iga concentrations Search Results


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  • 94
    Millipore iga
    Second-site immunization in the presence of PRO 2000 increases systemic and mucosal antibody responses. BALB/c mice (n = 7) were primed intranasally with 10 µg gp140 per animal in polycation and were boosted three times vaginally with gp140 +/− PRO 2000. Samples were collected 14 days after the final vaginal immunization. Serum <t>IgG</t> endpoint titres ( A ), or vaginal lavage IgG ( B ) and <t>IgA</t> ( C ) endpoint titres normalized to the total IgG or IgA content or each sample, respectively, are displayed. gp140 + PRO , combination of gp140 and PRO 2000.* p
    Iga, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2032 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Salimetrics LLC immunoglobulin a iga concentrations
    Second-site immunization in the presence of PRO 2000 increases systemic and mucosal antibody responses. BALB/c mice (n = 7) were primed intranasally with 10 µg gp140 per animal in polycation and were boosted three times vaginally with gp140 +/− PRO 2000. Samples were collected 14 days after the final vaginal immunization. Serum <t>IgG</t> endpoint titres ( A ), or vaginal lavage IgG ( B ) and <t>IgA</t> ( C ) endpoint titres normalized to the total IgG or IgA content or each sample, respectively, are displayed. gp140 + PRO , combination of gp140 and PRO 2000.* p
    Immunoglobulin A Iga Concentrations, supplied by Salimetrics LLC, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    EUROIMMUN vca iga concentration
    Second-site immunization in the presence of PRO 2000 increases systemic and mucosal antibody responses. BALB/c mice (n = 7) were primed intranasally with 10 µg gp140 per animal in polycation and were boosted three times vaginally with gp140 +/− PRO 2000. Samples were collected 14 days after the final vaginal immunization. Serum <t>IgG</t> endpoint titres ( A ), or vaginal lavage IgG ( B ) and <t>IgA</t> ( C ) endpoint titres normalized to the total IgG or IgA content or each sample, respectively, are displayed. gp140 + PRO , combination of gp140 and PRO 2000.* p
    Vca Iga Concentration, supplied by EUROIMMUN, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson biotinylated mouse anti human iga1 iga2
    Modulatory effect of various human plasma-derived <t>IgA/SIgA</t> preparations on Caco-2 cells infected by S. flexneri . A , internalization of S. flexneri alone or associated with SIgAC5 monoclonal Ab and with plasma-derived pIgA, reconstituted SIgA, mIgA, or IgG to polarized Caco-2 cell monolayers, as determined after O/N incubation, with addition of gentamicin for the last 30 min. Data are expressed on a “per filter” basis and correspond to a pool of two independent experiments ( n = 4 or 5) for each tested condition. B , basolateral secretion of TNF-α, CXCL8, and CCL3 by polarized Caco-2 cell monolayers after O/N incubation with S. flexneri alone or associated to plasma-derived pIgA, SIgA, mIgA, IgG, or anti- Shigella LPS-specific SIgAC5 monoclonal Ab. The concentration of proinflammatory mediators was determined by ELISA. Non-infected Caco-2 cell monolayers ( No bacteria ) served as a control. Data are the pool of two experiments performed in triplicates ( n = 6). When obtained, significant statistical differences were calculated by comparison with the condition Sf. **, p
    Biotinylated Mouse Anti Human Iga1 Iga2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    IGAN Bio immunoglobulin a iga nephropathy
    A. Saturation isotherm and Scatchard analysis of 125 I-UG binding to Fn . Binding isotherms of hUG to Fn were performed by incubating increasing concentrations of 125 I-UG. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of hUG, from the total binding (■). The data shown are the mean of triplicate measurements±SD. B. Scatchard plots of hUG binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 15 nM ± 2.6 nM (n = 3). C. Binding isotherms of <t>IgA</t> to Fn. Binding isotherms of IgA to Fn were performed by incubating increasing concentration of 125 I-IgA. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of IgA, from the total binding (■). The data shown are the mean of triplicate measurements. D. Scatchard plots of IgA binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 65 nM ± 5.2 nM (n = 3).
    Immunoglobulin A Iga Nephropathy, supplied by IGAN Bio, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    ALPCO immunoglobulin a sciga
    Normalized matricellular proteins and risk of developing BPD or death. Normalized SPARC levels were significantly higher in babies developing BPD or death prior to 36 weeks postmenstrual age (*p = 0.0185, Mann Whitney U test). There were no other significant differences in normalized matricellular protein concentration between infants who did not develop BPD and infants who developed BPD or died prior to 36 weeks postmenstrual age. Tracheal aspirate matricellular protein and secretory component of <t>IgA</t> <t>(scIgA)</t> concentrations were measured by ELISA or multiplex immune assay. The data show individual matricellular protein levels normalized to scIgA concentration. Medians and interquartile ranges are shown.
    Immunoglobulin A Sciga, supplied by ALPCO, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Kent Laboratories human myeloma serum immunoglobulin a iga
    The rate of G. vaginalis sialic acid (Neu5Ac) consumption from sialoglycans is substrate-dependent and inhibited by N -glycolylneuraminic acid (Neu5Gc). Washed G. vaginalis JCP8151B was incubated with 0.2 mg/ml of human <t>IgA</t> or 0.3 mg/ml of BSM, followed
    Human Myeloma Serum Immunoglobulin A Iga, supplied by Kent Laboratories, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Nordic-Mubio iga1
    Arithmetic means (± standard deviations) of the percentages of <t>IgA1-</t> and IgA2-secreting cells in peripheral blood expressing various lymphocyte markers. The numbers of unimmunized healthy volunteers from whom the data were pooled are given under the bars. Statistically significant differences between IgA1 and IgA2 ISC in paired Student's t test are indicated by an asterisk (*, 0.01
    Iga1, supplied by Nordic-Mubio, used in various techniques. Bioz Stars score: 91/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    SouthernBiotech iga2
    <t>IgA</t> + CCR10 + cells are present in tonsil germinal centers (A) Fixed frozen tonsil sections were stained with DAPI (Blue), anti-IgA (Green) and anti-CCR10 (Red). The three-color overview picture (10×) shows the presence of IgA + CCR10 + cells in the germinal center (GC). The rectangle indicates the magnified area (40×). (B) Fixed frozen tonsil sections were stained with anti-IgM (Blue), anti-IgA (Green) and anti-CCR10 (Red) showing the presence of IgA + CCR10 + cells in the subepithelial area (Ep) as well as in the GC (10×). (C) Adjacent tonsil sections stained with control antibodies.
    Iga2, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 90/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Bethyl immunoglobulin a iga elisa kits
    The mean (±SEM) concentrations (mg/mL) of serum IgG, IgM, <t>IgA</t> and sIgA in piglets between maternal COS supplementation and control groups at weaning (postnatal d 19). ∗, P
    Immunoglobulin A Iga Elisa Kits, supplied by Bethyl, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam natural human iga2 protein
    TPLTA N 205 ITK (H5N5S1F1) and (H5N4S2F1) in normal control-HBV-LC-HCC cascade. ( A and B ) Crude serum of 10 independent HCC, 10 independent LC patients, 10 HBV patients, and 10 normal subjects were enrolled using MRM and profound increases of the two glycopeptides were found from HBV infection. ( C and D ) Purified <t>IgA</t> of 10 independent HCC, 10 independent LC patients, 10 HBV patients, and 10 normal subjects were enrolled using MRM, and similar trend was found. * p
    Natural Human Iga2 Protein, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Athens Research iga2
    Env antigen-specific <t>IgA</t> and IgG responses in milk and plasma seldom correlate with other Env antigen-specific responses in HIV-infected lactating women. To determine whether immune responses to the various HIV-1 antigens were associated with each other, correlations between binding scores for selected antigen specificities were tested for plasma IgA (A), plasma IgG (B), breast milk (BM) IgA (C), and breast milk IgG (D). Kendall’s tau values and corresponding corrected P values (see Materials and Methods) are reported. Boldface P values indicate significant correlations at a P value of
    Iga2, supplied by Athens Research, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Beckman Coulter immunoglobulin a reagent kit
    Significant increase of galectin-8N non-binding <t>IgA</t> in sera from IgAN patients compared to healthy controls. a Total IgA (mean/median for each group (mg/ml sera): 3.4/3.3, 1,7/2.1, 2.3/2.4), b galectin-8N unbound (total bound) IgA (mean/median for each group (mg/ml sera): 2.6/2.4, 1.5/1.0, 1.6/1.7), and c ratio of galectin-8N bound/unbound serum IgA (logarithmic scale on the Y -axis) in sera from 98 IgAN patients, 17 healthy, and 76 kidney disease controls. d Scatter plot of ratio from c ( Y -axis) vs. total IgA ( X -axis). IgA in unfractionated serum (total) or galectin-8N bound fractions was quantitated using nephelometry. Horizontal lines mark the average for each group in a – c . The difference between groups was statistically significant ( p
    Immunoglobulin A Reagent Kit, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    IGAN Bio immunoglobulin a
    A. Saturation isotherm and Scatchard analysis of 125 I-UG binding to Fn . Binding isotherms of hUG to Fn were performed by incubating increasing concentrations of 125 I-UG. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of hUG, from the total binding (■). The data shown are the mean of triplicate measurements±SD. B. Scatchard plots of hUG binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 15 nM ± 2.6 nM (n = 3). C. Binding isotherms of <t>IgA</t> to Fn. Binding isotherms of IgA to Fn were performed by incubating increasing concentration of 125 I-IgA. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of IgA, from the total binding (■). The data shown are the mean of triplicate measurements. D. Scatchard plots of IgA binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 65 nM ± 5.2 nM (n = 3).
    Immunoglobulin A, supplied by IGAN Bio, used in various techniques. Bioz Stars score: 90/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore human iga
    Fluorescence flow cytometric analysis of the expression of sIgM, sIgG, sIgA, and sIgE by CL-01 cells and analysis of secreted IgM, <t>IgG,</t> <t>IgA,</t> and IgE. Dot plots depict the expression of sIgM, sIgD, sIgG, and sIgA by CL-01 cells (day 0) and by CL-01 cells cocultured with CD40L-293 cells and IL-4 (100 U/ml) for 5 or 8 days. Numbers inside quadrants are percentages of the total cells. Results are representative of three independent experiments. Histograms show the concentration of IgM (■), IgG ( ), IgA (▨), and IgE (□) accumulated in the fluids of CL-01 cells incubated for 8 days with or without IL-4, and cultured alone, with CD8-293 cells, with CD40L-293 cells, and with CD40L-293 cells in the presence of neutralizing anti-IL-10 and/or anti-TGF- β Abs (the concentrations of IgM and IgE were from cultures with both anti-IL-10 and anti-TGF- β Abs, whereas the concentrations of IgG and IgA were from cultures with anti-IL-10 and anti-TGF- β Abs, respectively). Values are mean ± SD of four determinations from three independent experiments.
    Human Iga, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Nordic-Mubio iga1 standard serum
    <t>IgA1</t> O-glycosylation assay in supernatant of IgAN patients and normal controls peripheral B lymphocytes.
    Iga1 Standard Serum, supplied by Nordic-Mubio, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    iga  (Bethyl)
    94
    Bethyl iga
    Concentration of immunoglobulins <t>(IgA,</t> <t>IgG</t> and IgM) in chickens infected with C. jejuni Samples were analyzed at days 15, 22 and 30 of age; a ) IgA b ) IgG and c ) IgM. Error bars represent the mean values and standard errors of 6 isolators, each with 15 birds. Interactions between treatment and age effect significantly ( p
    Iga, supplied by Bethyl, used in various techniques. Bioz Stars score: 94/100, based on 1286 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Jackson Immuno iga
    Binding interactions of Fog-1 and B2 IgG variants with human FcγR. (A) Binding of monomeric B2 IgGs was measured for the high-affinity FcγRI using the B2KA cell line and flow cytometry. (B–H) Binding of (B, G, H) pre-complexed B2 or (C–F) Fog-1 IgGs was measured using CHO cell lines expressing FcγRIIa of allotypes (B) 131R and (C) 131H, (D) FcγRIIb and FcγRIIIa of allotypes (E, G) 158F and (F, H) 158 V. The level of background binding is given by the negative control Ab, <t>IgA,κ</t> or <t>IgA,λ</t> as appropriate. Graphs show mean fluorescence of ≥12 000 cells at each Ab concentration and are typical of the results obtained in at least three experiments with each receptor.
    Iga, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 440 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    SouthernBiotech iga standard
    Binding interactions of Fog-1 and B2 IgG variants with human FcγR. (A) Binding of monomeric B2 IgGs was measured for the high-affinity FcγRI using the B2KA cell line and flow cytometry. (B–H) Binding of (B, G, H) pre-complexed B2 or (C–F) Fog-1 IgGs was measured using CHO cell lines expressing FcγRIIa of allotypes (B) 131R and (C) 131H, (D) FcγRIIb and FcγRIIIa of allotypes (E, G) 158F and (F, H) 158 V. The level of background binding is given by the negative control Ab, <t>IgA,κ</t> or <t>IgA,λ</t> as appropriate. Graphs show mean fluorescence of ≥12 000 cells at each Ab concentration and are typical of the results obtained in at least three experiments with each receptor.
    Iga Standard, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore standard human iga1
    <t>IgA1</t> production in response to cytokine stimulation. IgA1 concentration of supernatant was determined by sandwich ELISA. ( A ) IgA concentration of supernatant from cytokine stimulated cells. IgA1 content of supernatant from IL-4 stimulated cells was significantly higher than that of cells incubated with other cytokines, except for IL-2, and control (P
    Standard Human Iga1, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    iga  (Bio-Rad)
    85
    Bio-Rad iga
    Concentration of anti-Neu5Gc <t>IgA</t> (A: yellow), IgM (B: orange), and <t>IgG</t> (C: blue) antibodies in patients suffering from Hashimoto thyroiditis.
    Iga, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    IGAN Bio introduction immunoglobulin a nephritis
    Significant increase of galectin-8N non-binding <t>IgA</t> in sera from IgAN patients compared to healthy controls. a Total IgA (mean/median for each group (mg/ml sera): 3.4/3.3, 1,7/2.1, 2.3/2.4), b galectin-8N unbound (total bound) IgA (mean/median for each group (mg/ml sera): 2.6/2.4, 1.5/1.0, 1.6/1.7), and c ratio of galectin-8N bound/unbound serum IgA (logarithmic scale on the Y -axis) in sera from 98 IgAN patients, 17 healthy, and 76 kidney disease controls. d Scatter plot of ratio from c ( Y -axis) vs. total IgA ( X -axis). IgA in unfractionated serum (total) or galectin-8N bound fractions was quantitated using nephelometry. Horizontal lines mark the average for each group in a – c . The difference between groups was statistically significant ( p
    Introduction Immunoglobulin A Nephritis, supplied by IGAN Bio, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam iga isotype
    Decreased clearance of IgAN patient <t>IgA</t> by CD89-expressing Kupffer cells. (A) IgA, purified from a normal donor and from a patient with IgAN, was stained with Cy3-anti-human IgA Ab, and subsequently incubated with Kupffer cells from CD89 transgenic mice at a concentration of 1 μg/mL and a temperature of <t>37°C.</t> After 1-h-incubation, intracellular vesicles containing IgA were diminished when IgAN patient-derived IgA endocytosis was assessed and compared with normal human IgA. (B) sCD89-IgA complexes in cell supernatant of (A) were detected by ELISA using A3 and anti-mouse IgA mAb, using 1 μg/mL human IgA binding to 5 μg/mL recCD89 as an internal control. The sCD89-IgA complexes were detected in the Kupffer cell supernatant after 1h of incubation with either control IgA or patient IgA. (C) Decreased clearance of 10 μg IgAN patient-derived IgA in C57BL/6-Tg mice when compared to normal human IgA. A total of 10 μg of each of the proteins was injected into the tail vein of C57BL/6-Tg (n = 5 per group). At the indicated times after injection, sera were obtained and IgA levels were determined. IgA from IgAN patients was cleared less quickly in Tg mice compared with normal IgA. (D) Induction of mesangial IgA/sCD89 deposits by injecting patient IgA into 6-wk-old C57BL/6-Tg mice. Double staining by anti-human IgA and anti-CD89 in kidney 48 h after constant injection of patient IgA into C57BL/6-Tg mice(n = 4) is shown. Bar = 10μm.
    Iga Isotype, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BlueGene Biotech intestinal immunoglobulin a
    Decreased clearance of IgAN patient <t>IgA</t> by CD89-expressing Kupffer cells. (A) IgA, purified from a normal donor and from a patient with IgAN, was stained with Cy3-anti-human IgA Ab, and subsequently incubated with Kupffer cells from CD89 transgenic mice at a concentration of 1 μg/mL and a temperature of <t>37°C.</t> After 1-h-incubation, intracellular vesicles containing IgA were diminished when IgAN patient-derived IgA endocytosis was assessed and compared with normal human IgA. (B) sCD89-IgA complexes in cell supernatant of (A) were detected by ELISA using A3 and anti-mouse IgA mAb, using 1 μg/mL human IgA binding to 5 μg/mL recCD89 as an internal control. The sCD89-IgA complexes were detected in the Kupffer cell supernatant after 1h of incubation with either control IgA or patient IgA. (C) Decreased clearance of 10 μg IgAN patient-derived IgA in C57BL/6-Tg mice when compared to normal human IgA. A total of 10 μg of each of the proteins was injected into the tail vein of C57BL/6-Tg (n = 5 per group). At the indicated times after injection, sera were obtained and IgA levels were determined. IgA from IgAN patients was cleared less quickly in Tg mice compared with normal IgA. (D) Induction of mesangial IgA/sCD89 deposits by injecting patient IgA into 6-wk-old C57BL/6-Tg mice. Double staining by anti-human IgA and anti-CD89 in kidney 48 h after constant injection of patient IgA into C57BL/6-Tg mice(n = 4) is shown. Bar = 10μm.
    Intestinal Immunoglobulin A, supplied by BlueGene Biotech, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Second-site immunization in the presence of PRO 2000 increases systemic and mucosal antibody responses. BALB/c mice (n = 7) were primed intranasally with 10 µg gp140 per animal in polycation and were boosted three times vaginally with gp140 +/− PRO 2000. Samples were collected 14 days after the final vaginal immunization. Serum IgG endpoint titres ( A ), or vaginal lavage IgG ( B ) and IgA ( C ) endpoint titres normalized to the total IgG or IgA content or each sample, respectively, are displayed. gp140 + PRO , combination of gp140 and PRO 2000.* p

    Journal: PLoS ONE

    Article Title: A Novel Strategy for Inducing Enhanced Mucosal HIV-1 Antibody Responses in an Anti-Inflammatory Environment

    doi: 10.1371/journal.pone.0015861

    Figure Lengend Snippet: Second-site immunization in the presence of PRO 2000 increases systemic and mucosal antibody responses. BALB/c mice (n = 7) were primed intranasally with 10 µg gp140 per animal in polycation and were boosted three times vaginally with gp140 +/− PRO 2000. Samples were collected 14 days after the final vaginal immunization. Serum IgG endpoint titres ( A ), or vaginal lavage IgG ( B ) and IgA ( C ) endpoint titres normalized to the total IgG or IgA content or each sample, respectively, are displayed. gp140 + PRO , combination of gp140 and PRO 2000.* p

    Article Snippet: Total Ig determination by multiplex immunoassay The total IgG and IgA concentration of mouse vaginal lavage was determined using a multiplex mouse immunoglobulin isotyping kit (Millipore, Billerica, MA) following the manufacturer's instructions.

    Techniques: Mouse Assay

    PRO 2000 co-application induces increased systemic and mucosal antibody responses in rabbits. New Zealand White rabbits (n = 6) received six vaginal immunizations using 50 mg antigen per dose in the presence or absence of 1% PRO 2000. ( A ) Time course of rabbit serum IgG endpoint titres. ( B ) Endpoint IgG titres of week 12 vaginal lavage samples. ( C ) Endpoint IgA titres of week 12 vaginal lavage samples. ( D ) MW965 pseudovirus neutralization of week 12 vaginal lavage samples. Horizontal bars represent geometric means. Samples were tested at 1∶8 dilution; 100% infection level was defined by pre-immune samples. ( E ) Correlation of the rabbit serum- and vaginal gp140-specific IgG responses with the MW965 pseudovirus neutralization ( D ) of week 12 samples. Red symbols represent data from animals immunized with gp140 + PRO 2000. Pre-immune indicates samples collected prior to the first immunization; gp140 + PRO indicates combined gp140 and PRO 2000. Titre data were log-transformed and are shown as mean ± SEM. The grey dashed line indicates the ELISA detection limit. * p

    Journal: PLoS ONE

    Article Title: A Novel Strategy for Inducing Enhanced Mucosal HIV-1 Antibody Responses in an Anti-Inflammatory Environment

    doi: 10.1371/journal.pone.0015861

    Figure Lengend Snippet: PRO 2000 co-application induces increased systemic and mucosal antibody responses in rabbits. New Zealand White rabbits (n = 6) received six vaginal immunizations using 50 mg antigen per dose in the presence or absence of 1% PRO 2000. ( A ) Time course of rabbit serum IgG endpoint titres. ( B ) Endpoint IgG titres of week 12 vaginal lavage samples. ( C ) Endpoint IgA titres of week 12 vaginal lavage samples. ( D ) MW965 pseudovirus neutralization of week 12 vaginal lavage samples. Horizontal bars represent geometric means. Samples were tested at 1∶8 dilution; 100% infection level was defined by pre-immune samples. ( E ) Correlation of the rabbit serum- and vaginal gp140-specific IgG responses with the MW965 pseudovirus neutralization ( D ) of week 12 samples. Red symbols represent data from animals immunized with gp140 + PRO 2000. Pre-immune indicates samples collected prior to the first immunization; gp140 + PRO indicates combined gp140 and PRO 2000. Titre data were log-transformed and are shown as mean ± SEM. The grey dashed line indicates the ELISA detection limit. * p

    Article Snippet: Total Ig determination by multiplex immunoassay The total IgG and IgA concentration of mouse vaginal lavage was determined using a multiplex mouse immunoglobulin isotyping kit (Millipore, Billerica, MA) following the manufacturer's instructions.

    Techniques: Neutralization, Infection, Transformation Assay, Enzyme-linked Immunosorbent Assay

    PRO 2000 co-application induces increased mucosal antibody responses in mice. BALB/c mice (n = 7) were immunized four times vaginally with 12.5 µg gp140 +/− 1% PRO 2000; Time courses of the vaginal lavage IgA ( A ) and IgG ( B ) endpoint titres. gp140 + PRO indicates combined gp140 and PRO 2000. Titre data were log-transformed and are shown as mean ± SEM. The grey dashed line indicates the ELISA detection limit. * p

    Journal: PLoS ONE

    Article Title: A Novel Strategy for Inducing Enhanced Mucosal HIV-1 Antibody Responses in an Anti-Inflammatory Environment

    doi: 10.1371/journal.pone.0015861

    Figure Lengend Snippet: PRO 2000 co-application induces increased mucosal antibody responses in mice. BALB/c mice (n = 7) were immunized four times vaginally with 12.5 µg gp140 +/− 1% PRO 2000; Time courses of the vaginal lavage IgA ( A ) and IgG ( B ) endpoint titres. gp140 + PRO indicates combined gp140 and PRO 2000. Titre data were log-transformed and are shown as mean ± SEM. The grey dashed line indicates the ELISA detection limit. * p

    Article Snippet: Total Ig determination by multiplex immunoassay The total IgG and IgA concentration of mouse vaginal lavage was determined using a multiplex mouse immunoglobulin isotyping kit (Millipore, Billerica, MA) following the manufacturer's instructions.

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

    Modulatory effect of various human plasma-derived IgA/SIgA preparations on Caco-2 cells infected by S. flexneri . A , internalization of S. flexneri alone or associated with SIgAC5 monoclonal Ab and with plasma-derived pIgA, reconstituted SIgA, mIgA, or IgG to polarized Caco-2 cell monolayers, as determined after O/N incubation, with addition of gentamicin for the last 30 min. Data are expressed on a “per filter” basis and correspond to a pool of two independent experiments ( n = 4 or 5) for each tested condition. B , basolateral secretion of TNF-α, CXCL8, and CCL3 by polarized Caco-2 cell monolayers after O/N incubation with S. flexneri alone or associated to plasma-derived pIgA, SIgA, mIgA, IgG, or anti- Shigella LPS-specific SIgAC5 monoclonal Ab. The concentration of proinflammatory mediators was determined by ELISA. Non-infected Caco-2 cell monolayers ( No bacteria ) served as a control. Data are the pool of two experiments performed in triplicates ( n = 6). When obtained, significant statistical differences were calculated by comparison with the condition Sf. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstituted Human Polyclonal Plasma-derived Secretory-like IgM and IgA Maintain the Barrier Function of Epithelial Cells Infected with an Enteropathogen *

    doi: 10.1074/jbc.M114.549139

    Figure Lengend Snippet: Modulatory effect of various human plasma-derived IgA/SIgA preparations on Caco-2 cells infected by S. flexneri . A , internalization of S. flexneri alone or associated with SIgAC5 monoclonal Ab and with plasma-derived pIgA, reconstituted SIgA, mIgA, or IgG to polarized Caco-2 cell monolayers, as determined after O/N incubation, with addition of gentamicin for the last 30 min. Data are expressed on a “per filter” basis and correspond to a pool of two independent experiments ( n = 4 or 5) for each tested condition. B , basolateral secretion of TNF-α, CXCL8, and CCL3 by polarized Caco-2 cell monolayers after O/N incubation with S. flexneri alone or associated to plasma-derived pIgA, SIgA, mIgA, IgG, or anti- Shigella LPS-specific SIgAC5 monoclonal Ab. The concentration of proinflammatory mediators was determined by ELISA. Non-infected Caco-2 cell monolayers ( No bacteria ) served as a control. Data are the pool of two experiments performed in triplicates ( n = 6). When obtained, significant statistical differences were calculated by comparison with the condition Sf. **, p

    Article Snippet: The formation of immune complexes was verified after incubation with biotinylated mouse anti-human IgA1/IgA2 (1:10, BD Biosciences) for 30 min at room temperature under gentle agitation, followed by cyanine 5-conjugated streptavidin (1:400, GE Healthcare) for 30 min at room temperature under gentle agitation.

    Techniques: Derivative Assay, Infection, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Impact of human plasma-derived IgA and IgM preparations on the secretion of virulence factors IpaB and IpaC by S. flexneri . Immune complexes between bacteria and human plasma-derived pIgA, SIgA, mIgA, IgG, IgM, SigM, or anti- Shigella LPS-specific SIgAC5 monoclonal Ab were formed for 1 h as described under “Experimental procedures,” and the expression of virulence factors was induced by Congo red. A and B , the secretion of IpaB ( A ) and IpaC ( B ) was examined by immunoblot analysis using monoclonal Abs directed against IpaB and IpaC, with immunodetection of OspF serving as a loading control. The images are representative of one individual experiment performed three times in duplicates. C , densitometric analysis of replicated immunoblots ( n = 6) exposed for optimal times to avoid saturation of the photographic film. The intensity of the signals reached with S. flexneri alone was fixed arbitrarily at 100%. When obtained, significant statistical differences were calculated by comparison with the condition Sf. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstituted Human Polyclonal Plasma-derived Secretory-like IgM and IgA Maintain the Barrier Function of Epithelial Cells Infected with an Enteropathogen *

    doi: 10.1074/jbc.M114.549139

    Figure Lengend Snippet: Impact of human plasma-derived IgA and IgM preparations on the secretion of virulence factors IpaB and IpaC by S. flexneri . Immune complexes between bacteria and human plasma-derived pIgA, SIgA, mIgA, IgG, IgM, SigM, or anti- Shigella LPS-specific SIgAC5 monoclonal Ab were formed for 1 h as described under “Experimental procedures,” and the expression of virulence factors was induced by Congo red. A and B , the secretion of IpaB ( A ) and IpaC ( B ) was examined by immunoblot analysis using monoclonal Abs directed against IpaB and IpaC, with immunodetection of OspF serving as a loading control. The images are representative of one individual experiment performed three times in duplicates. C , densitometric analysis of replicated immunoblots ( n = 6) exposed for optimal times to avoid saturation of the photographic film. The intensity of the signals reached with S. flexneri alone was fixed arbitrarily at 100%. When obtained, significant statistical differences were calculated by comparison with the condition Sf. *, p

    Article Snippet: The formation of immune complexes was verified after incubation with biotinylated mouse anti-human IgA1/IgA2 (1:10, BD Biosciences) for 30 min at room temperature under gentle agitation, followed by cyanine 5-conjugated streptavidin (1:400, GE Healthcare) for 30 min at room temperature under gentle agitation.

    Techniques: Derivative Assay, Expressing, Immunodetection, Western Blot

    A. Saturation isotherm and Scatchard analysis of 125 I-UG binding to Fn . Binding isotherms of hUG to Fn were performed by incubating increasing concentrations of 125 I-UG. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of hUG, from the total binding (■). The data shown are the mean of triplicate measurements±SD. B. Scatchard plots of hUG binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 15 nM ± 2.6 nM (n = 3). C. Binding isotherms of IgA to Fn. Binding isotherms of IgA to Fn were performed by incubating increasing concentration of 125 I-IgA. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of IgA, from the total binding (■). The data shown are the mean of triplicate measurements. D. Scatchard plots of IgA binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 65 nM ± 5.2 nM (n = 3).

    Journal: FEBS letters

    Article Title: Uteroglobin interacts with the heparin-binding site of fibronectin and prevents fibronectin-IgA complex formation found in IgA-nephropathy

    doi: 10.1016/j.febslet.2008.01.025

    Figure Lengend Snippet: A. Saturation isotherm and Scatchard analysis of 125 I-UG binding to Fn . Binding isotherms of hUG to Fn were performed by incubating increasing concentrations of 125 I-UG. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of hUG, from the total binding (■). The data shown are the mean of triplicate measurements±SD. B. Scatchard plots of hUG binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 15 nM ± 2.6 nM (n = 3). C. Binding isotherms of IgA to Fn. Binding isotherms of IgA to Fn were performed by incubating increasing concentration of 125 I-IgA. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of IgA, from the total binding (■). The data shown are the mean of triplicate measurements. D. Scatchard plots of IgA binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 65 nM ± 5.2 nM (n = 3).

    Article Snippet: Immunoglobulin A (IgA)-nephropathy (IgAN) is the most common primary renal glomerular disease in the world that has no effective treatment.

    Techniques: Binding Assay, Concentration Assay

    Normalized matricellular proteins and risk of developing BPD or death. Normalized SPARC levels were significantly higher in babies developing BPD or death prior to 36 weeks postmenstrual age (*p = 0.0185, Mann Whitney U test). There were no other significant differences in normalized matricellular protein concentration between infants who did not develop BPD and infants who developed BPD or died prior to 36 weeks postmenstrual age. Tracheal aspirate matricellular protein and secretory component of IgA (scIgA) concentrations were measured by ELISA or multiplex immune assay. The data show individual matricellular protein levels normalized to scIgA concentration. Medians and interquartile ranges are shown.

    Journal: PLoS ONE

    Article Title: Tracheal Aspirate Levels of the Matricellular Protein SPARC Predict Development of Bronchopulmonary Dysplasia

    doi: 10.1371/journal.pone.0144122

    Figure Lengend Snippet: Normalized matricellular proteins and risk of developing BPD or death. Normalized SPARC levels were significantly higher in babies developing BPD or death prior to 36 weeks postmenstrual age (*p = 0.0185, Mann Whitney U test). There were no other significant differences in normalized matricellular protein concentration between infants who did not develop BPD and infants who developed BPD or died prior to 36 weeks postmenstrual age. Tracheal aspirate matricellular protein and secretory component of IgA (scIgA) concentrations were measured by ELISA or multiplex immune assay. The data show individual matricellular protein levels normalized to scIgA concentration. Medians and interquartile ranges are shown.

    Article Snippet: To control for sample dilution, matricellular protein levels were normalized to the secretory component of immunoglobulin A (scIgA) (ALPCO, Salem, NH) [ ].

    Techniques: MANN-WHITNEY, Protein Concentration, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Concentration Assay

    Mesenchymal stromal cell (MSC) isolation does not influence tracheal aspirate matricellular protein concentration. Tracheal aspirate matricellular protein and secretory component of IgA (scIgA) concentrations were measured by ELISA or multiplex immune assay. The isolation of MSCs from tracheal aspirates had no influence on matricellular protein concentration. The data show individual matricellular protein levels normalized to scIgA concentration. Medians and interquartile ranges are shown. Statistical significance was determined by Mann-Whitney U test.

    Journal: PLoS ONE

    Article Title: Tracheal Aspirate Levels of the Matricellular Protein SPARC Predict Development of Bronchopulmonary Dysplasia

    doi: 10.1371/journal.pone.0144122

    Figure Lengend Snippet: Mesenchymal stromal cell (MSC) isolation does not influence tracheal aspirate matricellular protein concentration. Tracheal aspirate matricellular protein and secretory component of IgA (scIgA) concentrations were measured by ELISA or multiplex immune assay. The isolation of MSCs from tracheal aspirates had no influence on matricellular protein concentration. The data show individual matricellular protein levels normalized to scIgA concentration. Medians and interquartile ranges are shown. Statistical significance was determined by Mann-Whitney U test.

    Article Snippet: To control for sample dilution, matricellular protein levels were normalized to the secretory component of immunoglobulin A (scIgA) (ALPCO, Salem, NH) [ ].

    Techniques: Isolation, Protein Concentration, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Concentration Assay, MANN-WHITNEY

    The rate of G. vaginalis sialic acid (Neu5Ac) consumption from sialoglycans is substrate-dependent and inhibited by N -glycolylneuraminic acid (Neu5Gc). Washed G. vaginalis JCP8151B was incubated with 0.2 mg/ml of human IgA or 0.3 mg/ml of BSM, followed

    Journal: The Journal of Biological Chemistry

    Article Title: Degradation, Foraging, and Depletion of Mucus Sialoglycans by the Vagina-adapted Actinobacterium Gardnerella vaginalis *

    doi: 10.1074/jbc.M113.453654

    Figure Lengend Snippet: The rate of G. vaginalis sialic acid (Neu5Ac) consumption from sialoglycans is substrate-dependent and inhibited by N -glycolylneuraminic acid (Neu5Gc). Washed G. vaginalis JCP8151B was incubated with 0.2 mg/ml of human IgA or 0.3 mg/ml of BSM, followed

    Article Snippet: The bacterial suspensions were supplemented with human myeloma serum immunoglobulin A-IgA (Kent Laboratories) or bovine submaxillary mucin-BSM (Sigma) at a final concentration of 400 μg/ml.

    Techniques: Incubation

    A , extracellular hydrolysis of human IgA sialic acids by G. vaginalis precedes consumption. Clinical isolates JCP8151B (sialidase-positive) and JCP8481A (sialidase-negative) were incubated with purified human serum IgA in acetate buffer and the concentration

    Journal: The Journal of Biological Chemistry

    Article Title: Degradation, Foraging, and Depletion of Mucus Sialoglycans by the Vagina-adapted Actinobacterium Gardnerella vaginalis *

    doi: 10.1074/jbc.M113.453654

    Figure Lengend Snippet: A , extracellular hydrolysis of human IgA sialic acids by G. vaginalis precedes consumption. Clinical isolates JCP8151B (sialidase-positive) and JCP8481A (sialidase-negative) were incubated with purified human serum IgA in acetate buffer and the concentration

    Article Snippet: The bacterial suspensions were supplemented with human myeloma serum immunoglobulin A-IgA (Kent Laboratories) or bovine submaxillary mucin-BSM (Sigma) at a final concentration of 400 μg/ml.

    Techniques: Incubation, Purification, Concentration Assay

    Arithmetic means (± standard deviations) of the percentages of IgA1- and IgA2-secreting cells in peripheral blood expressing various lymphocyte markers. The numbers of unimmunized healthy volunteers from whom the data were pooled are given under the bars. Statistically significant differences between IgA1 and IgA2 ISC in paired Student's t test are indicated by an asterisk (*, 0.01

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Expression of Homing Receptors on IgA1 and IgA2 Plasmablasts in Blood Reflects Differential Distribution of IgA1 and IgA2 in Various Body Fluids ▿

    doi: 10.1128/CVI.00475-09

    Figure Lengend Snippet: Arithmetic means (± standard deviations) of the percentages of IgA1- and IgA2-secreting cells in peripheral blood expressing various lymphocyte markers. The numbers of unimmunized healthy volunteers from whom the data were pooled are given under the bars. Statistically significant differences between IgA1 and IgA2 ISC in paired Student's t test are indicated by an asterisk (*, 0.01

    Article Snippet: In the present study, the only other mucosal site where IgA2 reached the concentration of IgA1 was the vaginal fluid; the female genital tract is colonized by a significant Gram-negative bacterial population.

    Techniques: Expressing

    Arithmetic means (± standard deviations) of the percentages of antigen-specific IgA1- and IgA2-secreting cells expressing the indicated HR in healthy volunteers after oral vaccination with live attenuated Salmonella serovar Typhi Ty21a (A) or parenteral vaccination with an unconjugated pneumococcal polysaccharide vaccine (B). The numbers of volunteers from whom the data were pooled are given under the bars. There were no statistically significant differences between IgA1 and IgA2 ASC in the paired Student's t test.

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Expression of Homing Receptors on IgA1 and IgA2 Plasmablasts in Blood Reflects Differential Distribution of IgA1 and IgA2 in Various Body Fluids ▿

    doi: 10.1128/CVI.00475-09

    Figure Lengend Snippet: Arithmetic means (± standard deviations) of the percentages of antigen-specific IgA1- and IgA2-secreting cells expressing the indicated HR in healthy volunteers after oral vaccination with live attenuated Salmonella serovar Typhi Ty21a (A) or parenteral vaccination with an unconjugated pneumococcal polysaccharide vaccine (B). The numbers of volunteers from whom the data were pooled are given under the bars. There were no statistically significant differences between IgA1 and IgA2 ASC in the paired Student's t test.

    Article Snippet: In the present study, the only other mucosal site where IgA2 reached the concentration of IgA1 was the vaginal fluid; the female genital tract is colonized by a significant Gram-negative bacterial population.

    Techniques: Expressing

    IgA + CCR10 + cells are present in tonsil germinal centers (A) Fixed frozen tonsil sections were stained with DAPI (Blue), anti-IgA (Green) and anti-CCR10 (Red). The three-color overview picture (10×) shows the presence of IgA + CCR10 + cells in the germinal center (GC). The rectangle indicates the magnified area (40×). (B) Fixed frozen tonsil sections were stained with anti-IgM (Blue), anti-IgA (Green) and anti-CCR10 (Red) showing the presence of IgA + CCR10 + cells in the subepithelial area (Ep) as well as in the GC (10×). (C) Adjacent tonsil sections stained with control antibodies.

    Journal: Immunity

    Article Title: A T cell-dependent mechanism for the induction of human mucosal homing IgA-secreting plasmablasts

    doi: 10.1016/j.immuni.2008.11.008

    Figure Lengend Snippet: IgA + CCR10 + cells are present in tonsil germinal centers (A) Fixed frozen tonsil sections were stained with DAPI (Blue), anti-IgA (Green) and anti-CCR10 (Red). The three-color overview picture (10×) shows the presence of IgA + CCR10 + cells in the germinal center (GC). The rectangle indicates the magnified area (40×). (B) Fixed frozen tonsil sections were stained with anti-IgM (Blue), anti-IgA (Green) and anti-CCR10 (Red) showing the presence of IgA + CCR10 + cells in the subepithelial area (Ep) as well as in the GC (10×). (C) Adjacent tonsil sections stained with control antibodies.

    Article Snippet: Sandwich ELISAs were performed to measure total IgM, IgG, IgA, IgA1 and IgA2 in the culture supernatant.

    Techniques: Staining

    TGFβ1 skews IL-21 mediated class switching towards IgA1 and IgA2 Naive B cells activated with anti-IgM and anti-CD40 were cultured in the presence of IL-2 and CpG with either IL-21 alone or a combination of IL-21 and TGFβ1. (A, B) On day 6, surface immunoglobulin expression was determined by staining the cells with anti-IgG, IgA, IgA1 and IgA2 antibodies. The left panels show the dot plots. The right panels show the results of multiple experiments with mean. (C, D) On day 12, IgG, IgA, IgA1 and IgA2 secreted in the culture supernatants were measured by ELIS A. (E) IgM secreted in the culture supernatants on day 12 as measured by ELISA.

    Journal: Immunity

    Article Title: A T cell-dependent mechanism for the induction of human mucosal homing IgA-secreting plasmablasts

    doi: 10.1016/j.immuni.2008.11.008

    Figure Lengend Snippet: TGFβ1 skews IL-21 mediated class switching towards IgA1 and IgA2 Naive B cells activated with anti-IgM and anti-CD40 were cultured in the presence of IL-2 and CpG with either IL-21 alone or a combination of IL-21 and TGFβ1. (A, B) On day 6, surface immunoglobulin expression was determined by staining the cells with anti-IgG, IgA, IgA1 and IgA2 antibodies. The left panels show the dot plots. The right panels show the results of multiple experiments with mean. (C, D) On day 12, IgG, IgA, IgA1 and IgA2 secreted in the culture supernatants were measured by ELIS A. (E) IgM secreted in the culture supernatants on day 12 as measured by ELISA.

    Article Snippet: Sandwich ELISAs were performed to measure total IgM, IgG, IgA, IgA1 and IgA2 in the culture supernatant.

    Techniques: Cell Culture, Expressing, Staining, Enzyme-linked Immunosorbent Assay

    Surface IgA expression is directly related to TGFβ1 concentration Naive B cells were activated with anti-IgM, anti-CD40 and CpG and cultured with 20 ng/mL IL-21 and increasing concentrations of TGFβ1. Surface immunoglobulin expression was determined on day 6. Average ±SEM of 3 independent experiments is shown.

    Journal: Immunity

    Article Title: A T cell-dependent mechanism for the induction of human mucosal homing IgA-secreting plasmablasts

    doi: 10.1016/j.immuni.2008.11.008

    Figure Lengend Snippet: Surface IgA expression is directly related to TGFβ1 concentration Naive B cells were activated with anti-IgM, anti-CD40 and CpG and cultured with 20 ng/mL IL-21 and increasing concentrations of TGFβ1. Surface immunoglobulin expression was determined on day 6. Average ±SEM of 3 independent experiments is shown.

    Article Snippet: Sandwich ELISAs were performed to measure total IgM, IgG, IgA, IgA1 and IgA2 in the culture supernatant.

    Techniques: Expressing, Concentration Assay, Cell Culture

    Molecular events in IL-21/TGFβ1 induced IgA class switching CD40/BCR/TLR9-activated naive B cells cultured with the indicated cytokines were harvested on day 4. mRNA was isolated and cDNA synthesized. Real time RT-PCR was performed for activation-induced cytidine deaminase (AICDA) (A) , germline transcripts Iγ 3 -Cγ 3 , Iαγ-Cα 1 and Iα-Cα 2 (B) and switch circle transcripts Iγ-Cμ. and Iα-Cμ (C) . The expression levels of AICDA, Iγ 3 -Cγ 3 , Iα 1 -Cα 1 and Iα 2 -Cα 2 ). Below C , agarose gel-electrophoresis of RT-PCR amplified Iγ-Cμ and Iα-μ transcipts compared to Iμ-Cμ, (loading control). Data shown are mean ± SEM of three independent experiments using cells from three different healthy donors. (D) Conventional RT-PCR was used to detect mature Ig transcripts. One out of three similar results is shown. *, P

    Journal: Immunity

    Article Title: A T cell-dependent mechanism for the induction of human mucosal homing IgA-secreting plasmablasts

    doi: 10.1016/j.immuni.2008.11.008

    Figure Lengend Snippet: Molecular events in IL-21/TGFβ1 induced IgA class switching CD40/BCR/TLR9-activated naive B cells cultured with the indicated cytokines were harvested on day 4. mRNA was isolated and cDNA synthesized. Real time RT-PCR was performed for activation-induced cytidine deaminase (AICDA) (A) , germline transcripts Iγ 3 -Cγ 3 , Iαγ-Cα 1 and Iα-Cα 2 (B) and switch circle transcripts Iγ-Cμ. and Iα-Cμ (C) . The expression levels of AICDA, Iγ 3 -Cγ 3 , Iα 1 -Cα 1 and Iα 2 -Cα 2 ). Below C , agarose gel-electrophoresis of RT-PCR amplified Iγ-Cμ and Iα-μ transcipts compared to Iμ-Cμ, (loading control). Data shown are mean ± SEM of three independent experiments using cells from three different healthy donors. (D) Conventional RT-PCR was used to detect mature Ig transcripts. One out of three similar results is shown. *, P

    Article Snippet: Sandwich ELISAs were performed to measure total IgM, IgG, IgA, IgA1 and IgA2 in the culture supernatant.

    Techniques: Cell Culture, Isolation, Synthesized, Quantitative RT-PCR, Activation Assay, Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification

    Tonsil T fh induce IgA + CCR10 + plasma blasts in an IL-21-dependent manner (A) Sorted peripheral blood CXCR5 + and CXCR5 − CD4 + T cells (10 6 /mL) were cultured with PMA + ionomycin. After 48 hours, concentrations of IL-21 and TGFβ1 in the supernatant were measured by Luminex. (B) Total peripheral blood CD4 + T cells were cultured with or without PMA and ionomycin for 20 hours, the last 4 hours in the presence of brefeldin A. Cells were stained for CXCR5, intracellular IL-21 and TGFβ1. Plots shown are gated on CXCR5 + and CXCR5 − populations. One out of 2 similar experiments is shown. (C, D, E, F) Naive B cells (5*10 4 ) were co-cultured with autologous CXCR5 + CD4 + T cells (2*10 4 ) in the presence of IL-21R-Fc or control IgGl-Fc. (C) B cell proliferation, CD38 and CCR10 expression were measured on day 8. (D) On day 4, cells were harvested, RNA isolated and cDNA synthesized. Real time RT-PCR was performed for switch circle transcripts Iγ-Cμ, and Iα-Cμ. Expression was normalized to Iμ-Cμ expression. (E) Agarose gel-electrophoresis showing absolute expression levels of Iμ-Cμ, Iγ-Cμ, and Iα-Cμ. (F) On day 8, the supernatants from the cocultures were harvested and IgM and IgA were measured by ELISA. IgA is normalized for IgM secretion by using IgA/IgM ratio.

    Journal: Immunity

    Article Title: A T cell-dependent mechanism for the induction of human mucosal homing IgA-secreting plasmablasts

    doi: 10.1016/j.immuni.2008.11.008

    Figure Lengend Snippet: Tonsil T fh induce IgA + CCR10 + plasma blasts in an IL-21-dependent manner (A) Sorted peripheral blood CXCR5 + and CXCR5 − CD4 + T cells (10 6 /mL) were cultured with PMA + ionomycin. After 48 hours, concentrations of IL-21 and TGFβ1 in the supernatant were measured by Luminex. (B) Total peripheral blood CD4 + T cells were cultured with or without PMA and ionomycin for 20 hours, the last 4 hours in the presence of brefeldin A. Cells were stained for CXCR5, intracellular IL-21 and TGFβ1. Plots shown are gated on CXCR5 + and CXCR5 − populations. One out of 2 similar experiments is shown. (C, D, E, F) Naive B cells (5*10 4 ) were co-cultured with autologous CXCR5 + CD4 + T cells (2*10 4 ) in the presence of IL-21R-Fc or control IgGl-Fc. (C) B cell proliferation, CD38 and CCR10 expression were measured on day 8. (D) On day 4, cells were harvested, RNA isolated and cDNA synthesized. Real time RT-PCR was performed for switch circle transcripts Iγ-Cμ, and Iα-Cμ. Expression was normalized to Iμ-Cμ expression. (E) Agarose gel-electrophoresis showing absolute expression levels of Iμ-Cμ, Iγ-Cμ, and Iα-Cμ. (F) On day 8, the supernatants from the cocultures were harvested and IgM and IgA were measured by ELISA. IgA is normalized for IgM secretion by using IgA/IgM ratio.

    Article Snippet: Sandwich ELISAs were performed to measure total IgM, IgG, IgA, IgA1 and IgA2 in the culture supernatant.

    Techniques: Cell Culture, Luminex, Staining, Expressing, Isolation, Synthesized, Quantitative RT-PCR, Agarose Gel Electrophoresis, Enzyme-linked Immunosorbent Assay

    The mean (±SEM) concentrations (mg/mL) of serum IgG, IgM, IgA and sIgA in piglets between maternal COS supplementation and control groups at weaning (postnatal d 19). ∗, P

    Journal: Animal Nutrition

    Article Title: Maternal chitosan oligosaccharide intervention optimizes the production performance and health status of gilts and their offspring

    doi: 10.1016/j.aninu.2020.02.001

    Figure Lengend Snippet: The mean (±SEM) concentrations (mg/mL) of serum IgG, IgM, IgA and sIgA in piglets between maternal COS supplementation and control groups at weaning (postnatal d 19). ∗, P

    Article Snippet: A pig immunoglobulin M (IgM) ELISA kit (Life Diagnostics, Inc. USA, Catalog no. 5015-9), immunoglobulin G (IgG) and immunoglobulin A (IgA) ELISA kits (Bethyl Laboratories, Inc. USA, Catalog no. E101-104 and Catalog no. E100-102) were used to determine the serum concentrations of IgM, IgG, and IgA, respectively.

    Techniques:

    Expression levels of IgM, IgG, IgA, sIgA in serum and sIgA in faecal samples of the COS treatment and control gilts at 19 d after giving birth and 90 d of gestation, respectively. (A) The mean (±SEM) concentrations (mg/mL) of immune markers of IgM, IgG, IgA and sIgA in blood serum samples of the COS treatment and control gilts at weaning d 19 after giving birth. Samples without any hemolysis were used in this assay. (B) The mean (±SEM) of sIgA concentration (mg/mL) in faecal samples of the COS treatment and control gilts at d 90 of gestation. ∗, P

    Journal: Animal Nutrition

    Article Title: Maternal chitosan oligosaccharide intervention optimizes the production performance and health status of gilts and their offspring

    doi: 10.1016/j.aninu.2020.02.001

    Figure Lengend Snippet: Expression levels of IgM, IgG, IgA, sIgA in serum and sIgA in faecal samples of the COS treatment and control gilts at 19 d after giving birth and 90 d of gestation, respectively. (A) The mean (±SEM) concentrations (mg/mL) of immune markers of IgM, IgG, IgA and sIgA in blood serum samples of the COS treatment and control gilts at weaning d 19 after giving birth. Samples without any hemolysis were used in this assay. (B) The mean (±SEM) of sIgA concentration (mg/mL) in faecal samples of the COS treatment and control gilts at d 90 of gestation. ∗, P

    Article Snippet: A pig immunoglobulin M (IgM) ELISA kit (Life Diagnostics, Inc. USA, Catalog no. 5015-9), immunoglobulin G (IgG) and immunoglobulin A (IgA) ELISA kits (Bethyl Laboratories, Inc. USA, Catalog no. E101-104 and Catalog no. E100-102) were used to determine the serum concentrations of IgM, IgG, and IgA, respectively.

    Techniques: Expressing, Concentration Assay

    TPLTA N 205 ITK (H5N5S1F1) and (H5N4S2F1) in normal control-HBV-LC-HCC cascade. ( A and B ) Crude serum of 10 independent HCC, 10 independent LC patients, 10 HBV patients, and 10 normal subjects were enrolled using MRM and profound increases of the two glycopeptides were found from HBV infection. ( C and D ) Purified IgA of 10 independent HCC, 10 independent LC patients, 10 HBV patients, and 10 normal subjects were enrolled using MRM, and similar trend was found. * p

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: N-glycopeptide Signatures of IgA2 in Serum from Patients with Hepatitis B Virus-related Liver Diseases *

    doi: 10.1074/mcp.RA119.001722

    Figure Lengend Snippet: TPLTA N 205 ITK (H5N5S1F1) and (H5N4S2F1) in normal control-HBV-LC-HCC cascade. ( A and B ) Crude serum of 10 independent HCC, 10 independent LC patients, 10 HBV patients, and 10 normal subjects were enrolled using MRM and profound increases of the two glycopeptides were found from HBV infection. ( C and D ) Purified IgA of 10 independent HCC, 10 independent LC patients, 10 HBV patients, and 10 normal subjects were enrolled using MRM, and similar trend was found. * p

    Article Snippet: Standard curves were prepared with natural human IgA2 protein (ab91021, Abcam, Cambridge, UK) from 0.025 mg/ml to 0.4 mg/ml, with subsequent regression analysis showing acceptable linearity.

    Techniques: Infection, Purification

    MRM validation of TPLTA N 205 ITK (H5N5S1F1) and (H5N4S2F1) in LC and HCC. ( A ) Target glycopeptides and unique peptide DASGATFTWTPSSGK of IgA 2 were fitted linearly with R 2 of 0. 99. ( B ) Glycopeptide abundance was divided by unique peptide abundance to separate out the contribution of protein concentration, named as Normalized Abund. Crude serum (10 HCC individuals and 10 LC individuals) and purified IgA (10 HCC individuals and 10 LC individuals) were applied to this approach, respectively. TPLTA N 205 ITK (H5N5S1F1) and (H5N4S2F1) were significantly decreased in HCC compared with LC patients. * p

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: N-glycopeptide Signatures of IgA2 in Serum from Patients with Hepatitis B Virus-related Liver Diseases *

    doi: 10.1074/mcp.RA119.001722

    Figure Lengend Snippet: MRM validation of TPLTA N 205 ITK (H5N5S1F1) and (H5N4S2F1) in LC and HCC. ( A ) Target glycopeptides and unique peptide DASGATFTWTPSSGK of IgA 2 were fitted linearly with R 2 of 0. 99. ( B ) Glycopeptide abundance was divided by unique peptide abundance to separate out the contribution of protein concentration, named as Normalized Abund. Crude serum (10 HCC individuals and 10 LC individuals) and purified IgA (10 HCC individuals and 10 LC individuals) were applied to this approach, respectively. TPLTA N 205 ITK (H5N5S1F1) and (H5N4S2F1) were significantly decreased in HCC compared with LC patients. * p

    Article Snippet: Standard curves were prepared with natural human IgA2 protein (ab91021, Abcam, Cambridge, UK) from 0.025 mg/ml to 0.4 mg/ml, with subsequent regression analysis showing acceptable linearity.

    Techniques: Protein Concentration, Purification

    TPLTA N 205 ITK (H5N5S1F1) and (H5N4S2F1) of IgA 2 in LC and HCC. ( A ) Based on 45 biological repeats, 60 N-glycopeptides that occurred at least 10 times (QC1) and passed through stringent filtering criteria (QC2, FDR

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: N-glycopeptide Signatures of IgA2 in Serum from Patients with Hepatitis B Virus-related Liver Diseases *

    doi: 10.1074/mcp.RA119.001722

    Figure Lengend Snippet: TPLTA N 205 ITK (H5N5S1F1) and (H5N4S2F1) of IgA 2 in LC and HCC. ( A ) Based on 45 biological repeats, 60 N-glycopeptides that occurred at least 10 times (QC1) and passed through stringent filtering criteria (QC2, FDR

    Article Snippet: Standard curves were prepared with natural human IgA2 protein (ab91021, Abcam, Cambridge, UK) from 0.025 mg/ml to 0.4 mg/ml, with subsequent regression analysis showing acceptable linearity.

    Techniques:

    Env antigen-specific IgA and IgG responses in milk and plasma seldom correlate with other Env antigen-specific responses in HIV-infected lactating women. To determine whether immune responses to the various HIV-1 antigens were associated with each other, correlations between binding scores for selected antigen specificities were tested for plasma IgA (A), plasma IgG (B), breast milk (BM) IgA (C), and breast milk IgG (D). Kendall’s tau values and corresponding corrected P values (see Materials and Methods) are reported. Boldface P values indicate significant correlations at a P value of

    Journal: Journal of Virology

    Article Title: Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1

    doi: 10.1128/JVI.02084-18

    Figure Lengend Snippet: Env antigen-specific IgA and IgG responses in milk and plasma seldom correlate with other Env antigen-specific responses in HIV-infected lactating women. To determine whether immune responses to the various HIV-1 antigens were associated with each other, correlations between binding scores for selected antigen specificities were tested for plasma IgA (A), plasma IgG (B), breast milk (BM) IgA (C), and breast milk IgG (D). Kendall’s tau values and corresponding corrected P values (see Materials and Methods) are reported. Boldface P values indicate significant correlations at a P value of

    Article Snippet: To test IgA binding to TZM-bl/FcαRI-expressing cells, human IgA1(κ), IgA2(κ) (Athens Research and Technology, Athens GA), purified human secretory IgA (MP Biomedicals, LLC, Solon, Ohio), and FITC-conjugated goat anti-human IgA (Sigma) were used.

    Techniques: Infection, Binding Assay

    Lack of detectable milk IgA-mediated phagocytosis despite increasing IgA concentration. To determine if lack of milk IgA phagocytosis activity was due to lower HIV-1 specific activity in IgA than that in IgG, higher concentrations of IgA and IgG were purified from the milk of an HIV-positive lactating woman recruited in the United states. Breast milk (BM) IgG, breast milk IgA, and a control antibody, CH31 mIgA2, were tested for phagocytosis of HIV ConS gp140-coated beads at 5-fold dilutions as indicated, and flow cytometry diagrams indicative of the phagocytosis results are shown. The red traces indicate sample antibody setup while black traces indicate the no-antibody control setup, and gray fill indicates the no-target control setup. Antibody-mediated phagocytosis is indicated by a greater area under the curve for the red trace than that for the black trace. Milk IgG and CH31 mIgA2 showed antibody-mediated phagocytosis activity down to 2 μg/ml, while milk IgA showed no antibody-mediated phagocytosis activity even at 250 μg/ml.

    Journal: Journal of Virology

    Article Title: Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1

    doi: 10.1128/JVI.02084-18

    Figure Lengend Snippet: Lack of detectable milk IgA-mediated phagocytosis despite increasing IgA concentration. To determine if lack of milk IgA phagocytosis activity was due to lower HIV-1 specific activity in IgA than that in IgG, higher concentrations of IgA and IgG were purified from the milk of an HIV-positive lactating woman recruited in the United states. Breast milk (BM) IgG, breast milk IgA, and a control antibody, CH31 mIgA2, were tested for phagocytosis of HIV ConS gp140-coated beads at 5-fold dilutions as indicated, and flow cytometry diagrams indicative of the phagocytosis results are shown. The red traces indicate sample antibody setup while black traces indicate the no-antibody control setup, and gray fill indicates the no-target control setup. Antibody-mediated phagocytosis is indicated by a greater area under the curve for the red trace than that for the black trace. Milk IgG and CH31 mIgA2 showed antibody-mediated phagocytosis activity down to 2 μg/ml, while milk IgA showed no antibody-mediated phagocytosis activity even at 250 μg/ml.

    Article Snippet: To test IgA binding to TZM-bl/FcαRI-expressing cells, human IgA1(κ), IgA2(κ) (Athens Research and Technology, Athens GA), purified human secretory IgA (MP Biomedicals, LLC, Solon, Ohio), and FITC-conjugated goat anti-human IgA (Sigma) were used.

    Techniques: Concentration Assay, Activity Assay, Purification, Flow Cytometry, Cytometry

    Breast milk and plasma IgAs may inhibit C.1086 HIV-1 virion binding to epithelial cells. Inhibition of binding to the colonic epithelial cell line HT-29 was assessed using the tier 2 clade C virus HIV-1 C.1086, with 7 to 12 replicates performed over three independent experiments. VRC01 IgA was used as a positive control, and HIV-negative colostrum IgA and anti-influenza virus CH65 IgA were used as negative controls. A dotted line indicates the mean percent inhibition (MPI) cutoff of 31%, calculated as 2 standard deviations plus the MPI of anti-influenza virus hemagglutinin MAb CH65 IgA relative to that of the no-antibody condition.

    Journal: Journal of Virology

    Article Title: Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1

    doi: 10.1128/JVI.02084-18

    Figure Lengend Snippet: Breast milk and plasma IgAs may inhibit C.1086 HIV-1 virion binding to epithelial cells. Inhibition of binding to the colonic epithelial cell line HT-29 was assessed using the tier 2 clade C virus HIV-1 C.1086, with 7 to 12 replicates performed over three independent experiments. VRC01 IgA was used as a positive control, and HIV-negative colostrum IgA and anti-influenza virus CH65 IgA were used as negative controls. A dotted line indicates the mean percent inhibition (MPI) cutoff of 31%, calculated as 2 standard deviations plus the MPI of anti-influenza virus hemagglutinin MAb CH65 IgA relative to that of the no-antibody condition.

    Article Snippet: To test IgA binding to TZM-bl/FcαRI-expressing cells, human IgA1(κ), IgA2(κ) (Athens Research and Technology, Athens GA), purified human secretory IgA (MP Biomedicals, LLC, Solon, Ohio), and FITC-conjugated goat anti-human IgA (Sigma) were used.

    Techniques: Binding Assay, Inhibition, Positive Control

    Milk and plasma IgG, but not IgA, mediate tier 1 HIV MW965 neutralization. To identify HIV-1 neutralization function in antibody fractions of milk and plasma, we determined the neutralization IC 50 in TZM-bl cells for IgA and IgG antibodies isolated from milk and plasma samples of 20 participants. HIV-specific neutralization activities against the clade C tier 1 HIV variants MW965 (and also S0032 for selected samples) are shown, as well as nonspecific antiviral neutralization activities against murine leukemia virus (MLV). Eight HIV-negative controls are also shown, as well as positive-control recombinant monoclonal antibodies (b12 and VRC01, CD4 binding site broadly neutralizing antibodies) in IgA and IgG backbones. No milk IgA samples neutralized HIV-1 MW965 or HIV-1 S0032 , whereas three plasma IgA samples had detectable neutralization.

    Journal: Journal of Virology

    Article Title: Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1

    doi: 10.1128/JVI.02084-18

    Figure Lengend Snippet: Milk and plasma IgG, but not IgA, mediate tier 1 HIV MW965 neutralization. To identify HIV-1 neutralization function in antibody fractions of milk and plasma, we determined the neutralization IC 50 in TZM-bl cells for IgA and IgG antibodies isolated from milk and plasma samples of 20 participants. HIV-specific neutralization activities against the clade C tier 1 HIV variants MW965 (and also S0032 for selected samples) are shown, as well as nonspecific antiviral neutralization activities against murine leukemia virus (MLV). Eight HIV-negative controls are also shown, as well as positive-control recombinant monoclonal antibodies (b12 and VRC01, CD4 binding site broadly neutralizing antibodies) in IgA and IgG backbones. No milk IgA samples neutralized HIV-1 MW965 or HIV-1 S0032 , whereas three plasma IgA samples had detectable neutralization.

    Article Snippet: To test IgA binding to TZM-bl/FcαRI-expressing cells, human IgA1(κ), IgA2(κ) (Athens Research and Technology, Athens GA), purified human secretory IgA (MP Biomedicals, LLC, Solon, Ohio), and FITC-conjugated goat anti-human IgA (Sigma) were used.

    Techniques: Neutralization, Isolation, Positive Control, Recombinant, Binding Assay

    Env antigen specificities of milk IgG and IgA and plasma and milk IgAs do not correlate, while milk and plasma IgG Env antigen-specificities are strongly correlated. (A, C, D, F and H) For 16 HIV-1 + lactating women, milk and plasma samples were obtained, and IgA and IgG were purified from both sample types. For IgA (A) and sIgA (D), binding scores to gp41, gp41 PID, gp140, and gp120 antigens are shown (see Materials and Methods for antigens and calculation). For IgA, scores for binding to gp70 V1/V2, linear V2, linear V3, and linear C5 antigen subspecificities are also shown (C). For IgG, scores are shown for binding to Env gp41, gp41 PID, gp140, and gp120, as well as Gag p24 antigens (F), and to gp70 V1/V2, linear V2, gp70 V3, and linear C5 antigen subspecificities (H). Background-subtracted MFI values below 0 are shown with a value of 0.01. (B, E, G, I, J and K) Correlations between compartments for each antigen specificity or antigen specificity ratio. Kendall’s tau values and corresponding corrected P values (see Materials and Methods) are reported. Boldface P values indicate significant correlations at a P value of

    Journal: Journal of Virology

    Article Title: Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1

    doi: 10.1128/JVI.02084-18

    Figure Lengend Snippet: Env antigen specificities of milk IgG and IgA and plasma and milk IgAs do not correlate, while milk and plasma IgG Env antigen-specificities are strongly correlated. (A, C, D, F and H) For 16 HIV-1 + lactating women, milk and plasma samples were obtained, and IgA and IgG were purified from both sample types. For IgA (A) and sIgA (D), binding scores to gp41, gp41 PID, gp140, and gp120 antigens are shown (see Materials and Methods for antigens and calculation). For IgA, scores for binding to gp70 V1/V2, linear V2, linear V3, and linear C5 antigen subspecificities are also shown (C). For IgG, scores are shown for binding to Env gp41, gp41 PID, gp140, and gp120, as well as Gag p24 antigens (F), and to gp70 V1/V2, linear V2, gp70 V3, and linear C5 antigen subspecificities (H). Background-subtracted MFI values below 0 are shown with a value of 0.01. (B, E, G, I, J and K) Correlations between compartments for each antigen specificity or antigen specificity ratio. Kendall’s tau values and corresponding corrected P values (see Materials and Methods) are reported. Boldface P values indicate significant correlations at a P value of

    Article Snippet: To test IgA binding to TZM-bl/FcαRI-expressing cells, human IgA1(κ), IgA2(κ) (Athens Research and Technology, Athens GA), purified human secretory IgA (MP Biomedicals, LLC, Solon, Ohio), and FITC-conjugated goat anti-human IgA (Sigma) were used.

    Techniques: Purification, Binding Assay

    Breast milk and plasma IgGs, but not IgAs, mediate phagocytosis of HIV-1 virions and Env-coated beads. To determine the phagocytosis function in antibody fractions of breast milk (BM) and plasma (PL), we tested IgA and IgG from milk and plasma of 16 HIV + women for phagocytosis of beads coated with HIV-1 Env ConS gp140 (A), beads coated with HIV-1 Env 1086.C gp140 (B), and fully infectious fluorescent HIV-1 virions (HIV-1 92Th023 -Tomato) (C). Antibodies from an additional five HIV-negative women were also tested as negative controls, as well as the anti-respiratory syncytial virus antibody palivizumab. The black dotted line indicates the positivity cutoff, determined using the mean +3 standard deviations of values of the negative controls used in the assay.

    Journal: Journal of Virology

    Article Title: Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1

    doi: 10.1128/JVI.02084-18

    Figure Lengend Snippet: Breast milk and plasma IgGs, but not IgAs, mediate phagocytosis of HIV-1 virions and Env-coated beads. To determine the phagocytosis function in antibody fractions of breast milk (BM) and plasma (PL), we tested IgA and IgG from milk and plasma of 16 HIV + women for phagocytosis of beads coated with HIV-1 Env ConS gp140 (A), beads coated with HIV-1 Env 1086.C gp140 (B), and fully infectious fluorescent HIV-1 virions (HIV-1 92Th023 -Tomato) (C). Antibodies from an additional five HIV-negative women were also tested as negative controls, as well as the anti-respiratory syncytial virus antibody palivizumab. The black dotted line indicates the positivity cutoff, determined using the mean +3 standard deviations of values of the negative controls used in the assay.

    Article Snippet: To test IgA binding to TZM-bl/FcαRI-expressing cells, human IgA1(κ), IgA2(κ) (Athens Research and Technology, Athens GA), purified human secretory IgA (MP Biomedicals, LLC, Solon, Ohio), and FITC-conjugated goat anti-human IgA (Sigma) were used.

    Techniques:

    IgA-mediated tier 1 HIV-1 neutralization is not enhanced in FcαRI-expressing cells or peripheral blood mononuclear cells. (A) To gain insight into the role of CD89 in HIV infectivity and neutralization, the cDNAs for the human FcαRI and the γ chain of the FcεR were transduced into TZM-bl cells using lentiviral constructs. Specific monoclonal antibodies and flow cytometry detected surface expression of FcαRI on this new TZM-bl cell line. (B) Flow cytometry diagrams show the capability of the TZM-bl/FcαRI cell line to bind to human monomeric IgA1 and monomeric IgA2, human colostrum secretory IgA, and monoclonal 2F5 IgA. (C) To determine whether neutralization activity is enhanced in the presence of Fc alpha receptor 1 (FcαRI), we examined the neutralization IC 50 using TZM-bl cells transduced with FcαRI and also in human peripheral blood mononuclear cells (PBMCs). Milk and plasma IgAs were tested for six participants with a variety of neutralization phenotypes in TZM-bl cells. The positive controls HIVIG (clade C) and b12 IgA MAb and the negative-control 2F5 MAb (broadly neutralizing, but does not neutralize MW965) were also tested. ND, not done for the indicated sample. Neutralization was not enhanced in either FcαRI-transduced TZM-bl cells or in peripheral blood mononuclear cells.

    Journal: Journal of Virology

    Article Title: Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1

    doi: 10.1128/JVI.02084-18

    Figure Lengend Snippet: IgA-mediated tier 1 HIV-1 neutralization is not enhanced in FcαRI-expressing cells or peripheral blood mononuclear cells. (A) To gain insight into the role of CD89 in HIV infectivity and neutralization, the cDNAs for the human FcαRI and the γ chain of the FcεR were transduced into TZM-bl cells using lentiviral constructs. Specific monoclonal antibodies and flow cytometry detected surface expression of FcαRI on this new TZM-bl cell line. (B) Flow cytometry diagrams show the capability of the TZM-bl/FcαRI cell line to bind to human monomeric IgA1 and monomeric IgA2, human colostrum secretory IgA, and monoclonal 2F5 IgA. (C) To determine whether neutralization activity is enhanced in the presence of Fc alpha receptor 1 (FcαRI), we examined the neutralization IC 50 using TZM-bl cells transduced with FcαRI and also in human peripheral blood mononuclear cells (PBMCs). Milk and plasma IgAs were tested for six participants with a variety of neutralization phenotypes in TZM-bl cells. The positive controls HIVIG (clade C) and b12 IgA MAb and the negative-control 2F5 MAb (broadly neutralizing, but does not neutralize MW965) were also tested. ND, not done for the indicated sample. Neutralization was not enhanced in either FcαRI-transduced TZM-bl cells or in peripheral blood mononuclear cells.

    Article Snippet: To test IgA binding to TZM-bl/FcαRI-expressing cells, human IgA1(κ), IgA2(κ) (Athens Research and Technology, Athens GA), purified human secretory IgA (MP Biomedicals, LLC, Solon, Ohio), and FITC-conjugated goat anti-human IgA (Sigma) were used.

    Techniques: Neutralization, Expressing, Infection, Construct, Flow Cytometry, Cytometry, Activity Assay, Transduction, Negative Control

    Significant increase of galectin-8N non-binding IgA in sera from IgAN patients compared to healthy controls. a Total IgA (mean/median for each group (mg/ml sera): 3.4/3.3, 1,7/2.1, 2.3/2.4), b galectin-8N unbound (total bound) IgA (mean/median for each group (mg/ml sera): 2.6/2.4, 1.5/1.0, 1.6/1.7), and c ratio of galectin-8N bound/unbound serum IgA (logarithmic scale on the Y -axis) in sera from 98 IgAN patients, 17 healthy, and 76 kidney disease controls. d Scatter plot of ratio from c ( Y -axis) vs. total IgA ( X -axis). IgA in unfractionated serum (total) or galectin-8N bound fractions was quantitated using nephelometry. Horizontal lines mark the average for each group in a – c . The difference between groups was statistically significant ( p

    Journal: Journal of Clinical Immunology

    Article Title: Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

    doi: 10.1007/s10875-011-9618-3

    Figure Lengend Snippet: Significant increase of galectin-8N non-binding IgA in sera from IgAN patients compared to healthy controls. a Total IgA (mean/median for each group (mg/ml sera): 3.4/3.3, 1,7/2.1, 2.3/2.4), b galectin-8N unbound (total bound) IgA (mean/median for each group (mg/ml sera): 2.6/2.4, 1.5/1.0, 1.6/1.7), and c ratio of galectin-8N bound/unbound serum IgA (logarithmic scale on the Y -axis) in sera from 98 IgAN patients, 17 healthy, and 76 kidney disease controls. d Scatter plot of ratio from c ( Y -axis) vs. total IgA ( X -axis). IgA in unfractionated serum (total) or galectin-8N bound fractions was quantitated using nephelometry. Horizontal lines mark the average for each group in a – c . The difference between groups was statistically significant ( p

    Article Snippet: IgA Measurements IgA content of sera (diluted 1/10 in PBS) or pooled galectin-8N binding fractions from 98 IgAN patients, 20 healthy donors, and 76 controls with IgAN symptoms were analyzed with nephelometry, IMMAGE 800 (Beckman Coulter) using a low concentration Immunoglobulin A Reagent kit (Beckman Coulter).

    Techniques: Binding Assay

    Indication of increased rate of disease progress to end stage renal disease for IgAN patients with low galectin-8 bound/unbound IgA ratio. Kaplan–Meier cumulative analysis of end stage renal disease for 87 IgAN patients, divided according to the ratio of galectin-8N bound/unbound IgA with a cutoff at a ratio 0.50 based on the mean in the control group. Patients with a ratio > 0.50 may have better early (the first 9 years) renal survival than patients with a binding ratio

    Journal: Journal of Clinical Immunology

    Article Title: Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

    doi: 10.1007/s10875-011-9618-3

    Figure Lengend Snippet: Indication of increased rate of disease progress to end stage renal disease for IgAN patients with low galectin-8 bound/unbound IgA ratio. Kaplan–Meier cumulative analysis of end stage renal disease for 87 IgAN patients, divided according to the ratio of galectin-8N bound/unbound IgA with a cutoff at a ratio 0.50 based on the mean in the control group. Patients with a ratio > 0.50 may have better early (the first 9 years) renal survival than patients with a binding ratio

    Article Snippet: IgA Measurements IgA content of sera (diluted 1/10 in PBS) or pooled galectin-8N binding fractions from 98 IgAN patients, 20 healthy donors, and 76 controls with IgAN symptoms were analyzed with nephelometry, IMMAGE 800 (Beckman Coulter) using a low concentration Immunoglobulin A Reagent kit (Beckman Coulter).

    Techniques: Binding Assay

    Simplified schematic of pathological O-glycosylation changes of IgA1 in IgAN and predicted binding of galectin-8. The two top O-glycans NeuAcα2,3Galβ1,3GalNAcα and Galβ1,3GalNAc are predominant (30–40% each) in IgA1 of normal sera [ 35 ], whereas the bottom two, GalNAcα and Neuα2,6GalNAcα, increase in IgA1 from IgAN patients, due to decreased activity of the galactosyltransferase adding the Galβ1,3 ( black circle in top two glycans) and increased activity of the sialyltransferase adding the NeuAcα2,6 ( vertical diamond in bottom glycan) to GalNAc ( open square ). Galectin-8N binds the top O-glycan with high affinity, but not the others [ 24 ]. The NeuAcα2,6 sialyltransferase may also act on the top structure to make NeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAc, but also this structure has strongly reduced affinity for galectin-8N [ 24 ]. Thus, galectin-8N binding of IgA1 is expected to decrease in IgAN

    Journal: Journal of Clinical Immunology

    Article Title: Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

    doi: 10.1007/s10875-011-9618-3

    Figure Lengend Snippet: Simplified schematic of pathological O-glycosylation changes of IgA1 in IgAN and predicted binding of galectin-8. The two top O-glycans NeuAcα2,3Galβ1,3GalNAcα and Galβ1,3GalNAc are predominant (30–40% each) in IgA1 of normal sera [ 35 ], whereas the bottom two, GalNAcα and Neuα2,6GalNAcα, increase in IgA1 from IgAN patients, due to decreased activity of the galactosyltransferase adding the Galβ1,3 ( black circle in top two glycans) and increased activity of the sialyltransferase adding the NeuAcα2,6 ( vertical diamond in bottom glycan) to GalNAc ( open square ). Galectin-8N binds the top O-glycan with high affinity, but not the others [ 24 ]. The NeuAcα2,6 sialyltransferase may also act on the top structure to make NeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAc, but also this structure has strongly reduced affinity for galectin-8N [ 24 ]. Thus, galectin-8N binding of IgA1 is expected to decrease in IgAN

    Article Snippet: IgA Measurements IgA content of sera (diluted 1/10 in PBS) or pooled galectin-8N binding fractions from 98 IgAN patients, 20 healthy donors, and 76 controls with IgAN symptoms were analyzed with nephelometry, IMMAGE 800 (Beckman Coulter) using a low concentration Immunoglobulin A Reagent kit (Beckman Coulter).

    Techniques: Binding Assay, Activity Assay, Activated Clotting Time Assay

    Identification of major galectin-8N binding serum glycoproteins. a SDS-PAGE (4–20% stained with Coomassie) of galectin-8N bound glycoproteins with major proteins indicated as previously identified by Western blot and MALDI-TOF-MS [ 25 ]. b Estimated relative amounts of galectin-8N bound serum proteins in four samples by LC–MS/MS analysis of pooled tryptic peptides. The serum concentrations of galectin-8N bound total protein and IgA were determined by protein assay and nephelometry as given in Table S I . The relative amounts of the remaining galectin-8N bound proteins were determined by LC–MS/MS assuming a linear relationship between sample protein concentration and the summed abundances for the peptides uniquely mapping to each protein (Tables S II and S III ). c SDS-PAGE of galectin-8N unbound ( UB ) and bound ( B ) fractions with the highest protein concentrations from four healthy individuals, four IgAN patients, and three controls with other histological patterns of glomerulonephritis. Indicated to the left are the mobilities of known size markers. The major visible bands of the unbound fractions correspond to albumin (at 67 kDa), transferrin (above the albumin), and IgG heavy chain (below the albumin)

    Journal: Journal of Clinical Immunology

    Article Title: Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

    doi: 10.1007/s10875-011-9618-3

    Figure Lengend Snippet: Identification of major galectin-8N binding serum glycoproteins. a SDS-PAGE (4–20% stained with Coomassie) of galectin-8N bound glycoproteins with major proteins indicated as previously identified by Western blot and MALDI-TOF-MS [ 25 ]. b Estimated relative amounts of galectin-8N bound serum proteins in four samples by LC–MS/MS analysis of pooled tryptic peptides. The serum concentrations of galectin-8N bound total protein and IgA were determined by protein assay and nephelometry as given in Table S I . The relative amounts of the remaining galectin-8N bound proteins were determined by LC–MS/MS assuming a linear relationship between sample protein concentration and the summed abundances for the peptides uniquely mapping to each protein (Tables S II and S III ). c SDS-PAGE of galectin-8N unbound ( UB ) and bound ( B ) fractions with the highest protein concentrations from four healthy individuals, four IgAN patients, and three controls with other histological patterns of glomerulonephritis. Indicated to the left are the mobilities of known size markers. The major visible bands of the unbound fractions correspond to albumin (at 67 kDa), transferrin (above the albumin), and IgG heavy chain (below the albumin)

    Article Snippet: IgA Measurements IgA content of sera (diluted 1/10 in PBS) or pooled galectin-8N binding fractions from 98 IgAN patients, 20 healthy donors, and 76 controls with IgAN symptoms were analyzed with nephelometry, IMMAGE 800 (Beckman Coulter) using a low concentration Immunoglobulin A Reagent kit (Beckman Coulter).

    Techniques: Binding Assay, SDS Page, Staining, Western Blot, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Protein Concentration

    A. Saturation isotherm and Scatchard analysis of 125 I-UG binding to Fn . Binding isotherms of hUG to Fn were performed by incubating increasing concentrations of 125 I-UG. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of hUG, from the total binding (■). The data shown are the mean of triplicate measurements±SD. B. Scatchard plots of hUG binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 15 nM ± 2.6 nM (n = 3). C. Binding isotherms of IgA to Fn. Binding isotherms of IgA to Fn were performed by incubating increasing concentration of 125 I-IgA. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of IgA, from the total binding (■). The data shown are the mean of triplicate measurements. D. Scatchard plots of IgA binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 65 nM ± 5.2 nM (n = 3).

    Journal: FEBS letters

    Article Title: Uteroglobin interacts with the heparin-binding site of fibronectin and prevents fibronectin-IgA complex formation found in IgA-nephropathy

    doi: 10.1016/j.febslet.2008.01.025

    Figure Lengend Snippet: A. Saturation isotherm and Scatchard analysis of 125 I-UG binding to Fn . Binding isotherms of hUG to Fn were performed by incubating increasing concentrations of 125 I-UG. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of hUG, from the total binding (■). The data shown are the mean of triplicate measurements±SD. B. Scatchard plots of hUG binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 15 nM ± 2.6 nM (n = 3). C. Binding isotherms of IgA to Fn. Binding isotherms of IgA to Fn were performed by incubating increasing concentration of 125 I-IgA. Specific binding (■) was calculated by subtracting nonspecific binding (▲), measured as the residual binding in the presence of IgA, from the total binding (■). The data shown are the mean of triplicate measurements. D. Scatchard plots of IgA binding to Fn. The apparent dissociation constant (K d ) estimated from the slope of the line was 65 nM ± 5.2 nM (n = 3).

    Article Snippet: We previously reported that UG-knockout (UG-KO) mice( ) develop abnormal deposition of immunoglobulin A (IgA) and fibronectin (Fn) in the renal glomeruli, reminiscent of the pathologic finding characteristic of human IgA-nephropathy (IgAN)( ).

    Techniques: Binding Assay, Concentration Assay

    Fluorescence flow cytometric analysis of the expression of sIgM, sIgG, sIgA, and sIgE by CL-01 cells and analysis of secreted IgM, IgG, IgA, and IgE. Dot plots depict the expression of sIgM, sIgD, sIgG, and sIgA by CL-01 cells (day 0) and by CL-01 cells cocultured with CD40L-293 cells and IL-4 (100 U/ml) for 5 or 8 days. Numbers inside quadrants are percentages of the total cells. Results are representative of three independent experiments. Histograms show the concentration of IgM (■), IgG ( ), IgA (▨), and IgE (□) accumulated in the fluids of CL-01 cells incubated for 8 days with or without IL-4, and cultured alone, with CD8-293 cells, with CD40L-293 cells, and with CD40L-293 cells in the presence of neutralizing anti-IL-10 and/or anti-TGF- β Abs (the concentrations of IgM and IgE were from cultures with both anti-IL-10 and anti-TGF- β Abs, whereas the concentrations of IgG and IgA were from cultures with anti-IL-10 and anti-TGF- β Abs, respectively). Values are mean ± SD of four determinations from three independent experiments.

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

    Article Title: CD40 Ligand and Appropriate Cytokines Induce Switching to IgG, IgA, and IgE and Coordinated Germinal Center and Plasmacytoid Phenotypic Differentiation in a Human Monoclonal IgM+IgD+ B Cell Line

    doi:

    Figure Lengend Snippet: Fluorescence flow cytometric analysis of the expression of sIgM, sIgG, sIgA, and sIgE by CL-01 cells and analysis of secreted IgM, IgG, IgA, and IgE. Dot plots depict the expression of sIgM, sIgD, sIgG, and sIgA by CL-01 cells (day 0) and by CL-01 cells cocultured with CD40L-293 cells and IL-4 (100 U/ml) for 5 or 8 days. Numbers inside quadrants are percentages of the total cells. Results are representative of three independent experiments. Histograms show the concentration of IgM (■), IgG ( ), IgA (▨), and IgE (□) accumulated in the fluids of CL-01 cells incubated for 8 days with or without IL-4, and cultured alone, with CD8-293 cells, with CD40L-293 cells, and with CD40L-293 cells in the presence of neutralizing anti-IL-10 and/or anti-TGF- β Abs (the concentrations of IgM and IgE were from cultures with both anti-IL-10 and anti-TGF- β Abs, whereas the concentrations of IgG and IgA were from cultures with anti-IL-10 and anti-TGF- β Abs, respectively). Values are mean ± SD of four determinations from three independent experiments.

    Article Snippet: Following rinsing with TBST buffer (10 mM Tris-HCl (pH 8), 150 mM NaCl, 0.02% Tween-20, and 0.02% NaN3 ) and incubation with TBST containing 3% BSA, cells were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (Boehringer Mannheim Corp., Indianapolis, IN), rhodamine-conjugated goat Ab to human IgG (ICN Pharmaceuticals, Inc., Costa Mesa, CA), and FITC-conjugated mouse mAb to human IgA (Sigma Chemical Co., St. Louis, MO).

    Techniques: Fluorescence, Flow Cytometry, Expressing, Concentration Assay, Incubation, Cell Culture

    IL-6 induces plasmacytoid differentiation in IgG-switched CL-01 cells. CL-01 cells were incubated with medium only, with CD40L-293 cells, IL-4, and IL-10, and with CD40L-293 cells, IL-4, IL-10, and IL-6 for 8 days. IL-6 was added at day 5 of culture. 4′,6-Diamidine-2′-phenylindole dihydrochloride (blue), rhodamine-conjugated mouse Abs to human IgG (red), and FITC-conjugated mouse mAb to human IgA were used to visualize cellular nuclei, IgG, and IgA, respectively. Arrows indicate CL-01 cells that show accumulation of cIgG and cIgA as well as plasmacytoid morphology (×1000). Data were obtained from one of four experiments yielding similar results.

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

    Article Title: CD40 Ligand and Appropriate Cytokines Induce Switching to IgG, IgA, and IgE and Coordinated Germinal Center and Plasmacytoid Phenotypic Differentiation in a Human Monoclonal IgM+IgD+ B Cell Line

    doi:

    Figure Lengend Snippet: IL-6 induces plasmacytoid differentiation in IgG-switched CL-01 cells. CL-01 cells were incubated with medium only, with CD40L-293 cells, IL-4, and IL-10, and with CD40L-293 cells, IL-4, IL-10, and IL-6 for 8 days. IL-6 was added at day 5 of culture. 4′,6-Diamidine-2′-phenylindole dihydrochloride (blue), rhodamine-conjugated mouse Abs to human IgG (red), and FITC-conjugated mouse mAb to human IgA were used to visualize cellular nuclei, IgG, and IgA, respectively. Arrows indicate CL-01 cells that show accumulation of cIgG and cIgA as well as plasmacytoid morphology (×1000). Data were obtained from one of four experiments yielding similar results.

    Article Snippet: Following rinsing with TBST buffer (10 mM Tris-HCl (pH 8), 150 mM NaCl, 0.02% Tween-20, and 0.02% NaN3 ) and incubation with TBST containing 3% BSA, cells were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (Boehringer Mannheim Corp., Indianapolis, IN), rhodamine-conjugated goat Ab to human IgG (ICN Pharmaceuticals, Inc., Costa Mesa, CA), and FITC-conjugated mouse mAb to human IgA (Sigma Chemical Co., St. Louis, MO).

    Techniques: Incubation

    IgA1 O-glycosylation assay in supernatant of IgAN patients and normal controls peripheral B lymphocytes.

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Astragalus membranaceus up-regulate Cosmc expression and reverse IgA dys-glycosylation in IgA nephropathy

    doi: 10.1186/1472-6882-14-195

    Figure Lengend Snippet: IgA1 O-glycosylation assay in supernatant of IgAN patients and normal controls peripheral B lymphocytes.

    Article Snippet: Standard curve constructed with a serial dilution of IgA1 standard serum (Nordic Immunological Laboratories, Netherlands) was used to calculate IgA1 concentration.

    Techniques:

    IgA1 (ng/mL) concentration in supernatant from IgAN patients and normal controls peripheral B lymphocytes. After LPS stimulation, IgA1 was remarkably increased, especially in IgAN group. Treatment with AMI could inhibit the secretion of IgA1.

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Astragalus membranaceus up-regulate Cosmc expression and reverse IgA dys-glycosylation in IgA nephropathy

    doi: 10.1186/1472-6882-14-195

    Figure Lengend Snippet: IgA1 (ng/mL) concentration in supernatant from IgAN patients and normal controls peripheral B lymphocytes. After LPS stimulation, IgA1 was remarkably increased, especially in IgAN group. Treatment with AMI could inhibit the secretion of IgA1.

    Article Snippet: Standard curve constructed with a serial dilution of IgA1 standard serum (Nordic Immunological Laboratories, Netherlands) was used to calculate IgA1 concentration.

    Techniques: Concentration Assay

    Concentration of immunoglobulins (IgA, IgG and IgM) in chickens infected with C. jejuni Samples were analyzed at days 15, 22 and 30 of age; a ) IgA b ) IgG and c ) IgM. Error bars represent the mean values and standard errors of 6 isolators, each with 15 birds. Interactions between treatment and age effect significantly ( p

    Journal: BMC Veterinary Research

    Article Title: Influence of silver nanoparticles on growth and health of broiler chickens after infection with Campylobacter jejuni

    doi: 10.1186/s12917-017-1323-x

    Figure Lengend Snippet: Concentration of immunoglobulins (IgA, IgG and IgM) in chickens infected with C. jejuni Samples were analyzed at days 15, 22 and 30 of age; a ) IgA b ) IgG and c ) IgM. Error bars represent the mean values and standard errors of 6 isolators, each with 15 birds. Interactions between treatment and age effect significantly ( p

    Article Snippet: Chicken plasma specific antibodies such as IgA (Bethyl laboratories, cat. no. E33–103), IgG (Bethyl laboratories, cat. no. E33–104) and IgM (Bethyl laboratories, cat. no. E33–102) concentrations were determined in diluted samples (1:100) by enzyme-linked immunosorbent assay (ELISA) using microtiter plates (NuncImmunoplate 96-well, cat. no. 446612) as per manufacturer’s ELISA quantitation kits (Bethyl Laboratories Inc., Montgomery, TX, USA).

    Techniques: Concentration Assay, Infection

    Binding interactions of Fog-1 and B2 IgG variants with human FcγR. (A) Binding of monomeric B2 IgGs was measured for the high-affinity FcγRI using the B2KA cell line and flow cytometry. (B–H) Binding of (B, G, H) pre-complexed B2 or (C–F) Fog-1 IgGs was measured using CHO cell lines expressing FcγRIIa of allotypes (B) 131R and (C) 131H, (D) FcγRIIb and FcγRIIIa of allotypes (E, G) 158F and (F, H) 158 V. The level of background binding is given by the negative control Ab, IgA,κ or IgA,λ as appropriate. Graphs show mean fluorescence of ≥12 000 cells at each Ab concentration and are typical of the results obtained in at least three experiments with each receptor.

    Journal: European Journal of Immunology

    Article Title: Low-affinity FcγR interactions can decide the fate of novel human IgG-sensitised red blood cells and platelets

    doi: 10.1002/eji.201343825

    Figure Lengend Snippet: Binding interactions of Fog-1 and B2 IgG variants with human FcγR. (A) Binding of monomeric B2 IgGs was measured for the high-affinity FcγRI using the B2KA cell line and flow cytometry. (B–H) Binding of (B, G, H) pre-complexed B2 or (C–F) Fog-1 IgGs was measured using CHO cell lines expressing FcγRIIa of allotypes (B) 131R and (C) 131H, (D) FcγRIIb and FcγRIIIa of allotypes (E, G) 158F and (F, H) 158 V. The level of background binding is given by the negative control Ab, IgA,κ or IgA,λ as appropriate. Graphs show mean fluorescence of ≥12 000 cells at each Ab concentration and are typical of the results obtained in at least three experiments with each receptor.

    Article Snippet: Human IgA1,κ purified myeloma protein (The Binding Site, Birmingham, UK) or IgA,λ (Jackson ImmunoResearch, Newmarket, UK) were used as negative control test Abs.

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Expressing, Negative Control, Fluorescence, Concentration Assay

    IgA1 production in response to cytokine stimulation. IgA1 concentration of supernatant was determined by sandwich ELISA. ( A ) IgA concentration of supernatant from cytokine stimulated cells. IgA1 content of supernatant from IL-4 stimulated cells was significantly higher than that of cells incubated with other cytokines, except for IL-2, and control (P

    Journal: Nephrology Dialysis Transplantation

    Article Title: Down-regulation of core 1 ?1,3-galactosyltransferase and Cosmc by Th2 cytokine alters O-glycosylation of IgA1

    doi: 10.1093/ndt/gfq325

    Figure Lengend Snippet: IgA1 production in response to cytokine stimulation. IgA1 concentration of supernatant was determined by sandwich ELISA. ( A ) IgA concentration of supernatant from cytokine stimulated cells. IgA1 content of supernatant from IL-4 stimulated cells was significantly higher than that of cells incubated with other cytokines, except for IL-2, and control (P

    Article Snippet: After three washes with PBS containing 0.05% Tween-20 (PBST), plates were blocked by adding PBS containing 1% bovine serum albumin (BSA) to the wells for 90 min. Next, 50 µL of supernatant sample or standard human IgA1 (CALBIOCHEM, La Jolla, CA, USA) was added to the reaction wells and then incubated for 90 min. After three washes, 0.75 µg/mL alkaline phosphatase conjugated goat anti-human IgA (Southern Biotechnology Associates, Birmingham, AL, USA) in 1% BSA/PBS was added to the reaction wells and then incubated for 90 min. Plates were washed three times and developed with a substrate solution of 1 mg/mL p -nitrophenyl phosphate disodium salt (SIGMA, St. Louis, MO, USA) in 0.1 M glycine buffer containing 1 mM MgCl2 , 1 mM ZnCl2 , pH 10.4.

    Techniques: Concentration Assay, Sandwich ELISA, Incubation

    Lectin binding to IgA1 derived from DAKIKI cells. Glycosylation of IgA1 was assessed by enzyme-linked lectin binding assay. Lectin binding to GalNAc of IgA1 was significantly higher after IL-4 stimulation than after stimulation by either other cytokines or medium control (P

    Journal: Nephrology Dialysis Transplantation

    Article Title: Down-regulation of core 1 ?1,3-galactosyltransferase and Cosmc by Th2 cytokine alters O-glycosylation of IgA1

    doi: 10.1093/ndt/gfq325

    Figure Lengend Snippet: Lectin binding to IgA1 derived from DAKIKI cells. Glycosylation of IgA1 was assessed by enzyme-linked lectin binding assay. Lectin binding to GalNAc of IgA1 was significantly higher after IL-4 stimulation than after stimulation by either other cytokines or medium control (P

    Article Snippet: After three washes with PBS containing 0.05% Tween-20 (PBST), plates were blocked by adding PBS containing 1% bovine serum albumin (BSA) to the wells for 90 min. Next, 50 µL of supernatant sample or standard human IgA1 (CALBIOCHEM, La Jolla, CA, USA) was added to the reaction wells and then incubated for 90 min. After three washes, 0.75 µg/mL alkaline phosphatase conjugated goat anti-human IgA (Southern Biotechnology Associates, Birmingham, AL, USA) in 1% BSA/PBS was added to the reaction wells and then incubated for 90 min. Plates were washed three times and developed with a substrate solution of 1 mg/mL p -nitrophenyl phosphate disodium salt (SIGMA, St. Louis, MO, USA) in 0.1 M glycine buffer containing 1 mM MgCl2 , 1 mM ZnCl2 , pH 10.4.

    Techniques: Binding Assay, Derivative Assay

    Concentration of anti-Neu5Gc IgA (A: yellow), IgM (B: orange), and IgG (C: blue) antibodies in patients suffering from Hashimoto thyroiditis.

    Journal: BioMed Research International

    Article Title: Prevalence of Anti-Neu5Gc Antibodies in Patients with Hypothyroidism

    doi: 10.1155/2014/963230

    Figure Lengend Snippet: Concentration of anti-Neu5Gc IgA (A: yellow), IgM (B: orange), and IgG (C: blue) antibodies in patients suffering from Hashimoto thyroiditis.

    Article Snippet: Peroxidase-conjugated, goat, and anti-human IgG or IgM or IgA were purchased by AbD Serotec.

    Techniques: Concentration Assay

    Significant increase of galectin-8N non-binding IgA in sera from IgAN patients compared to healthy controls. a Total IgA (mean/median for each group (mg/ml sera): 3.4/3.3, 1,7/2.1, 2.3/2.4), b galectin-8N unbound (total bound) IgA (mean/median for each group (mg/ml sera): 2.6/2.4, 1.5/1.0, 1.6/1.7), and c ratio of galectin-8N bound/unbound serum IgA (logarithmic scale on the Y -axis) in sera from 98 IgAN patients, 17 healthy, and 76 kidney disease controls. d Scatter plot of ratio from c ( Y -axis) vs. total IgA ( X -axis). IgA in unfractionated serum (total) or galectin-8N bound fractions was quantitated using nephelometry. Horizontal lines mark the average for each group in a – c . The difference between groups was statistically significant ( p

    Journal: Journal of Clinical Immunology

    Article Title: Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

    doi: 10.1007/s10875-011-9618-3

    Figure Lengend Snippet: Significant increase of galectin-8N non-binding IgA in sera from IgAN patients compared to healthy controls. a Total IgA (mean/median for each group (mg/ml sera): 3.4/3.3, 1,7/2.1, 2.3/2.4), b galectin-8N unbound (total bound) IgA (mean/median for each group (mg/ml sera): 2.6/2.4, 1.5/1.0, 1.6/1.7), and c ratio of galectin-8N bound/unbound serum IgA (logarithmic scale on the Y -axis) in sera from 98 IgAN patients, 17 healthy, and 76 kidney disease controls. d Scatter plot of ratio from c ( Y -axis) vs. total IgA ( X -axis). IgA in unfractionated serum (total) or galectin-8N bound fractions was quantitated using nephelometry. Horizontal lines mark the average for each group in a – c . The difference between groups was statistically significant ( p

    Article Snippet: Introduction Immunoglobulin A nephritis (IgAN) was first described in 1968 by Berger and colleagues; in an immunofluorescence study of renal biopsies from an unknown form of glomerulonephritis, mesangial deposits with IgA as their main component were found [ ].

    Techniques: Binding Assay

    Indication of increased rate of disease progress to end stage renal disease for IgAN patients with low galectin-8 bound/unbound IgA ratio. Kaplan–Meier cumulative analysis of end stage renal disease for 87 IgAN patients, divided according to the ratio of galectin-8N bound/unbound IgA with a cutoff at a ratio 0.50 based on the mean in the control group. Patients with a ratio > 0.50 may have better early (the first 9 years) renal survival than patients with a binding ratio

    Journal: Journal of Clinical Immunology

    Article Title: Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

    doi: 10.1007/s10875-011-9618-3

    Figure Lengend Snippet: Indication of increased rate of disease progress to end stage renal disease for IgAN patients with low galectin-8 bound/unbound IgA ratio. Kaplan–Meier cumulative analysis of end stage renal disease for 87 IgAN patients, divided according to the ratio of galectin-8N bound/unbound IgA with a cutoff at a ratio 0.50 based on the mean in the control group. Patients with a ratio > 0.50 may have better early (the first 9 years) renal survival than patients with a binding ratio

    Article Snippet: Introduction Immunoglobulin A nephritis (IgAN) was first described in 1968 by Berger and colleagues; in an immunofluorescence study of renal biopsies from an unknown form of glomerulonephritis, mesangial deposits with IgA as their main component were found [ ].

    Techniques: Binding Assay

    Simplified schematic of pathological O-glycosylation changes of IgA1 in IgAN and predicted binding of galectin-8. The two top O-glycans NeuAcα2,3Galβ1,3GalNAcα and Galβ1,3GalNAc are predominant (30–40% each) in IgA1 of normal sera [ 35 ], whereas the bottom two, GalNAcα and Neuα2,6GalNAcα, increase in IgA1 from IgAN patients, due to decreased activity of the galactosyltransferase adding the Galβ1,3 ( black circle in top two glycans) and increased activity of the sialyltransferase adding the NeuAcα2,6 ( vertical diamond in bottom glycan) to GalNAc ( open square ). Galectin-8N binds the top O-glycan with high affinity, but not the others [ 24 ]. The NeuAcα2,6 sialyltransferase may also act on the top structure to make NeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAc, but also this structure has strongly reduced affinity for galectin-8N [ 24 ]. Thus, galectin-8N binding of IgA1 is expected to decrease in IgAN

    Journal: Journal of Clinical Immunology

    Article Title: Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

    doi: 10.1007/s10875-011-9618-3

    Figure Lengend Snippet: Simplified schematic of pathological O-glycosylation changes of IgA1 in IgAN and predicted binding of galectin-8. The two top O-glycans NeuAcα2,3Galβ1,3GalNAcα and Galβ1,3GalNAc are predominant (30–40% each) in IgA1 of normal sera [ 35 ], whereas the bottom two, GalNAcα and Neuα2,6GalNAcα, increase in IgA1 from IgAN patients, due to decreased activity of the galactosyltransferase adding the Galβ1,3 ( black circle in top two glycans) and increased activity of the sialyltransferase adding the NeuAcα2,6 ( vertical diamond in bottom glycan) to GalNAc ( open square ). Galectin-8N binds the top O-glycan with high affinity, but not the others [ 24 ]. The NeuAcα2,6 sialyltransferase may also act on the top structure to make NeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAc, but also this structure has strongly reduced affinity for galectin-8N [ 24 ]. Thus, galectin-8N binding of IgA1 is expected to decrease in IgAN

    Article Snippet: Introduction Immunoglobulin A nephritis (IgAN) was first described in 1968 by Berger and colleagues; in an immunofluorescence study of renal biopsies from an unknown form of glomerulonephritis, mesangial deposits with IgA as their main component were found [ ].

    Techniques: Binding Assay, Activity Assay, Activated Clotting Time Assay

    Identification of major galectin-8N binding serum glycoproteins. a SDS-PAGE (4–20% stained with Coomassie) of galectin-8N bound glycoproteins with major proteins indicated as previously identified by Western blot and MALDI-TOF-MS [ 25 ]. b Estimated relative amounts of galectin-8N bound serum proteins in four samples by LC–MS/MS analysis of pooled tryptic peptides. The serum concentrations of galectin-8N bound total protein and IgA were determined by protein assay and nephelometry as given in Table S I . The relative amounts of the remaining galectin-8N bound proteins were determined by LC–MS/MS assuming a linear relationship between sample protein concentration and the summed abundances for the peptides uniquely mapping to each protein (Tables S II and S III ). c SDS-PAGE of galectin-8N unbound ( UB ) and bound ( B ) fractions with the highest protein concentrations from four healthy individuals, four IgAN patients, and three controls with other histological patterns of glomerulonephritis. Indicated to the left are the mobilities of known size markers. The major visible bands of the unbound fractions correspond to albumin (at 67 kDa), transferrin (above the albumin), and IgG heavy chain (below the albumin)

    Journal: Journal of Clinical Immunology

    Article Title: Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

    doi: 10.1007/s10875-011-9618-3

    Figure Lengend Snippet: Identification of major galectin-8N binding serum glycoproteins. a SDS-PAGE (4–20% stained with Coomassie) of galectin-8N bound glycoproteins with major proteins indicated as previously identified by Western blot and MALDI-TOF-MS [ 25 ]. b Estimated relative amounts of galectin-8N bound serum proteins in four samples by LC–MS/MS analysis of pooled tryptic peptides. The serum concentrations of galectin-8N bound total protein and IgA were determined by protein assay and nephelometry as given in Table S I . The relative amounts of the remaining galectin-8N bound proteins were determined by LC–MS/MS assuming a linear relationship between sample protein concentration and the summed abundances for the peptides uniquely mapping to each protein (Tables S II and S III ). c SDS-PAGE of galectin-8N unbound ( UB ) and bound ( B ) fractions with the highest protein concentrations from four healthy individuals, four IgAN patients, and three controls with other histological patterns of glomerulonephritis. Indicated to the left are the mobilities of known size markers. The major visible bands of the unbound fractions correspond to albumin (at 67 kDa), transferrin (above the albumin), and IgG heavy chain (below the albumin)

    Article Snippet: Introduction Immunoglobulin A nephritis (IgAN) was first described in 1968 by Berger and colleagues; in an immunofluorescence study of renal biopsies from an unknown form of glomerulonephritis, mesangial deposits with IgA as their main component were found [ ].

    Techniques: Binding Assay, SDS Page, Staining, Western Blot, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Protein Concentration

    Decreased clearance of IgAN patient IgA by CD89-expressing Kupffer cells. (A) IgA, purified from a normal donor and from a patient with IgAN, was stained with Cy3-anti-human IgA Ab, and subsequently incubated with Kupffer cells from CD89 transgenic mice at a concentration of 1 μg/mL and a temperature of 37°C. After 1-h-incubation, intracellular vesicles containing IgA were diminished when IgAN patient-derived IgA endocytosis was assessed and compared with normal human IgA. (B) sCD89-IgA complexes in cell supernatant of (A) were detected by ELISA using A3 and anti-mouse IgA mAb, using 1 μg/mL human IgA binding to 5 μg/mL recCD89 as an internal control. The sCD89-IgA complexes were detected in the Kupffer cell supernatant after 1h of incubation with either control IgA or patient IgA. (C) Decreased clearance of 10 μg IgAN patient-derived IgA in C57BL/6-Tg mice when compared to normal human IgA. A total of 10 μg of each of the proteins was injected into the tail vein of C57BL/6-Tg (n = 5 per group). At the indicated times after injection, sera were obtained and IgA levels were determined. IgA from IgAN patients was cleared less quickly in Tg mice compared with normal IgA. (D) Induction of mesangial IgA/sCD89 deposits by injecting patient IgA into 6-wk-old C57BL/6-Tg mice. Double staining by anti-human IgA and anti-CD89 in kidney 48 h after constant injection of patient IgA into C57BL/6-Tg mice(n = 4) is shown. Bar = 10μm.

    Journal: PLoS ONE

    Article Title: Critical Role of Kupffer Cell CD89 Expression in Experimental IgA Nephropathy

    doi: 10.1371/journal.pone.0159426

    Figure Lengend Snippet: Decreased clearance of IgAN patient IgA by CD89-expressing Kupffer cells. (A) IgA, purified from a normal donor and from a patient with IgAN, was stained with Cy3-anti-human IgA Ab, and subsequently incubated with Kupffer cells from CD89 transgenic mice at a concentration of 1 μg/mL and a temperature of 37°C. After 1-h-incubation, intracellular vesicles containing IgA were diminished when IgAN patient-derived IgA endocytosis was assessed and compared with normal human IgA. (B) sCD89-IgA complexes in cell supernatant of (A) were detected by ELISA using A3 and anti-mouse IgA mAb, using 1 μg/mL human IgA binding to 5 μg/mL recCD89 as an internal control. The sCD89-IgA complexes were detected in the Kupffer cell supernatant after 1h of incubation with either control IgA or patient IgA. (C) Decreased clearance of 10 μg IgAN patient-derived IgA in C57BL/6-Tg mice when compared to normal human IgA. A total of 10 μg of each of the proteins was injected into the tail vein of C57BL/6-Tg (n = 5 per group). At the indicated times after injection, sera were obtained and IgA levels were determined. IgA from IgAN patients was cleared less quickly in Tg mice compared with normal IgA. (D) Induction of mesangial IgA/sCD89 deposits by injecting patient IgA into 6-wk-old C57BL/6-Tg mice. Double staining by anti-human IgA and anti-CD89 in kidney 48 h after constant injection of patient IgA into C57BL/6-Tg mice(n = 4) is shown. Bar = 10μm.

    Article Snippet: Raji cells (5×103 /well) were also transferred to the same plates and cultures were incubated with a recombinant anti-CD20 IgA antibody generated at our lab in different concentrations (0, 0.1, 1.0, 10.0 μg/mL) at 37°C for 20 h, an IgA isotype (Abcam) was used as a negative control.

    Techniques: Expressing, Purification, Staining, Incubation, Transgenic Assay, Mouse Assay, Concentration Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Injection, Double Staining

    IgA clearance induced by CD89. (A) Enhanced efficiency of a 60-min serum clearance challenge by 10 μg human mIgA1 in C57BL/6-Tg mice. A total of 10 μg of human mIgA1 was injected into the tail vein of C57BL/6-Tg or WT mice (n = 5 per group). At the indicated times post-injection, sera were obtained and IgA levels were determined. IgA cleared more quickly in Tg mice(*). (B) IgA clearance by CD89 on Kupffer cells of CD89 transgenic mice. A total of 10 μg of FITC-labeled human mIgA was injected into the tail vein of C57BL/6-Tg mice (n = 5 per group). Mice were sacrificed after 10 min or 1 h, livers were stained with rabbit anti–human CD89 mAb and Cy3-goat anti-rabbit IgG, as indicated. (C) IgA clearance by CD89 on Kupffer cells from CD89 transgenic mice analyzed in vitro . Cy3-labeled human mIgA (1 μg/mL) was incubated with Kupffer cells from CD89 transgenic mice for 1h at 37°C.

    Journal: PLoS ONE

    Article Title: Critical Role of Kupffer Cell CD89 Expression in Experimental IgA Nephropathy

    doi: 10.1371/journal.pone.0159426

    Figure Lengend Snippet: IgA clearance induced by CD89. (A) Enhanced efficiency of a 60-min serum clearance challenge by 10 μg human mIgA1 in C57BL/6-Tg mice. A total of 10 μg of human mIgA1 was injected into the tail vein of C57BL/6-Tg or WT mice (n = 5 per group). At the indicated times post-injection, sera were obtained and IgA levels were determined. IgA cleared more quickly in Tg mice(*). (B) IgA clearance by CD89 on Kupffer cells of CD89 transgenic mice. A total of 10 μg of FITC-labeled human mIgA was injected into the tail vein of C57BL/6-Tg mice (n = 5 per group). Mice were sacrificed after 10 min or 1 h, livers were stained with rabbit anti–human CD89 mAb and Cy3-goat anti-rabbit IgG, as indicated. (C) IgA clearance by CD89 on Kupffer cells from CD89 transgenic mice analyzed in vitro . Cy3-labeled human mIgA (1 μg/mL) was incubated with Kupffer cells from CD89 transgenic mice for 1h at 37°C.

    Article Snippet: Raji cells (5×103 /well) were also transferred to the same plates and cultures were incubated with a recombinant anti-CD20 IgA antibody generated at our lab in different concentrations (0, 0.1, 1.0, 10.0 μg/mL) at 37°C for 20 h, an IgA isotype (Abcam) was used as a negative control.

    Techniques: Mouse Assay, Injection, Transgenic Assay, Labeling, Staining, In Vitro, Incubation