecular weight determination (lane M); numbers on the left side indicate the apparent molecular weights (10 3 ). These MAbs reacted with all VLPs of the NLV genus whose major polypeptide had an apparent molecular weight of 58,000 but not with rSV. Oligomers or possible breakdown bands (short arrows) were seen in some VLP preparations.
Figure Legend Snippet: Western blot of polypeptides from VLPs reacting with group A MAbs. The results shown are for MAbs NV23, NV37, F8, and F120 and VLPs rSV, rNV, rSeV, rCV, rFUV, rSMA, rGV, rKAV, rNAV, rCHV, and rUEV. Proteins were boiled for 2 min and lysed with sample buffer (about 1 to 2 μg/well), and SDS-polyacrylamide gel electrophoresis (PAGE) was conducted in 10% polyacrylamide gels. The separated proteins were transferred to nitrocellulose sheets, which were incubated overnight at room temperature with the indicated MAbs (at a 1:1,000 dilution of ascites) and then incubated with HRPO-conjugated goat anti-mouse IgG, IgM, and IgA for 1 h at 37°C and treated with substrate (4-chloro-1-naphthol). Calibration kits were used for molecular weight determination (lane M); numbers on the left side indicate the apparent molecular weights (10 3 ). These MAbs reacted with all VLPs of the NLV genus whose major polypeptide had an apparent molecular weight of 58,000 but not with rSV. Oligomers or possible breakdown bands (short arrows) were seen in some VLP preparations.

Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis, Incubation, Molecular Weight

4) Product Images from "Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae"

Article Title: Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt1281

Bmt6 interacts with Nop2 in vivo . To demonstrate the interaction of Bmt6 with the preribosomal particles and its minor nuclear localization, we immunoprecipitated the total cell extract from Bmt6-TAP–tagged strain using IgG sepharose and analyzed its interaction for Nop2 by western blotting. ( A ) Western blot with PAP antibodies. ( B ) Western blot with anti-Nop2 (EnCor Biotechnology, Florida, USA) followed by anti-mouse IgG-conjugated horseradish peroxidase (Bio-Rad; 1:10 000 dilution). The samples in the different lanes of the gels are crude extract (total cell extract), flow through (unbound cell extract) and wash fraction 6 (the beads were washed six times with IPP-150 buffer) and eluate.
Figure Legend Snippet: Bmt6 interacts with Nop2 in vivo . To demonstrate the interaction of Bmt6 with the preribosomal particles and its minor nuclear localization, we immunoprecipitated the total cell extract from Bmt6-TAP–tagged strain using IgG sepharose and analyzed its interaction for Nop2 by western blotting. ( A ) Western blot with PAP antibodies. ( B ) Western blot with anti-Nop2 (EnCor Biotechnology, Florida, USA) followed by anti-mouse IgG-conjugated horseradish peroxidase (Bio-Rad; 1:10 000 dilution). The samples in the different lanes of the gels are crude extract (total cell extract), flow through (unbound cell extract) and wash fraction 6 (the beads were washed six times with IPP-150 buffer) and eluate.

Techniques Used: In Vivo, Immunoprecipitation, Western Blot, Flow Cytometry

5) Product Images from "Passive Protection against Anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region "

Article Title: Passive Protection against Anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region

Journal: Infection and Immunity

doi: 10.1128/IAI.73.12.8362-8368.2005

Pharmacokinetic analysis of Hartley guinea pigs injected subcutaneously with 14B7 IgG (black), M18 scAb conjugated to 20- kDa PEG (red), or M18 scAb conjugated to 40-kDa PEG (green). scAb-20-kDa PEG displayed a half-life of 23.2 h, 14B7 displayed a half-life
Figure Legend Snippet: Pharmacokinetic analysis of Hartley guinea pigs injected subcutaneously with 14B7 IgG (black), M18 scAb conjugated to 20- kDa PEG (red), or M18 scAb conjugated to 40-kDa PEG (green). scAb-20-kDa PEG displayed a half-life of 23.2 h, 14B7 displayed a half-life

Techniques Used: Injection

Related Articles

Incubation:

Article Title: Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422
Article Snippet: .. Antigen-coated wells were incubated with 0.5 μg/ml of appropriate antibodies at 37°C for 60 min, washed as described above, and then further incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:1,000; Bio-Rad) at 37°C for 60 min. After washing, the bound antibody was detected by adding peroxidase substrate solution (prepared by dissolving o -phenylenediamine dihydrochloride at 0.4 mg/ml in 0.1 M citric acid and 0.2 M Na2 HPO4 buffer [pH 4.8] and adding 0.2 μl/ml of 30% H2 O2 ). .. Following incubation at room temperature in the dark, the reaction was stopped with a 2.5 M H2 SO4 solution, and the optical density at 492 nm was determined using a microplate reader (BioTek, Winooski, VT, USA).

Article Title: Protein and modified vaccinia virus Ankara‐based influenza virus nucleoprotein vaccines are differentially immunogenic in BALB/c mice
Article Snippet: A three‐ or fivefold dilution series of the sera was prepared in blocking buffer starting at 1 : 30 and incubated on the coated plates for 1 h. Blocking buffer and anti‐NP positive serum were used as a negative or positive control, respectively. .. Plates were washed with PBST and incubated with anti‐IgG1 (Star 132P; AbD Serotec, Raleigh, NC, USA) or anti‐IgG2a (Star 133P; AbD Serotec) horseradish peroxidase (HRP)‐conjugated antibodies – indicative for a Th2 or Th1 response, respectively – for 1 h. Subsequently, plates were washed with PBST and 100 μl 3,3′,5,5′‐tetramethylbenzidine (TMB) peroxidase substrate (Svanova Biotech, Uppsala, Sweden) was added. .. Reactions were stopped by adding 1·8 M H2 SO4 and absorbance was measured subsequently at 450 nm using a SpectraMax Plus 384 Microplate Reader (Molecular Devices Corporation, Sunnyvale, CA, USA).

Article Title: Thymic stromal lymphopoietin (TSLP) acts as a potent mucosal adjuvant for HIV-1 gp140 vaccination in mice
Article Snippet: .. Bound IgA and IgG was detected by incubation for 1 h at 37°C with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgE, IgG1 , IgG2a (AbD Serotec, UK) or biotin-conjugated goat anti-mouse IgA and IgE (AbD Serotec, UK). .. For IgA and IgE detection, plates were incubated with Streptavidin-HRP (R & D Systems) for 1 h at 37°C.

Recombinant:

Article Title: A Nonhuman Primate Scrub Typhus Model: Protective Immune Responses Induced by pKarp47 DNA Vaccination in Cynomolgus Macaques
Article Snippet: The ELISA assays used a recombinant protein, 47-kDa Ag strain Karp (Kp r47b), supplied by Invitrogen (0.05 μg/well; Carlsbad, CA), or O. tsutsugamushi Karp whole-cell Ag (at 0.1 μg/well) as previously described ( , ). .. Isotyping was performed using recombinant protein Kpr47b as the Ag detected with HRP-conjugated anti-human IgG1, IgG2, IgG3, and IgG4 (Serotec, Raleigh, NC) as described previously ( ). ..

Western Blot:

Article Title: Human NUMB6 Induces Epithelial-Mesenchymal Transition and Enhances Breast Cancer Cells Migration and Invasion
Article Snippet: Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). .. Membranes were probed for GFP (Invitrogen), and β-actin (Sigma-Aldrich), followed by anti-mouse IgG horseradish peroxidase-linked whole antibody (Bio-Rad Laboratories) and detected using Luminata Crescendo Western HRP Substrate (Millipore, Billerica, MA, USA). ..

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  • 99
    Bio-Rad anti igg2a
    Nucleoprotein (NP)‐specific antibody responses induced by vaccination with NP wild‐type (NPwt) protein + Matrix‐M TM adjuvant or the respective recombinant Modified Vaccinia virus Ankara (rMVA)–NP constructs. Immunoglobulin (Ig)G1 and <t>IgG2a</t> NP‐specific antibody responses in mice 21 days after the primary vaccination (a,b) or 14 days after booster vaccination (c,d). IgG1 (a,c) or IgG2a (b,d) serum antibodies were detected using purified NPwt protein and anti‐IgG1 or anti‐IgG2a horseradish peroxidase (HRP)‐conjugated antibodies. Mean for each group is indicated. MM = Matrix‐M™ adjuvant. * P
    Anti Igg2a, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti igg2a/product/Bio-Rad
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti igg2a - by Bioz Stars, 2021-06
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    98
    Bio-Rad horseradish peroxidase hrp conjugated goat anti mouse igg
    Establishment of MAbs against M161Ag. (A) Western blot of M. fermentans PG18 with MAbs against M161Ag. Ten microliters of mycoplasma cell lysate (1.0 mg/ml) was applied to each lane. SDS-PAGE was performed under reducing conditions. Lane 1, Coomassie blue staining of M. fermentans PG18 proteins. Samples were transblotted onto nitrocellulose sheets and detected with the indicated MAbs (lanes 2 to 5). Mouse <t>IgG</t> was used as a control (lane 6). Molecular mass markers are shown to the left. (B) MAbs recognized the 43-kDa protein in P39(+) cells but not P39(−) cells. P39(+) and -(−) cell lysates were subjected to SDS-PAGE (10% gel) under reducing or nonreducing conditions and transferred onto nitrocellulose sheets. Each sheet was blotted with M161, MK53, MK5, or MK36 and then with <t>HRP-conjugated</t> goat anti-mouse IgG. NR, nonreducing conditions; R, reducing conditions. The positions of molecular mass markers are shown on the right. (C) Flow cytometric analysis of M161Ag on the P39 sublines. Cell surface M161Ag was assessed using flow cytometry with each MAb followed by a FITC-labeled anti-mouse IgG using P39(+) and -(−) cells.
    Horseradish Peroxidase Hrp Conjugated Goat Anti Mouse Igg, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase hrp conjugated goat anti mouse igg/product/Bio-Rad
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase hrp conjugated goat anti mouse igg - by Bioz Stars, 2021-06
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    Nucleoprotein (NP)‐specific antibody responses induced by vaccination with NP wild‐type (NPwt) protein + Matrix‐M TM adjuvant or the respective recombinant Modified Vaccinia virus Ankara (rMVA)–NP constructs. Immunoglobulin (Ig)G1 and IgG2a NP‐specific antibody responses in mice 21 days after the primary vaccination (a,b) or 14 days after booster vaccination (c,d). IgG1 (a,c) or IgG2a (b,d) serum antibodies were detected using purified NPwt protein and anti‐IgG1 or anti‐IgG2a horseradish peroxidase (HRP)‐conjugated antibodies. Mean for each group is indicated. MM = Matrix‐M™ adjuvant. * P

    Journal: Clinical and Experimental Immunology

    Article Title: Protein and modified vaccinia virus Ankara‐based influenza virus nucleoprotein vaccines are differentially immunogenic in BALB/c mice

    doi: 10.1111/cei.13004

    Figure Lengend Snippet: Nucleoprotein (NP)‐specific antibody responses induced by vaccination with NP wild‐type (NPwt) protein + Matrix‐M TM adjuvant or the respective recombinant Modified Vaccinia virus Ankara (rMVA)–NP constructs. Immunoglobulin (Ig)G1 and IgG2a NP‐specific antibody responses in mice 21 days after the primary vaccination (a,b) or 14 days after booster vaccination (c,d). IgG1 (a,c) or IgG2a (b,d) serum antibodies were detected using purified NPwt protein and anti‐IgG1 or anti‐IgG2a horseradish peroxidase (HRP)‐conjugated antibodies. Mean for each group is indicated. MM = Matrix‐M™ adjuvant. * P

    Article Snippet: Plates were washed with PBST and incubated with anti‐IgG1 (Star 132P; AbD Serotec, Raleigh, NC, USA) or anti‐IgG2a (Star 133P; AbD Serotec) horseradish peroxidase (HRP)‐conjugated antibodies – indicative for a Th2 or Th1 response, respectively – for 1 h. Subsequently, plates were washed with PBST and 100 μl 3,3′,5,5′‐tetramethylbenzidine (TMB) peroxidase substrate (Svanova Biotech, Uppsala, Sweden) was added.

    Techniques: Recombinant, Modification, Construct, Mouse Assay, Purification

    Nucleoprotein (NP)‐specific antibody responses after NP wild‐type (NPwt) protein (with or without Matrix‐M™ adjuvant) or recombinant Modified Vaccinia virus Ankara (rMVA)–NPwt vaccination. The immunoglobulin (Ig)G1 and IgG2a NP‐specific antibody responses 21 days after the primary vaccination (a,b) or 14 days after the booster vaccination (c,d). IgG1 (a,c) or IgG2a (b,d) serum antibodies were detected using purified NPwt protein and anti‐IgG1 or anti‐IgG2a horseradish peroxidase (HRP)‐conjugated antibodies. Mean of each group is indicated. MM = Matrix‐M™ adjuvant. **** P

    Journal: Clinical and Experimental Immunology

    Article Title: Protein and modified vaccinia virus Ankara‐based influenza virus nucleoprotein vaccines are differentially immunogenic in BALB/c mice

    doi: 10.1111/cei.13004

    Figure Lengend Snippet: Nucleoprotein (NP)‐specific antibody responses after NP wild‐type (NPwt) protein (with or without Matrix‐M™ adjuvant) or recombinant Modified Vaccinia virus Ankara (rMVA)–NPwt vaccination. The immunoglobulin (Ig)G1 and IgG2a NP‐specific antibody responses 21 days after the primary vaccination (a,b) or 14 days after the booster vaccination (c,d). IgG1 (a,c) or IgG2a (b,d) serum antibodies were detected using purified NPwt protein and anti‐IgG1 or anti‐IgG2a horseradish peroxidase (HRP)‐conjugated antibodies. Mean of each group is indicated. MM = Matrix‐M™ adjuvant. **** P

    Article Snippet: Plates were washed with PBST and incubated with anti‐IgG1 (Star 132P; AbD Serotec, Raleigh, NC, USA) or anti‐IgG2a (Star 133P; AbD Serotec) horseradish peroxidase (HRP)‐conjugated antibodies – indicative for a Th2 or Th1 response, respectively – for 1 h. Subsequently, plates were washed with PBST and 100 μl 3,3′,5,5′‐tetramethylbenzidine (TMB) peroxidase substrate (Svanova Biotech, Uppsala, Sweden) was added.

    Techniques: Recombinant, Modification, Purification

    Humoral and cell-mediated immune responses of macaques. ( A ) Vaccination elicits an immune response reflected by earlier and higher levels of IgG in pKarp47 vaccine regimens than in Kp47/47-VRP vaccine regimens. The elicited humoral immune response dynamics differed between the cynomolgus vaccine groups; the triple-homologous plasmid pKarp47 vaccine regimen (group 3) was slow to produce high IgG titers ( left panel ), whereas the heterologous plasmid pKarp47 vaccine regimen using a boost with Kp47/47-VRP (group 4) induced earlier and higher levels of IgG ( right panel ). The Kp47/47-VRP vaccine regimen achieved the lowest IgG titers, which, however, increased rapidly upon natural challenge with O. tsutsugamushi . ( B ) Vaccination using a plasmid pKarp47-based vaccine regimen (group 3) elicits higher and more sustained cell-mediated immune responses than other vaccine candidates in this study. Time-course diagram of the cellular immune response after ex vivo stimulation of PBMCs depicting that vaccinees receiving the triple-homologous plasmid pKarp47 vaccine regimen (group 3) achieved the most pronounced cell-mediated immune response of all vaccine groups in this study, with sustained high numbers of IFN-γ–producing PBMCs, whereas macaques in the heterologous prime-boost regimen (group 4) showed a marked decrease of SFUs after the third vaccine, a boost with Kp47/47-VRP, was applied (B). Data are actual values. The number of IFN-γ–producing PBMCs is defined as SFUs per 1 million PBMCs. Time points include baseline, first, second, and third vaccine (V1, V2, and V3), respectively, and challenge (Ch), followed by days postvaccine or postchallenge (p.ex. V2 d14 corresponds to 14 d after second vaccination).

    Journal: The Journal of Immunology Author Choice

    Article Title: A Nonhuman Primate Scrub Typhus Model: Protective Immune Responses Induced by pKarp47 DNA Vaccination in Cynomolgus Macaques

    doi: 10.4049/jimmunol.1402244

    Figure Lengend Snippet: Humoral and cell-mediated immune responses of macaques. ( A ) Vaccination elicits an immune response reflected by earlier and higher levels of IgG in pKarp47 vaccine regimens than in Kp47/47-VRP vaccine regimens. The elicited humoral immune response dynamics differed between the cynomolgus vaccine groups; the triple-homologous plasmid pKarp47 vaccine regimen (group 3) was slow to produce high IgG titers ( left panel ), whereas the heterologous plasmid pKarp47 vaccine regimen using a boost with Kp47/47-VRP (group 4) induced earlier and higher levels of IgG ( right panel ). The Kp47/47-VRP vaccine regimen achieved the lowest IgG titers, which, however, increased rapidly upon natural challenge with O. tsutsugamushi . ( B ) Vaccination using a plasmid pKarp47-based vaccine regimen (group 3) elicits higher and more sustained cell-mediated immune responses than other vaccine candidates in this study. Time-course diagram of the cellular immune response after ex vivo stimulation of PBMCs depicting that vaccinees receiving the triple-homologous plasmid pKarp47 vaccine regimen (group 3) achieved the most pronounced cell-mediated immune response of all vaccine groups in this study, with sustained high numbers of IFN-γ–producing PBMCs, whereas macaques in the heterologous prime-boost regimen (group 4) showed a marked decrease of SFUs after the third vaccine, a boost with Kp47/47-VRP, was applied (B). Data are actual values. The number of IFN-γ–producing PBMCs is defined as SFUs per 1 million PBMCs. Time points include baseline, first, second, and third vaccine (V1, V2, and V3), respectively, and challenge (Ch), followed by days postvaccine or postchallenge (p.ex. V2 d14 corresponds to 14 d after second vaccination).

    Article Snippet: Isotyping was performed using recombinant protein Kpr47b as the Ag detected with HRP-conjugated anti-human IgG1, IgG2, IgG3, and IgG4 (Serotec, Raleigh, NC) as described previously ( ).

    Techniques: Plasmid Preparation, Ex Vivo

    ePCL-ELISA. Panel A: comparison between ePCL and conventional polystyrene ELISA: microwell plates are coated with decreasing quantities of E7-GST on polystyrene and on ePCL, incubated with monoclonal anti-HPV16 E7 antibody, and thereafter with horseradish peroxidase-conjugated goat antimouse IgG. Absorbance at 450 nm is measured after a colorimetric reaction with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. Panel B: ePCL-ELISA specificity: ePCL-ELISA microwells are coated with decreasing concentrations of E7-GST or GST protein. Experimental procedures are the same as in panel A. Data are means of triplicate samples OD ± SD.

    Journal: ACS Omega

    Article Title: ePCL Electrospun Microfibrous Layers for Immune Assays: Sensitive ELISA for the Detection of Serum Antibodies Against HPV16 E7 Oncoprotein

    doi: 10.1021/acsomega.0c03976

    Figure Lengend Snippet: ePCL-ELISA. Panel A: comparison between ePCL and conventional polystyrene ELISA: microwell plates are coated with decreasing quantities of E7-GST on polystyrene and on ePCL, incubated with monoclonal anti-HPV16 E7 antibody, and thereafter with horseradish peroxidase-conjugated goat antimouse IgG. Absorbance at 450 nm is measured after a colorimetric reaction with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. Panel B: ePCL-ELISA specificity: ePCL-ELISA microwells are coated with decreasing concentrations of E7-GST or GST protein. Experimental procedures are the same as in panel A. Data are means of triplicate samples OD ± SD.

    Article Snippet: Horseradish-labeled goat antimouse IgG (H+L-HRP conjugate; BioRad) is utilized as the secondary antibody (1:1000 dilution).

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation

    Analysis of mouse sera immunized with HPV16L2-E7 chimeric DNA vaccine. Panel A: comparison between ePCL and bare polystyrene: ELISA plates are coated with 100 ng of E7-GST protein either on bare polystyrene or on ePCL, incubated with decreasing dilution of preimmunized (Pre Imm) and immunized (Imm) mouse sera and thereafter with HRP conjugate goat antimouse. Absorbance at 450 nm is measured after a colorimetric reaction with TMB substrate. Data are means of triplicate samples OD ± SD. Panel B: ePCL-ELISA for E7 antibody detection in five different mouse sera (Imm): ePCL microwells are coated with 100 ng of E7-GST protein. Experimental procedures are the same as in panel A. Data are means of triplicate samples OD ± SD calculated after subtracting preimmunized serum value.

    Journal: ACS Omega

    Article Title: ePCL Electrospun Microfibrous Layers for Immune Assays: Sensitive ELISA for the Detection of Serum Antibodies Against HPV16 E7 Oncoprotein

    doi: 10.1021/acsomega.0c03976

    Figure Lengend Snippet: Analysis of mouse sera immunized with HPV16L2-E7 chimeric DNA vaccine. Panel A: comparison between ePCL and bare polystyrene: ELISA plates are coated with 100 ng of E7-GST protein either on bare polystyrene or on ePCL, incubated with decreasing dilution of preimmunized (Pre Imm) and immunized (Imm) mouse sera and thereafter with HRP conjugate goat antimouse. Absorbance at 450 nm is measured after a colorimetric reaction with TMB substrate. Data are means of triplicate samples OD ± SD. Panel B: ePCL-ELISA for E7 antibody detection in five different mouse sera (Imm): ePCL microwells are coated with 100 ng of E7-GST protein. Experimental procedures are the same as in panel A. Data are means of triplicate samples OD ± SD calculated after subtracting preimmunized serum value.

    Article Snippet: Horseradish-labeled goat antimouse IgG (H+L-HRP conjugate; BioRad) is utilized as the secondary antibody (1:1000 dilution).

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation

    Establishment of MAbs against M161Ag. (A) Western blot of M. fermentans PG18 with MAbs against M161Ag. Ten microliters of mycoplasma cell lysate (1.0 mg/ml) was applied to each lane. SDS-PAGE was performed under reducing conditions. Lane 1, Coomassie blue staining of M. fermentans PG18 proteins. Samples were transblotted onto nitrocellulose sheets and detected with the indicated MAbs (lanes 2 to 5). Mouse IgG was used as a control (lane 6). Molecular mass markers are shown to the left. (B) MAbs recognized the 43-kDa protein in P39(+) cells but not P39(−) cells. P39(+) and -(−) cell lysates were subjected to SDS-PAGE (10% gel) under reducing or nonreducing conditions and transferred onto nitrocellulose sheets. Each sheet was blotted with M161, MK53, MK5, or MK36 and then with HRP-conjugated goat anti-mouse IgG. NR, nonreducing conditions; R, reducing conditions. The positions of molecular mass markers are shown on the right. (C) Flow cytometric analysis of M161Ag on the P39 sublines. Cell surface M161Ag was assessed using flow cytometry with each MAb followed by a FITC-labeled anti-mouse IgG using P39(+) and -(−) cells.

    Journal: Infection and Immunity

    Article Title: Complement Activation in Mycoplasma fermentans-Induced Mycoplasma Clearance from Infected Cells: Probing of the Organism with Monoclonal Antibodies against M161Ag

    doi:

    Figure Lengend Snippet: Establishment of MAbs against M161Ag. (A) Western blot of M. fermentans PG18 with MAbs against M161Ag. Ten microliters of mycoplasma cell lysate (1.0 mg/ml) was applied to each lane. SDS-PAGE was performed under reducing conditions. Lane 1, Coomassie blue staining of M. fermentans PG18 proteins. Samples were transblotted onto nitrocellulose sheets and detected with the indicated MAbs (lanes 2 to 5). Mouse IgG was used as a control (lane 6). Molecular mass markers are shown to the left. (B) MAbs recognized the 43-kDa protein in P39(+) cells but not P39(−) cells. P39(+) and -(−) cell lysates were subjected to SDS-PAGE (10% gel) under reducing or nonreducing conditions and transferred onto nitrocellulose sheets. Each sheet was blotted with M161, MK53, MK5, or MK36 and then with HRP-conjugated goat anti-mouse IgG. NR, nonreducing conditions; R, reducing conditions. The positions of molecular mass markers are shown on the right. (C) Flow cytometric analysis of M161Ag on the P39 sublines. Cell surface M161Ag was assessed using flow cytometry with each MAb followed by a FITC-labeled anti-mouse IgG using P39(+) and -(−) cells.

    Article Snippet: Fluorescein isothiocyanate (FITC)-labeled goat F(ab′)2 anti-mouse IgG was from Cappel (West Chester, Pa.), and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-labeled anti-rabbit IgG were from Bio-Rad Laboratories (Hercules, Calif.).

    Techniques: Western Blot, SDS Page, Staining, Flow Cytometry, Cytometry, Labeling