hrp-conjugated goat anti-human igg Search Results


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  • 99
    Millipore anti human igg fc specific peroxidase antibody
    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 <t>(IgG)</t> 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
    Anti Human Igg Fc Specific Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 871 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 871 article reviews
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    99
    Bio-Rad hrp conjugated goat anti mouse
    Murine and rat <t>IGSF1-2</t> proteins do not traffic to the plasma membrane of transfected cells. A) HEK293 cells were transfected with expression plasmids for wild-type murine (M.) or rat (R.) IGSF1-2, murine BMP type IA receptor (BMPR1A), or empty vector (pcDNA4). Note, IGSF1-2 proteins were expressed with Myc/His tags at their C-termini, whereas BMPR1A had the Myc tag alone. Cell surface proteins were labeled with biotin prior to collection of whole cell lysates. Lysates were immunoprecipitated (IP) with Myc-beads and then subjected to SDS-PAGE and transferred to nitrocellulose membranes. Total proteins were immunoblotted with anti-Myc (bottom), whereas biotinylated proteins were identified with <t>streptavidin-HRP</t> (top). B) HEK293 cells were cultured on coverslips and transiently transfected with wild-type murine IGSF1-2-Myc/His. Cells were then fixed and subjected to immunofluorescence with a Myc antibody (green) under non-permeabilizing (top) and permeabilizing conditions (bottom). C) Cells were transfected as in panel B, premeabilized, and processed for double-label immunofluorescence with the Myc antibody (green) and an antibody against GRP-78/BiP (red). The overlay is shown in yellow. In B and C, nuclei were stained with DAPI. Images were captured by confocal microscopy. Scale bar, 10 μm.
    Hrp Conjugated Goat Anti Mouse, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad hrp conjugated goat anti rabbit
    Representative example of <t>OFA</t> immune response in vivo . The lanes contain protein extracts (40 μg per lane) from OFA-expressing 4T1 cells, incubated with sera from mice orally immunized with wild-type (wt) L. plantarum or L. plantarum harboring pLp_0373sOFAcwa2 (cwa2), or from mice subcutaneously immunized with His-tagged OFA in Freund's complete adjuvant (OFA-His). Anti-OFA monoclonal mouse IgG antibody (mAb) was used as a positive control. Antibody binding was visualized using <t>HRP</t> conjugated to anti-mouse IgG and the ECL system (Amersham Life Science, Buckinghamshire, United Kingdom). The arrow indicates OFA.
    Hrp Conjugated Goat Anti Rabbit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher hrp conjugated goat anti human igg
    Sero-positivity of anti-E2EP3 IgG3 antibodies in CHIKV-infected patients from other cohorts CHIK virion-based ELISA was used to assess anti-CHIKV <t>IgG</t> titer in CHIKV-infected patients from another Singaporean cohort collected at median 10 days pio ( n = 36). Healthy donors' plasma ( n = 11) were used as controls. Individual samples were subjected to virion-based ELISA at a dilution of 1:2000, followed by secondary human <t>anti-IgG-HRP.</t> *** p
    Hrp Conjugated Goat Anti Human Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 239 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated goat anti human igg/product/Thermo Fisher
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    99
    Bethyl goat anti human igg fc antibody hrp conjugated
    Confirmation of hIgG expression in the Tc goat with Western blotting. The purified hIgG from the Tc goat, positive control human IVIG, and negative control purified from commercial goat <t>IgG</t> was probed with anti-human IgG (heavy and light; H + L) <t>HRP.</t>
    Goat Anti Human Igg Fc Antibody Hrp Conjugated, supplied by Bethyl, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher horseradish peroxidase hrp conjugated goat anti human igg
    The profiles of the PV IgGs absorbed by recombinant Dsg 1 and Dsg 3 baculoproteins. Western blots of protein extracts of normal human keratinocytes resolved by 7.5% SDS-PAGE and stained with PV3 <t>IgG</t> affinity-purified on rDsg3-His (lane 1), and that of Dsg3 null keratinocytes stained with PV3 IgG affinity purified on rDsg3-Ig-His (lane 5), rDsg1-Ig-His (lane 4) or on the Fc IgG column (lane 9). Binding of PV3 IgGs to the immunoblotting membranes was visualized using secondary, <t>HRP-conjugated</t> goat anti-human IgG antibodies. Lane 6 shows a single protein band with apparent M r of 190 kDa among SDS-PAGE–resolved Dsg 3–positive keratinocyte proteins from BALB/c mouse. This band was visualized by the PV3 IgG that was eluted from the 190-kDa area of the Western blot of Dsg3 null keratinocyte proteins stained with the PV3 IgG adsorbed on rDsg3-Ig-His. Note: Only a 190-kDa but not 130- or 160-kDa band or any other keratinocyte protein was visualized, indicating that the antibody targeting the 190-kDa protein is a unique one, as it does not recognize the 130-kDa Dsg 3 or 160-kDa Dsg 1. Lanes 2, 3, and 7 are the negative controls omitting primary antibody. The Dsg3 null keratinocyte protein extract in lane 8 was blotted with normal human IgG-affinity purified on rDsg3-Ig-His. The lack of multiple bands in lane 8 as well as complete absence of specific staining in lanes 3 and 7 indicate that there were no nonspecific cross reactivities of human or goat IgGs with murine epidermal proteins. The positions of relative molecular mass markers run in parallel lanes of each blot are shown to the left of the respective blot. The apparent relative molecular mass of keratinocyte protein bands visualized due to PV antibody binding is shown to the right of lanes 2 and 5 in the columns designated M r .
    Horseradish Peroxidase Hrp Conjugated Goat Anti Human Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    SouthernBiotech hrp conjugated goat anti human immunoglobulin g
    Binding of c2A10G6 to ZIKV envelope glycoprotein. (A) Varying dilutions of clarified protein extract containing ZsE was directly bound to a polystyrene plate and probed with purified c2A10G6 to assess whether the antibody recognized the fusion loop epitope. Bound antibody was detected with goat anti-human <t>IgG</t> (kappa only) <t>HRP</t> conjugate. An uninfiltrated negative control was included to assess the level of any non-specific binding to native plant proteins. (B) The ability of c2A10G6 to recognize the fusion loop epitope was analyzed via a western blot. Clarified protein extracts containing ZprME or an uninfiltrated control were probed with purified c2A10G6 and detected with HRP-conjugated goat anti-human IgG antibody.
    Hrp Conjugated Goat Anti Human Immunoglobulin G, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology hrp conjugated goat anti human igg
    Screening of PCa patient sera for autoantibodies to candidate tumor associated proteins by Western blotting. Each number corresponds to a different patient serum probed against whole PC3 cell lysates in PVDF membrane strips. Serum antibody reactivity against individual proteins for each patient was detected using <t>HRP-labeled</t> anti-human <t>IgG/IgM/IgA</t> secondary antibody and enhanced chemiluminescence (ECL) on film. A , One-minute exposure of membrane strips to film using sera from AA men with PCa. B , One-minute exposure of membrane strips to film using sera from EA men with PCa. C , Exposure increased to 3 mins showing overexposed AA serum immunoreactivity. D , Exposure increased to 3 mins to visualize EA serum immunoreactivity. E , Western blot zoomed in to highlight common 50 kDa immunoreactivity of several AA-PCa sera. All membrane strips were simultaneously probed with sera and exposed to ECL under identical conditions in the same experiment. *AA-PCa sera with 50 kDa immunoreactivity were identified to have anti-ENO1 antibodies using SERPA.
    Hrp Conjugated Goat Anti Human Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 269 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Bio-Rad hrp conjugated goat anti human igg
    B6N region is sufficient for binding to hSiglec-5. A , Western blot experiment analyzing the ability of the recombinant B6N and IgABR fragments (single copy and tandem, respectively) to bind hSiglec-5. The B6N and IgABR constructs were separated by SDS-PAGE together with full-length protein β (positive control) and protein α (negative control). After blotting and blocking, the membranes were incubated with 5 μg/ml hSiglec-5 followed by <t>HRP-conjugated</t> anti-human <t>IgG</t> or with 5 μg/ml IgA followed by a rabbit anti-human IgA and HRP-conjugated anti-rabbit IgG. B , analysis of the interaction of hSiglec-5 with pure proteins; the recombinant proteins and protein β were coated on microtiter plates at increasing concentrations and incubated with hSiglec-5, which was detected with an HRP-conjugated anti-human IgG. The plates were developed and read at 450 nm. The mean values of three separate experiments are shown, and error bars correspond to S.D.
    Hrp Conjugated Goat Anti Human Igg, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam hrp conjugated goat anti human igg
    Antigenic specificity analysis of recombinant SARS-N protein against human serum. (A) Purified BrSARS-N and ErSARS-N (200 ng/well) were coated onto the each microtiter plates, respectively. SARS positive (upper panel) and negative sera (lower panel) were incubated in each coated well, followed by <t>HRP-conjugated</t> anti-human <t>IgG,</t> and detection using TMB substrate solution, which indicated that all five SARS negative sera were highly cross-responsive with ErSARS-N protein, but not with BrSARS-N. (B) Purified BrSARS-N and ErSARS-N proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane separately. The membranes were cut into strips and incubated with five SARS positive sera and five SARS negative sera, followed by HRP-conjugated anti-human IgG, and proteins were detected using DAB staining. All five SARS negative sera showed strong positive bands with ErSARS-N protein, but not with BrSARS-N, suggesting that BrSARS-N protein is more specific than ErSARS-N protein.
    Hrp Conjugated Goat Anti Human Igg, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated goat anti human igg/product/Abcam
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    94
    SouthernBiotech hrp conjugated goat anti mouse igg2a
    Antigenic specificity analysis of recombinant SARS-N protein against human serum. (A) Purified BrSARS-N and ErSARS-N (200 ng/well) were coated onto the each microtiter plates, respectively. SARS positive (upper panel) and negative sera (lower panel) were incubated in each coated well, followed by <t>HRP-conjugated</t> anti-human <t>IgG,</t> and detection using TMB substrate solution, which indicated that all five SARS negative sera were highly cross-responsive with ErSARS-N protein, but not with BrSARS-N. (B) Purified BrSARS-N and ErSARS-N proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane separately. The membranes were cut into strips and incubated with five SARS positive sera and five SARS negative sera, followed by HRP-conjugated anti-human IgG, and proteins were detected using DAB staining. All five SARS negative sera showed strong positive bands with ErSARS-N protein, but not with BrSARS-N, suggesting that BrSARS-N protein is more specific than ErSARS-N protein.
    Hrp Conjugated Goat Anti Mouse Igg2a, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 94/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated goat anti mouse igg2a/product/SouthernBiotech
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    99
    Bethyl hrp conjugated goat anti human igg fc antibody
    Antigenic specificity analysis of recombinant SARS-N protein against human serum. (A) Purified BrSARS-N and ErSARS-N (200 ng/well) were coated onto the each microtiter plates, respectively. SARS positive (upper panel) and negative sera (lower panel) were incubated in each coated well, followed by <t>HRP-conjugated</t> anti-human <t>IgG,</t> and detection using TMB substrate solution, which indicated that all five SARS negative sera were highly cross-responsive with ErSARS-N protein, but not with BrSARS-N. (B) Purified BrSARS-N and ErSARS-N proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane separately. The membranes were cut into strips and incubated with five SARS positive sera and five SARS negative sera, followed by HRP-conjugated anti-human IgG, and proteins were detected using DAB staining. All five SARS negative sera showed strong positive bands with ErSARS-N protein, but not with BrSARS-N, suggesting that BrSARS-N protein is more specific than ErSARS-N protein.
    Hrp Conjugated Goat Anti Human Igg Fc Antibody, supplied by Bethyl, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated goat anti human igg fc antibody/product/Bethyl
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    95
    Millipore anti human igg gamma chain specific peroxidase antibody
    Antigenic specificity analysis of recombinant SARS-N protein against human serum. (A) Purified BrSARS-N and ErSARS-N (200 ng/well) were coated onto the each microtiter plates, respectively. SARS positive (upper panel) and negative sera (lower panel) were incubated in each coated well, followed by <t>HRP-conjugated</t> anti-human <t>IgG,</t> and detection using TMB substrate solution, which indicated that all five SARS negative sera were highly cross-responsive with ErSARS-N protein, but not with BrSARS-N. (B) Purified BrSARS-N and ErSARS-N proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane separately. The membranes were cut into strips and incubated with five SARS positive sera and five SARS negative sera, followed by HRP-conjugated anti-human IgG, and proteins were detected using DAB staining. All five SARS negative sera showed strong positive bands with ErSARS-N protein, but not with BrSARS-N, suggesting that BrSARS-N protein is more specific than ErSARS-N protein.
    Anti Human Igg Gamma Chain Specific Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam hrp conjugated anti human igg
    Antigenic specificity analysis of recombinant SARS-N protein against human serum. (A) Purified BrSARS-N and ErSARS-N (200 ng/well) were coated onto the each microtiter plates, respectively. SARS positive (upper panel) and negative sera (lower panel) were incubated in each coated well, followed by <t>HRP-conjugated</t> anti-human <t>IgG,</t> and detection using TMB substrate solution, which indicated that all five SARS negative sera were highly cross-responsive with ErSARS-N protein, but not with BrSARS-N. (B) Purified BrSARS-N and ErSARS-N proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane separately. The membranes were cut into strips and incubated with five SARS positive sera and five SARS negative sera, followed by HRP-conjugated anti-human IgG, and proteins were detected using DAB staining. All five SARS negative sera showed strong positive bands with ErSARS-N protein, but not with BrSARS-N, suggesting that BrSARS-N protein is more specific than ErSARS-N protein.
    Hrp Conjugated Anti Human Igg, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated anti human igg/product/Abcam
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    Image Search Results


    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

    Murine and rat IGSF1-2 proteins do not traffic to the plasma membrane of transfected cells. A) HEK293 cells were transfected with expression plasmids for wild-type murine (M.) or rat (R.) IGSF1-2, murine BMP type IA receptor (BMPR1A), or empty vector (pcDNA4). Note, IGSF1-2 proteins were expressed with Myc/His tags at their C-termini, whereas BMPR1A had the Myc tag alone. Cell surface proteins were labeled with biotin prior to collection of whole cell lysates. Lysates were immunoprecipitated (IP) with Myc-beads and then subjected to SDS-PAGE and transferred to nitrocellulose membranes. Total proteins were immunoblotted with anti-Myc (bottom), whereas biotinylated proteins were identified with streptavidin-HRP (top). B) HEK293 cells were cultured on coverslips and transiently transfected with wild-type murine IGSF1-2-Myc/His. Cells were then fixed and subjected to immunofluorescence with a Myc antibody (green) under non-permeabilizing (top) and permeabilizing conditions (bottom). C) Cells were transfected as in panel B, premeabilized, and processed for double-label immunofluorescence with the Myc antibody (green) and an antibody against GRP-78/BiP (red). The overlay is shown in yellow. In B and C, nuclei were stained with DAPI. Images were captured by confocal microscopy. Scale bar, 10 μm.

    Journal: PLoS ONE

    Article Title: The short mRNA isoform of the immunoglobulin superfamily, member 1 gene encodes an intracellular glycoprotein

    doi: 10.1371/journal.pone.0180731

    Figure Lengend Snippet: Murine and rat IGSF1-2 proteins do not traffic to the plasma membrane of transfected cells. A) HEK293 cells were transfected with expression plasmids for wild-type murine (M.) or rat (R.) IGSF1-2, murine BMP type IA receptor (BMPR1A), or empty vector (pcDNA4). Note, IGSF1-2 proteins were expressed with Myc/His tags at their C-termini, whereas BMPR1A had the Myc tag alone. Cell surface proteins were labeled with biotin prior to collection of whole cell lysates. Lysates were immunoprecipitated (IP) with Myc-beads and then subjected to SDS-PAGE and transferred to nitrocellulose membranes. Total proteins were immunoblotted with anti-Myc (bottom), whereas biotinylated proteins were identified with streptavidin-HRP (top). B) HEK293 cells were cultured on coverslips and transiently transfected with wild-type murine IGSF1-2-Myc/His. Cells were then fixed and subjected to immunofluorescence with a Myc antibody (green) under non-permeabilizing (top) and permeabilizing conditions (bottom). C) Cells were transfected as in panel B, premeabilized, and processed for double-label immunofluorescence with the Myc antibody (green) and an antibody against GRP-78/BiP (red). The overlay is shown in yellow. In B and C, nuclei were stained with DAPI. Images were captured by confocal microscopy. Scale bar, 10 μm.

    Article Snippet: The following antibodies were used at the indicated concentrations: monoclonal anti-mouse c-Myc (1:500, M5546, Sigma-Aldrich, St. Louis, MO), polyclonal rabbit anti-mouse IGSF1 (1:1000; previously described [ ]), and HRP-conjugated goat anti-mouse (1:5000, BioRad, Mississauga, Ontario).

    Techniques: Transfection, Expressing, IA, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Cell Culture, Immunofluorescence, Staining, Confocal Microscopy

    Representative example of OFA immune response in vivo . The lanes contain protein extracts (40 μg per lane) from OFA-expressing 4T1 cells, incubated with sera from mice orally immunized with wild-type (wt) L. plantarum or L. plantarum harboring pLp_0373sOFAcwa2 (cwa2), or from mice subcutaneously immunized with His-tagged OFA in Freund's complete adjuvant (OFA-His). Anti-OFA monoclonal mouse IgG antibody (mAb) was used as a positive control. Antibody binding was visualized using HRP conjugated to anti-mouse IgG and the ECL system (Amersham Life Science, Buckinghamshire, United Kingdom). The arrow indicates OFA.

    Journal: Applied and Environmental Microbiology

    Article Title: Cell Wall Anchoring of the 37-Kilodalton Oncofetal Antigen by Lactobacillus plantarum for Mucosal Cancer Vaccine Delivery ▿

    doi: 10.1128/AEM.01031-10

    Figure Lengend Snippet: Representative example of OFA immune response in vivo . The lanes contain protein extracts (40 μg per lane) from OFA-expressing 4T1 cells, incubated with sera from mice orally immunized with wild-type (wt) L. plantarum or L. plantarum harboring pLp_0373sOFAcwa2 (cwa2), or from mice subcutaneously immunized with His-tagged OFA in Freund's complete adjuvant (OFA-His). Anti-OFA monoclonal mouse IgG antibody (mAb) was used as a positive control. Antibody binding was visualized using HRP conjugated to anti-mouse IgG and the ECL system (Amersham Life Science, Buckinghamshire, United Kingdom). The arrow indicates OFA.

    Article Snippet: System (Millipore, Billerica, MA), using anti-laminin R polyclonal antibodies (Santa Cruz Biotechnology, Inc., CA) or OFA monoclonal antibodies combined with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Bio-Rad) (Fig. A and B) or rabbit anti-mouse antibody (Dako, Denmark) (Fig. ), respectively.

    Techniques: In Vivo, Expressing, Incubation, Mouse Assay, Plasmid Purification, Positive Control, Binding Assay

    Sero-positivity of anti-E2EP3 IgG3 antibodies in CHIKV-infected patients from other cohorts CHIK virion-based ELISA was used to assess anti-CHIKV IgG titer in CHIKV-infected patients from another Singaporean cohort collected at median 10 days pio ( n = 36). Healthy donors' plasma ( n = 11) were used as controls. Individual samples were subjected to virion-based ELISA at a dilution of 1:2000, followed by secondary human anti-IgG-HRP. *** p

    Journal: EMBO Molecular Medicine

    Article Title: Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein

    doi: 10.1002/emmm.201200213

    Figure Lengend Snippet: Sero-positivity of anti-E2EP3 IgG3 antibodies in CHIKV-infected patients from other cohorts CHIK virion-based ELISA was used to assess anti-CHIKV IgG titer in CHIKV-infected patients from another Singaporean cohort collected at median 10 days pio ( n = 36). Healthy donors' plasma ( n = 11) were used as controls. Individual samples were subjected to virion-based ELISA at a dilution of 1:2000, followed by secondary human anti-IgG-HRP. *** p

    Article Snippet: HRP-conjugated goat anti-human IgG (Molecular Probes), mouse anti-human IgG3 (Molecular Probes), and rabbit anti-monkey IgG (Alpha Diagnostic) were used to detect human and NHP antibodies, respectively.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay

    Antigenic recognition by CHIKV-infected patients' samples Total cell lysates were prepared from transiently expressed capsid protein (Capsid plasmid), E2 glycoprotein (E2 plasmid) and E1 glycoprotein (E1 plasmid). Vector transfected (Vector plasmid) cell lysates were used as negative control. Lysates and purified CHIKV virions (SGP11 virion) were subjected to SDS-PAGE gel and probed with a representative CHIKV-infected patient's plasma at a dilution of 1:2000, followed by secondary human anti-IgG-HRP. Sizes of molecular weight markers are indicated in the left part of the diagram. Band intensities corresponding to CHIKV structural proteins (Capsid, E2 and E1) were analysed by densitometry for all patient samples ( n = 30). Results are expressed as mean-grey value (MGV) ± SD. Data are representative of 2 independent experiments with similar results. *** p

    Journal: EMBO Molecular Medicine

    Article Title: Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein

    doi: 10.1002/emmm.201200213

    Figure Lengend Snippet: Antigenic recognition by CHIKV-infected patients' samples Total cell lysates were prepared from transiently expressed capsid protein (Capsid plasmid), E2 glycoprotein (E2 plasmid) and E1 glycoprotein (E1 plasmid). Vector transfected (Vector plasmid) cell lysates were used as negative control. Lysates and purified CHIKV virions (SGP11 virion) were subjected to SDS-PAGE gel and probed with a representative CHIKV-infected patient's plasma at a dilution of 1:2000, followed by secondary human anti-IgG-HRP. Sizes of molecular weight markers are indicated in the left part of the diagram. Band intensities corresponding to CHIKV structural proteins (Capsid, E2 and E1) were analysed by densitometry for all patient samples ( n = 30). Results are expressed as mean-grey value (MGV) ± SD. Data are representative of 2 independent experiments with similar results. *** p

    Article Snippet: HRP-conjugated goat anti-human IgG (Molecular Probes), mouse anti-human IgG3 (Molecular Probes), and rabbit anti-monkey IgG (Alpha Diagnostic) were used to detect human and NHP antibodies, respectively.

    Techniques: Infection, Plasmid Preparation, Transfection, Negative Control, Purification, SDS Page, Molecular Weight

    Epitope mapping of the E2 glycoprotein CHIKV-infected patient plasma pools (Median 10 days pio) were subjected to peptide-based ELISA at a dilution of 1:2000, followed by secondary human anti-IgG-HRP using pooled peptides (P1–P11). The same set of patient plasma pools were subjected to peptide-based ELISA at a dilution of 1:2000, followed by secondary human anti-IgG-HRP using both selected peptide pools (P1, 2, 10 and 11) and individual peptides. Selected individual peptides were re-screened with patients' plasma pools at a dilution of 1:200, followed by secondary human anti-IgG3-HRP. Black solid line represents the mean value of the healthy donors and dotted line represents the value of mean ± 6 SD. Values above mean ± 6 SD are considered positive. Results represent an average of two independent experiments.

    Journal: EMBO Molecular Medicine

    Article Title: Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein

    doi: 10.1002/emmm.201200213

    Figure Lengend Snippet: Epitope mapping of the E2 glycoprotein CHIKV-infected patient plasma pools (Median 10 days pio) were subjected to peptide-based ELISA at a dilution of 1:2000, followed by secondary human anti-IgG-HRP using pooled peptides (P1–P11). The same set of patient plasma pools were subjected to peptide-based ELISA at a dilution of 1:2000, followed by secondary human anti-IgG-HRP using both selected peptide pools (P1, 2, 10 and 11) and individual peptides. Selected individual peptides were re-screened with patients' plasma pools at a dilution of 1:200, followed by secondary human anti-IgG3-HRP. Black solid line represents the mean value of the healthy donors and dotted line represents the value of mean ± 6 SD. Values above mean ± 6 SD are considered positive. Results represent an average of two independent experiments.

    Article Snippet: HRP-conjugated goat anti-human IgG (Molecular Probes), mouse anti-human IgG3 (Molecular Probes), and rabbit anti-monkey IgG (Alpha Diagnostic) were used to detect human and NHP antibodies, respectively.

    Techniques: Infection, Peptide ELISA

    Confirmation of hIgG expression in the Tc goat with Western blotting. The purified hIgG from the Tc goat, positive control human IVIG, and negative control purified from commercial goat IgG was probed with anti-human IgG (heavy and light; H + L) HRP.

    Journal: Scientific Reports

    Article Title: Generation of H7N9-specific human polyclonal antibodies from a transchromosomic goat (caprine) system

    doi: 10.1038/s41598-018-36961-5

    Figure Lengend Snippet: Confirmation of hIgG expression in the Tc goat with Western blotting. The purified hIgG from the Tc goat, positive control human IVIG, and negative control purified from commercial goat IgG was probed with anti-human IgG (heavy and light; H + L) HRP.

    Article Snippet: Goat anti-human IgG-Fc Antibody HRP conjugated (Bethyl, Montgomery, TX, USA) was used as a detection antibody.

    Techniques: Expressing, Western Blot, Purification, Positive Control, Negative Control

    The profiles of the PV IgGs absorbed by recombinant Dsg 1 and Dsg 3 baculoproteins. Western blots of protein extracts of normal human keratinocytes resolved by 7.5% SDS-PAGE and stained with PV3 IgG affinity-purified on rDsg3-His (lane 1), and that of Dsg3 null keratinocytes stained with PV3 IgG affinity purified on rDsg3-Ig-His (lane 5), rDsg1-Ig-His (lane 4) or on the Fc IgG column (lane 9). Binding of PV3 IgGs to the immunoblotting membranes was visualized using secondary, HRP-conjugated goat anti-human IgG antibodies. Lane 6 shows a single protein band with apparent M r of 190 kDa among SDS-PAGE–resolved Dsg 3–positive keratinocyte proteins from BALB/c mouse. This band was visualized by the PV3 IgG that was eluted from the 190-kDa area of the Western blot of Dsg3 null keratinocyte proteins stained with the PV3 IgG adsorbed on rDsg3-Ig-His. Note: Only a 190-kDa but not 130- or 160-kDa band or any other keratinocyte protein was visualized, indicating that the antibody targeting the 190-kDa protein is a unique one, as it does not recognize the 130-kDa Dsg 3 or 160-kDa Dsg 1. Lanes 2, 3, and 7 are the negative controls omitting primary antibody. The Dsg3 null keratinocyte protein extract in lane 8 was blotted with normal human IgG-affinity purified on rDsg3-Ig-His. The lack of multiple bands in lane 8 as well as complete absence of specific staining in lanes 3 and 7 indicate that there were no nonspecific cross reactivities of human or goat IgGs with murine epidermal proteins. The positions of relative molecular mass markers run in parallel lanes of each blot are shown to the left of the respective blot. The apparent relative molecular mass of keratinocyte protein bands visualized due to PV antibody binding is shown to the right of lanes 2 and 5 in the columns designated M r .

    Journal: Journal of Clinical Investigation

    Article Title: Antibodies against keratinocyte antigens other than desmogleins 1 and 3 can induce pemphigus vulgaris-like lesions

    doi:

    Figure Lengend Snippet: The profiles of the PV IgGs absorbed by recombinant Dsg 1 and Dsg 3 baculoproteins. Western blots of protein extracts of normal human keratinocytes resolved by 7.5% SDS-PAGE and stained with PV3 IgG affinity-purified on rDsg3-His (lane 1), and that of Dsg3 null keratinocytes stained with PV3 IgG affinity purified on rDsg3-Ig-His (lane 5), rDsg1-Ig-His (lane 4) or on the Fc IgG column (lane 9). Binding of PV3 IgGs to the immunoblotting membranes was visualized using secondary, HRP-conjugated goat anti-human IgG antibodies. Lane 6 shows a single protein band with apparent M r of 190 kDa among SDS-PAGE–resolved Dsg 3–positive keratinocyte proteins from BALB/c mouse. This band was visualized by the PV3 IgG that was eluted from the 190-kDa area of the Western blot of Dsg3 null keratinocyte proteins stained with the PV3 IgG adsorbed on rDsg3-Ig-His. Note: Only a 190-kDa but not 130- or 160-kDa band or any other keratinocyte protein was visualized, indicating that the antibody targeting the 190-kDa protein is a unique one, as it does not recognize the 130-kDa Dsg 3 or 160-kDa Dsg 1. Lanes 2, 3, and 7 are the negative controls omitting primary antibody. The Dsg3 null keratinocyte protein extract in lane 8 was blotted with normal human IgG-affinity purified on rDsg3-Ig-His. The lack of multiple bands in lane 8 as well as complete absence of specific staining in lanes 3 and 7 indicate that there were no nonspecific cross reactivities of human or goat IgGs with murine epidermal proteins. The positions of relative molecular mass markers run in parallel lanes of each blot are shown to the left of the respective blot. The apparent relative molecular mass of keratinocyte protein bands visualized due to PV antibody binding is shown to the right of lanes 2 and 5 in the columns designated M r .

    Article Snippet: Binding of primary antibody was visualized using horseradish peroxidase (HRP) conjugated goat anti-human IgG (Pierce Chemical Co.) with an HRP color developer (Bio-Rad Laboratories Inc., Hercules, California, USA).

    Techniques: Recombinant, Western Blot, SDS Page, Staining, Affinity Purification, Binding Assay

    Binding of c2A10G6 to ZIKV envelope glycoprotein. (A) Varying dilutions of clarified protein extract containing ZsE was directly bound to a polystyrene plate and probed with purified c2A10G6 to assess whether the antibody recognized the fusion loop epitope. Bound antibody was detected with goat anti-human IgG (kappa only) HRP conjugate. An uninfiltrated negative control was included to assess the level of any non-specific binding to native plant proteins. (B) The ability of c2A10G6 to recognize the fusion loop epitope was analyzed via a western blot. Clarified protein extracts containing ZprME or an uninfiltrated control were probed with purified c2A10G6 and detected with HRP-conjugated goat anti-human IgG antibody.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: High Level Production of Monoclonal Antibodies Using an Optimized Plant Expression System

    doi: 10.3389/fbioe.2019.00472

    Figure Lengend Snippet: Binding of c2A10G6 to ZIKV envelope glycoprotein. (A) Varying dilutions of clarified protein extract containing ZsE was directly bound to a polystyrene plate and probed with purified c2A10G6 to assess whether the antibody recognized the fusion loop epitope. Bound antibody was detected with goat anti-human IgG (kappa only) HRP conjugate. An uninfiltrated negative control was included to assess the level of any non-specific binding to native plant proteins. (B) The ability of c2A10G6 to recognize the fusion loop epitope was analyzed via a western blot. Clarified protein extracts containing ZprME or an uninfiltrated control were probed with purified c2A10G6 and detected with HRP-conjugated goat anti-human IgG antibody.

    Article Snippet: After a 2-h room temperature incubation, the membrane was washed in PBST and probed with a 1:5,000 dilution of a HRP-conjugated goat anti-human IgG antibody (Southern Biotech, Birmingham, AL, USA).

    Techniques: Binding Assay, Purification, Negative Control, Western Blot

    Characterization of c2A10G6. (A) A N. benthamina leaf at 4 DPI was examined for signs of chlorosis or necrosis. Faint chlorosis was visible, but there was no visible necrosis. (B) To test whether the clarified leaf extracts of the antibody constructs were stable upon acid-precipitation, 1N phosphoric acid was added to a final acid volume of 4% of the total soluble extract. Following a 6-min incubation, the samples were neutralized with 1M Tris base. For comparison, a sample of the leaf extract pre-acid precipitation was also included along with a control uninfiltrated leaf extract that was not treated with acid. All three samples were mixed with non-reducing sample buffer and loaded on a 4–15% polyacrylamide gel for analysis by Coomassie-staining. (C) The same samples described in part A were run on a 4–15% polyacrylamide gel for analysis by Western blot. The Western blot was detected with HRP-conjugated goat anti-human IgG (kappa only). (D) After protein G affinity purification, samples of the purified antibody were run on SDS-PAGE gels under non-reducing or reducing conditions as noted. The left panel shows the results following a silver stain. Only the c2A10G6 band is visible. The two panels on the right show the results of the purified antibody run under non-reducing and reducing conditions on a stain-free gel. S, soluble fraction pre-acid precipitation; AP, samples subjected to acid-precipitation; U, uninfiltrated clarified leaf extract; NR, non-reducing conditions; R, reducing conditions.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: High Level Production of Monoclonal Antibodies Using an Optimized Plant Expression System

    doi: 10.3389/fbioe.2019.00472

    Figure Lengend Snippet: Characterization of c2A10G6. (A) A N. benthamina leaf at 4 DPI was examined for signs of chlorosis or necrosis. Faint chlorosis was visible, but there was no visible necrosis. (B) To test whether the clarified leaf extracts of the antibody constructs were stable upon acid-precipitation, 1N phosphoric acid was added to a final acid volume of 4% of the total soluble extract. Following a 6-min incubation, the samples were neutralized with 1M Tris base. For comparison, a sample of the leaf extract pre-acid precipitation was also included along with a control uninfiltrated leaf extract that was not treated with acid. All three samples were mixed with non-reducing sample buffer and loaded on a 4–15% polyacrylamide gel for analysis by Coomassie-staining. (C) The same samples described in part A were run on a 4–15% polyacrylamide gel for analysis by Western blot. The Western blot was detected with HRP-conjugated goat anti-human IgG (kappa only). (D) After protein G affinity purification, samples of the purified antibody were run on SDS-PAGE gels under non-reducing or reducing conditions as noted. The left panel shows the results following a silver stain. Only the c2A10G6 band is visible. The two panels on the right show the results of the purified antibody run under non-reducing and reducing conditions on a stain-free gel. S, soluble fraction pre-acid precipitation; AP, samples subjected to acid-precipitation; U, uninfiltrated clarified leaf extract; NR, non-reducing conditions; R, reducing conditions.

    Article Snippet: After a 2-h room temperature incubation, the membrane was washed in PBST and probed with a 1:5,000 dilution of a HRP-conjugated goat anti-human IgG antibody (Southern Biotech, Birmingham, AL, USA).

    Techniques: Construct, Incubation, Staining, Western Blot, Affinity Purification, Purification, SDS Page, Silver Staining

    Screening of PCa patient sera for autoantibodies to candidate tumor associated proteins by Western blotting. Each number corresponds to a different patient serum probed against whole PC3 cell lysates in PVDF membrane strips. Serum antibody reactivity against individual proteins for each patient was detected using HRP-labeled anti-human IgG/IgM/IgA secondary antibody and enhanced chemiluminescence (ECL) on film. A , One-minute exposure of membrane strips to film using sera from AA men with PCa. B , One-minute exposure of membrane strips to film using sera from EA men with PCa. C , Exposure increased to 3 mins showing overexposed AA serum immunoreactivity. D , Exposure increased to 3 mins to visualize EA serum immunoreactivity. E , Western blot zoomed in to highlight common 50 kDa immunoreactivity of several AA-PCa sera. All membrane strips were simultaneously probed with sera and exposed to ECL under identical conditions in the same experiment. *AA-PCa sera with 50 kDa immunoreactivity were identified to have anti-ENO1 antibodies using SERPA.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Immunoseroproteomic Profiling in African American Men with Prostate Cancer: Evidence for an Autoantibody Response to Glycolysis and Plasminogen-Associated Proteins *

    doi: 10.1074/mcp.M116.060244

    Figure Lengend Snippet: Screening of PCa patient sera for autoantibodies to candidate tumor associated proteins by Western blotting. Each number corresponds to a different patient serum probed against whole PC3 cell lysates in PVDF membrane strips. Serum antibody reactivity against individual proteins for each patient was detected using HRP-labeled anti-human IgG/IgM/IgA secondary antibody and enhanced chemiluminescence (ECL) on film. A , One-minute exposure of membrane strips to film using sera from AA men with PCa. B , One-minute exposure of membrane strips to film using sera from EA men with PCa. C , Exposure increased to 3 mins showing overexposed AA serum immunoreactivity. D , Exposure increased to 3 mins to visualize EA serum immunoreactivity. E , Western blot zoomed in to highlight common 50 kDa immunoreactivity of several AA-PCa sera. All membrane strips were simultaneously probed with sera and exposed to ECL under identical conditions in the same experiment. *AA-PCa sera with 50 kDa immunoreactivity were identified to have anti-ENO1 antibodies using SERPA.

    Article Snippet: After several washes with PBS containing 0.05% Tween 20 (PBS-T), HRP-conjugated goat anti-human IgG (1:4000) (Santa Cruz Biotechnology) was added to the wells and incubated for 90 min at room temperature.

    Techniques: Western Blot, Labeling

    B6N region is sufficient for binding to hSiglec-5. A , Western blot experiment analyzing the ability of the recombinant B6N and IgABR fragments (single copy and tandem, respectively) to bind hSiglec-5. The B6N and IgABR constructs were separated by SDS-PAGE together with full-length protein β (positive control) and protein α (negative control). After blotting and blocking, the membranes were incubated with 5 μg/ml hSiglec-5 followed by HRP-conjugated anti-human IgG or with 5 μg/ml IgA followed by a rabbit anti-human IgA and HRP-conjugated anti-rabbit IgG. B , analysis of the interaction of hSiglec-5 with pure proteins; the recombinant proteins and protein β were coated on microtiter plates at increasing concentrations and incubated with hSiglec-5, which was detected with an HRP-conjugated anti-human IgG. The plates were developed and read at 450 nm. The mean values of three separate experiments are shown, and error bars correspond to S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Human Siglec-5 Inhibitory Receptor and Immunoglobulin A (IgA) Have Separate Binding Sites in Streptococcal ? Protein *

    doi: 10.1074/jbc.M111.251728

    Figure Lengend Snippet: B6N region is sufficient for binding to hSiglec-5. A , Western blot experiment analyzing the ability of the recombinant B6N and IgABR fragments (single copy and tandem, respectively) to bind hSiglec-5. The B6N and IgABR constructs were separated by SDS-PAGE together with full-length protein β (positive control) and protein α (negative control). After blotting and blocking, the membranes were incubated with 5 μg/ml hSiglec-5 followed by HRP-conjugated anti-human IgG or with 5 μg/ml IgA followed by a rabbit anti-human IgA and HRP-conjugated anti-rabbit IgG. B , analysis of the interaction of hSiglec-5 with pure proteins; the recombinant proteins and protein β were coated on microtiter plates at increasing concentrations and incubated with hSiglec-5, which was detected with an HRP-conjugated anti-human IgG. The plates were developed and read at 450 nm. The mean values of three separate experiments are shown, and error bars correspond to S.D.

    Article Snippet: HRP-conjugated goat anti-human IgG was purchased from AbD Serotec (Düsseldorf, Germany).

    Techniques: Binding Assay, Western Blot, Recombinant, Construct, SDS Page, Positive Control, Negative Control, Blocking Assay, Incubation

    Protein β binds to hSiglec-5 independently of GBS strain serotype. A , Purified protein β extracted from different Ia and Ib GBS strains bound hSiglec-5, whereas no binding was detected against the control protein α. Bacteria were grown overnight, washed and the β proteins were extracted by elevating the pH in the bacterial suspensions. The β-negative mutant Δ bac was used as negative control. After four h incubation, the supernatants were separated by SDS-PAGE and blotted on a nitrocellulose membrane. The membrane was incubated with 5 μg/ml hSiglec-5 followed by a HRP-conjugated anti-human IgG. B , The hSiglec-5 binding is correlated to the level of β-expression. GBS strains of serotypes Ia, Ib, II, III, IV and V were incubated with 125 I-labeled hSiglec-5, or rabbit anti-protein β serum followed by 125 I-labeled protein G. Bound 125 I-labeled protein was then measured in a γ-counter. The mean values of three independently experiments are shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Human Siglec-5 Inhibitory Receptor and Immunoglobulin A (IgA) Have Separate Binding Sites in Streptococcal ? Protein *

    doi: 10.1074/jbc.M111.251728

    Figure Lengend Snippet: Protein β binds to hSiglec-5 independently of GBS strain serotype. A , Purified protein β extracted from different Ia and Ib GBS strains bound hSiglec-5, whereas no binding was detected against the control protein α. Bacteria were grown overnight, washed and the β proteins were extracted by elevating the pH in the bacterial suspensions. The β-negative mutant Δ bac was used as negative control. After four h incubation, the supernatants were separated by SDS-PAGE and blotted on a nitrocellulose membrane. The membrane was incubated with 5 μg/ml hSiglec-5 followed by a HRP-conjugated anti-human IgG. B , The hSiglec-5 binding is correlated to the level of β-expression. GBS strains of serotypes Ia, Ib, II, III, IV and V were incubated with 125 I-labeled hSiglec-5, or rabbit anti-protein β serum followed by 125 I-labeled protein G. Bound 125 I-labeled protein was then measured in a γ-counter. The mean values of three independently experiments are shown.

    Article Snippet: HRP-conjugated goat anti-human IgG was purchased from AbD Serotec (Düsseldorf, Germany).

    Techniques: Purification, IA, Binding Assay, Mutagenesis, BAC Assay, Negative Control, Incubation, SDS Page, Expressing, Labeling

    B6N fragment and sialic acid have overlapping binding sites on hSiglec-5. A , addition of sialic acid-containing fetuin inhibits radiolabeled hSiglec-5 binding to A909 WT, whereas asialofetuin does not. Bacteria were grown overnight and washed, and aliquots were incubated with 125 I-hSiglec-5 together with increasing concentrations of fetuin or asialofetuin. Bound 125 I-labeled protein was then measured in a γ-counter. B , protein β and B6N tandem inhibit the interaction between fetuin and soluble hSiglec-5. hSiglec-5 was preincubated with increasing concentrations of protein β, B6N tandem, or IgABR tandem and thereafter added to a microtiter plate with immobilized fetuin. Bound hSiglec-5 was detected with an HRP-conjugated anti-human IgG. Mean values of three experiments are shown, and error bars indicate S.D. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

    Journal: The Journal of Biological Chemistry

    Article Title: Human Siglec-5 Inhibitory Receptor and Immunoglobulin A (IgA) Have Separate Binding Sites in Streptococcal ? Protein *

    doi: 10.1074/jbc.M111.251728

    Figure Lengend Snippet: B6N fragment and sialic acid have overlapping binding sites on hSiglec-5. A , addition of sialic acid-containing fetuin inhibits radiolabeled hSiglec-5 binding to A909 WT, whereas asialofetuin does not. Bacteria were grown overnight and washed, and aliquots were incubated with 125 I-hSiglec-5 together with increasing concentrations of fetuin or asialofetuin. Bound 125 I-labeled protein was then measured in a γ-counter. B , protein β and B6N tandem inhibit the interaction between fetuin and soluble hSiglec-5. hSiglec-5 was preincubated with increasing concentrations of protein β, B6N tandem, or IgABR tandem and thereafter added to a microtiter plate with immobilized fetuin. Bound hSiglec-5 was detected with an HRP-conjugated anti-human IgG. Mean values of three experiments are shown, and error bars indicate S.D. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

    Article Snippet: HRP-conjugated goat anti-human IgG was purchased from AbD Serotec (Düsseldorf, Germany).

    Techniques: Binding Assay, Incubation, Labeling

    Antigenic specificity analysis of recombinant SARS-N protein against human serum. (A) Purified BrSARS-N and ErSARS-N (200 ng/well) were coated onto the each microtiter plates, respectively. SARS positive (upper panel) and negative sera (lower panel) were incubated in each coated well, followed by HRP-conjugated anti-human IgG, and detection using TMB substrate solution, which indicated that all five SARS negative sera were highly cross-responsive with ErSARS-N protein, but not with BrSARS-N. (B) Purified BrSARS-N and ErSARS-N proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane separately. The membranes were cut into strips and incubated with five SARS positive sera and five SARS negative sera, followed by HRP-conjugated anti-human IgG, and proteins were detected using DAB staining. All five SARS negative sera showed strong positive bands with ErSARS-N protein, but not with BrSARS-N, suggesting that BrSARS-N protein is more specific than ErSARS-N protein.

    Journal: Virus Research

    Article Title: Antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: The effect of phosphorylation on immunoreactivity and specificity

    doi: 10.1016/j.virusres.2007.03.019

    Figure Lengend Snippet: Antigenic specificity analysis of recombinant SARS-N protein against human serum. (A) Purified BrSARS-N and ErSARS-N (200 ng/well) were coated onto the each microtiter plates, respectively. SARS positive (upper panel) and negative sera (lower panel) were incubated in each coated well, followed by HRP-conjugated anti-human IgG, and detection using TMB substrate solution, which indicated that all five SARS negative sera were highly cross-responsive with ErSARS-N protein, but not with BrSARS-N. (B) Purified BrSARS-N and ErSARS-N proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane separately. The membranes were cut into strips and incubated with five SARS positive sera and five SARS negative sera, followed by HRP-conjugated anti-human IgG, and proteins were detected using DAB staining. All five SARS negative sera showed strong positive bands with ErSARS-N protein, but not with BrSARS-N, suggesting that BrSARS-N protein is more specific than ErSARS-N protein.

    Article Snippet: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-human IgG were purchased from Abcam (Cambridgeshire, UK).

    Techniques: Recombinant, Purification, Incubation, SDS Page, Staining

    Phosphorylation of SARS-N protein is important for the preservation of specificity and immunoreactivity. (A) Specificity analysis of BrSARS-N proteins treated with or without PP1. The purified BrSARS-N protein treated with or without PP1 were separated by SDS-PAGE (upper panel) and transferred to a nitrocellulose membrane. The membrane was incubated with SARS-N mAbs (middle panel) or SARS negative serum (lower panel), followed by HRP-conjugated anti-mouse or human IgG, and proteins were detected by DAB staining. Data shown are representative of five SARS negative sera with similar results, suggesting that both dephosphorylated and nonphosphorylated proteins are cross-reacted with SARS negative serum. (B) Immunoreactivity analysis of BrSARS-N protein treated with or without PP1. Aliquots of the protein samples treated with or without PP1 used in specificity analysis were coated onto the microtiter plates and incubated with SARS-N mAb, followed by HRP-conjugated anti-mouse IgG, and detection was performed using TMB substrate solution. Finding indicated that dephosphorylation considerably reduced the immunoreactivity of SARS-N protein.

    Journal: Virus Research

    Article Title: Antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: The effect of phosphorylation on immunoreactivity and specificity

    doi: 10.1016/j.virusres.2007.03.019

    Figure Lengend Snippet: Phosphorylation of SARS-N protein is important for the preservation of specificity and immunoreactivity. (A) Specificity analysis of BrSARS-N proteins treated with or without PP1. The purified BrSARS-N protein treated with or without PP1 were separated by SDS-PAGE (upper panel) and transferred to a nitrocellulose membrane. The membrane was incubated with SARS-N mAbs (middle panel) or SARS negative serum (lower panel), followed by HRP-conjugated anti-mouse or human IgG, and proteins were detected by DAB staining. Data shown are representative of five SARS negative sera with similar results, suggesting that both dephosphorylated and nonphosphorylated proteins are cross-reacted with SARS negative serum. (B) Immunoreactivity analysis of BrSARS-N protein treated with or without PP1. Aliquots of the protein samples treated with or without PP1 used in specificity analysis were coated onto the microtiter plates and incubated with SARS-N mAb, followed by HRP-conjugated anti-mouse IgG, and detection was performed using TMB substrate solution. Finding indicated that dephosphorylation considerably reduced the immunoreactivity of SARS-N protein.

    Article Snippet: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-human IgG were purchased from Abcam (Cambridgeshire, UK).

    Techniques: Preserving, Purification, SDS Page, Incubation, Staining, De-Phosphorylation Assay

    Phosphorylation of SARS-N protein expressed in insect cells. (A) Purified BrSARS-N and ErSARS-N proteins were separated by SDS-PAGE. The proteins were visualized by Coomassie brilliant blue staining (upper panel) or the phosphorylated N proteins were detected using a GelCode phosphoprotein kit as described in Materials and Methods (lower panel). (B) The gel transferred to a nitrocellulose membrane and analyzed by Western blot using anti-phosphoserine antibody (upper panel) or SARS-N mAbs (lower panel), followed by HRP-conjugated anti-mouse IgG, and proteins were detected using chemiluminescence staining. This result indicated that BrSARS-N protein was highly phosphorylated, but ErSARS-N was not.

    Journal: Virus Research

    Article Title: Antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: The effect of phosphorylation on immunoreactivity and specificity

    doi: 10.1016/j.virusres.2007.03.019

    Figure Lengend Snippet: Phosphorylation of SARS-N protein expressed in insect cells. (A) Purified BrSARS-N and ErSARS-N proteins were separated by SDS-PAGE. The proteins were visualized by Coomassie brilliant blue staining (upper panel) or the phosphorylated N proteins were detected using a GelCode phosphoprotein kit as described in Materials and Methods (lower panel). (B) The gel transferred to a nitrocellulose membrane and analyzed by Western blot using anti-phosphoserine antibody (upper panel) or SARS-N mAbs (lower panel), followed by HRP-conjugated anti-mouse IgG, and proteins were detected using chemiluminescence staining. This result indicated that BrSARS-N protein was highly phosphorylated, but ErSARS-N was not.

    Article Snippet: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-human IgG were purchased from Abcam (Cambridgeshire, UK).

    Techniques: Purification, SDS Page, Staining, Western Blot

    Immunoreactivity analysis of recombinant SARS-N protein using mAbs. (A) Immunoreactivity analysis of BrSARS-N and ErSARS-N protein by Western blot under denaturing conditions. The serial diluents of both recombinant proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with SARS-N mAbs, followed by HRP-conjugated anti-mouse IgG, and proteins were detected using chemiluminescence staining. Findings suggested that BrSARS-N is more highly antigenic than ErSARS-N under denaturing conditions. (B) Immunoreactivity analysis of BrSARS-N and ErSARS-N protein by indirect ELISA under nondenaturing conditions. SARS-N mAb was incubated in each coated well with the rSARS-N proteins, followed by HRP-conjugated anti-mouse IgG, and proteins were detected using TMB substrate solution, which indicated that BrSARS-N protein has also higher antigenicity than ErSARS-N protein under nondenaturing conditions.

    Journal: Virus Research

    Article Title: Antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: The effect of phosphorylation on immunoreactivity and specificity

    doi: 10.1016/j.virusres.2007.03.019

    Figure Lengend Snippet: Immunoreactivity analysis of recombinant SARS-N protein using mAbs. (A) Immunoreactivity analysis of BrSARS-N and ErSARS-N protein by Western blot under denaturing conditions. The serial diluents of both recombinant proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with SARS-N mAbs, followed by HRP-conjugated anti-mouse IgG, and proteins were detected using chemiluminescence staining. Findings suggested that BrSARS-N is more highly antigenic than ErSARS-N under denaturing conditions. (B) Immunoreactivity analysis of BrSARS-N and ErSARS-N protein by indirect ELISA under nondenaturing conditions. SARS-N mAb was incubated in each coated well with the rSARS-N proteins, followed by HRP-conjugated anti-mouse IgG, and proteins were detected using TMB substrate solution, which indicated that BrSARS-N protein has also higher antigenicity than ErSARS-N protein under nondenaturing conditions.

    Article Snippet: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-human IgG were purchased from Abcam (Cambridgeshire, UK).

    Techniques: Recombinant, Western Blot, SDS Page, Incubation, Staining, Indirect ELISA

    Expression of SARS-N protein in insect cells using recombinant baculovirus. (A) Full-length SARS-N protein produced in Sf21 suspension cultures. Uninfected (lane 1) and infected cells with a recombinant baculovirus expressing the full-length SARS-N gene of the SARS-CoV Urbani strain (lane 2) were collected at 48 h post-infection. Cell lysates were analyzed in a 10% SDS-PAGE gel and revealed with Coomassie brilliant blue staining, revealing a major 46 and 48 kDa band expressed in insect cell-infected recombinant baculovirus. (B) Western blot analysis of purified SARS-N protein. The purified recombinant proteins were separated by SDS-PAGE (lane 1) and transferred to a nitrocellulose membrane (lane 2). The membrane was incubated with SARS-N mAbs, followed by HRP-conjugated anti-mouse IgG, and detected by chemiluminescence staining. A strong immunoreactive band and a minor band were detected at 48 and 46 kDa, respectively, suggesting that those proteins are SARS-N proteins. (C) The comparison of mass differences among BrSARS-N, ErSARS-N, and MrSARS-N protein by SDS-PAGE analysis. This result revealed that BrSARS-N was showed to a similar mass pattern with MrSARS-N protein, but molecular weight of ErSARS-N protein was slightly lower than BrSARS-N and MrSARS-N.

    Journal: Virus Research

    Article Title: Antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: The effect of phosphorylation on immunoreactivity and specificity

    doi: 10.1016/j.virusres.2007.03.019

    Figure Lengend Snippet: Expression of SARS-N protein in insect cells using recombinant baculovirus. (A) Full-length SARS-N protein produced in Sf21 suspension cultures. Uninfected (lane 1) and infected cells with a recombinant baculovirus expressing the full-length SARS-N gene of the SARS-CoV Urbani strain (lane 2) were collected at 48 h post-infection. Cell lysates were analyzed in a 10% SDS-PAGE gel and revealed with Coomassie brilliant blue staining, revealing a major 46 and 48 kDa band expressed in insect cell-infected recombinant baculovirus. (B) Western blot analysis of purified SARS-N protein. The purified recombinant proteins were separated by SDS-PAGE (lane 1) and transferred to a nitrocellulose membrane (lane 2). The membrane was incubated with SARS-N mAbs, followed by HRP-conjugated anti-mouse IgG, and detected by chemiluminescence staining. A strong immunoreactive band and a minor band were detected at 48 and 46 kDa, respectively, suggesting that those proteins are SARS-N proteins. (C) The comparison of mass differences among BrSARS-N, ErSARS-N, and MrSARS-N protein by SDS-PAGE analysis. This result revealed that BrSARS-N was showed to a similar mass pattern with MrSARS-N protein, but molecular weight of ErSARS-N protein was slightly lower than BrSARS-N and MrSARS-N.

    Article Snippet: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-human IgG were purchased from Abcam (Cambridgeshire, UK).

    Techniques: Expressing, Recombinant, Produced, Infection, SDS Page, Staining, Western Blot, Purification, Incubation, Molecular Weight