Journal:

Article Title: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies

doi:

Figure Lengend Snippet: Binding of IgG from normal human sera to IgA1 and IgA2 myeloma proteins and Fab IgA1 with intact and modified hinge region glycans

Article Snippet: Biotinylated polyclonal antibodies to IgM and F(ab′) 2 fragment of anti-IgA and anti-IgG (all heavy chain specific) were purchased from Southern Biotechnology Associates Inc. (Birmingham, Alabama, USA).

Techniques: Binding Assay, Modification

Journal:

Article Title: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies

doi:

Figure Lengend Snippet: Correlation of binding of HAA and normal serum IgG to IgA myeloma proteins. The binding of serum IgG to IgA1 (filled squares) and IgA2 (open circle) myeloma proteins. The binding of IgG to IgA1 significantly correlated with HAA binding (r = 0.875, P = 0.044), indicating requirement of terminal GalNAc residues for IgG binding.

Article Snippet: Biotinylated polyclonal antibodies to IgM and F(ab′) 2 fragment of anti-IgA and anti-IgG (all heavy chain specific) were purchased from Southern Biotechnology Associates Inc. (Birmingham, Alabama, USA).

Techniques: Binding Assay

Journal:

Article Title: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies

doi:

Figure Lengend Snippet: Binding of serum of IgG from IgAN patients and healthy controls to intact and modified IgA myeloma proteins and their Fab fragments

Article Snippet: Biotinylated polyclonal antibodies to IgM and F(ab′) 2 fragment of anti-IgA and anti-IgG (all heavy chain specific) were purchased from Southern Biotechnology Associates Inc. (Birmingham, Alabama, USA).

Techniques: Binding Assay, Modification

Journal:

Article Title: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies

doi:

Figure Lengend Snippet: The binding of serum IgG to Fab fragment of IgA1 (Ste) myeloma protein. Wells of microtiter plates were coated with Fab fragment of IgA1, incubated with diluted sera from 20 IgAN patients, 20 healthy controls, and 20 patients with non-IgA GN and subsequently with biotinylated mAb specific for IgG, avidin-alkaline phosphatase, and phosphatase substrate. Data shown are OD at 405 nm, mean and SD. Statistical significance is noted; NS, not significant.

Article Snippet: Biotinylated polyclonal antibodies to IgM and F(ab′) 2 fragment of anti-IgA and anti-IgG (all heavy chain specific) were purchased from Southern Biotechnology Associates Inc. (Birmingham, Alabama, USA).

Techniques: Binding Assay, Incubation, Avidin-Biotin Assay

Journal:

Article Title: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies

doi:

Figure Lengend Snippet: Subclass specificity of serum IgG binding to a,a-IgA1. Wells of microtiter plates were coated with myeloma a,a-IgA1, incubated with sera (diluted 1:10) from 30 IgAN patients and 30 healthy controls (C) and subsequently with biotinylated mAb specific for IgG1, IgG2, IgG3, and IgG4; avidin-alkaline phosphatase; and phosphatase substrate. Data shown are OD at 405 nm, mean and SD.

Article Snippet: Biotinylated polyclonal antibodies to IgM and F(ab′) 2 fragment of anti-IgA and anti-IgG (all heavy chain specific) were purchased from Southern Biotechnology Associates Inc. (Birmingham, Alabama, USA).

Techniques: Binding Assay, Incubation, Avidin-Biotin Assay

Journal:

Article Title: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies

doi:

Figure Lengend Snippet: Distribution of IgM, IgA, IgG, and reactivity with HAA in serum fractions from Superose 6 column. Serum from an IgAN patient was fractionated on a Superose 6 column (0.9 × 58.5 cm); 0.25-mL fractions were collected and analyzed by ELISA for the content of IgM (squares), IgA (circles), IgG (open triangles), and reactivity with HAA (filled triangles). Fractions containing aberrantly glycosylated IgA were pooled (HAA pool) and used in the inhibition experiment.

Article Snippet: Biotinylated polyclonal antibodies to IgM and F(ab′) 2 fragment of anti-IgA and anti-IgG (all heavy chain specific) were purchased from Southern Biotechnology Associates Inc. (Birmingham, Alabama, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition

Journal:

Article Title: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies

doi:

Figure Lengend Snippet: Inhibition of IgA1-IgG complex formation. IgA1-IgG CICs from an IgAN patient (left columns 1–6) and a healthy control (right columns 1–6) prepared by size-exclusion chromatography on Superose 6 were dissociated at pH 3.0. Aliquots of dissociated CICs were incubated with agarose- or Sepharose-immobilized inhibitors: 1, IgA1(Mce); 2, a,a-IgA1(Mce); 3, GalNAc; 4, GlcNAc; 5, a-OSM; and 6, HSA. The mixture was adjusted to neutral pH to allow re-formation of immune complexes, and the affinity of IgG toward the particular inhibitor was determined as described in Methods.

Article Snippet: Biotinylated polyclonal antibodies to IgM and F(ab′) 2 fragment of anti-IgA and anti-IgG (all heavy chain specific) were purchased from Southern Biotechnology Associates Inc. (Birmingham, Alabama, USA).

Techniques: Inhibition, Size-exclusion Chromatography, Incubation

Journal:

Article Title: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies

doi:

Figure Lengend Snippet: Binding of serum IgA1, IgM, and IgG from IgAN patients and healthy controls to Fab fragment of IgA1 (Ste) and partially de-O-glycosylated Fab fragment of IgA1 (Ste)

Article Snippet: Biotinylated polyclonal antibodies to IgM and F(ab′) 2 fragment of anti-IgA and anti-IgG (all heavy chain specific) were purchased from Southern Biotechnology Associates Inc. (Birmingham, Alabama, USA).

Techniques: Binding Assay

Journal: The EMBO Journal

Article Title: Memory IgM protects endogenous insulin from autoimmune destruction

doi: 10.15252/embj.2020107621

Figure Lengend Snippet: A Schematic illustration of proinsulin‐derived full‐length C‐peptide and insulin. B Table comparing human to murine C‐peptide amino acid sequences. Underlined: sequence used as peptide for immunization. C Schematic immunization schedule. D–H (D) Serum anti‐C‐peptide‐immunoglobulins (coating: C‐peptide) titers of mice immunized with cCP ( n = 9 for d14 and d28; n = 5 for d49) and (G) anti‐insulin‐immunoglobulins (coating: insulin) titers of mice immunized with complex native insulin (cInsulin, n = 5) and control‐immunized (CI: CpG only, n = 3) measured by ELISA at indicated days. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann–Whitney U‐test. (E, H) ELISpot assays showing CP‐specific IgG ( n = 4 mice/group) (E) and insulin‐specific IgG ( n = 3 mice/group) (H) producing splenocytes at day 14. Top lane showing representative images of wells. Mean ± SD, statistical significance was calculated by using Mann–Whitney U‐test, * P < 0.05, ** P < 0.01. (F) Schematic illustration of native insulin (cInsulin) and full‐length C‐peptide (cCP) tetrameric polyvalent complexes. I Flow cytometric analysis of splenocytes of mice immunized with cInsulin or control immunization on day 26. Left and middle panel showing germinal center (GC) B cells (GL‐7 + ) pre‐gated on B cells (CD19 + B220 + ). Right panel showing insulin‐reactive GC B cells pre‐gated on GL‐7 + cells in a histogram. Data are representative of two independent experiments with n = 4/group.

Article Snippet: Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standard.

Techniques: Derivative Assay, Sequencing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Enzyme-linked Immunospot

Journal: The EMBO Journal

Article Title: Memory IgM protects endogenous insulin from autoimmune destruction

doi: 10.15252/embj.2020107621

Figure Lengend Snippet: Flow cytometric analysis of peripheral blood showing B cells (CD19 + Thy1.2 ‐ ) and T cells (Thy1.2 + CD19 ‐ ) of wild‐type (left) and B‐cell‐deficient (right) mice. Cells were pre‐gated on lymphocytes. Representative of three independent experiments ( n = 5/group). Blood glucose levels of cInsulin‐immunized (red: WT, n = 5; yellow: B‐cell‐deficient, n = 5) and CI mice (gray, n = 8) were assessed at indicated days post‐immunization. Dots represent individual mice, mean ± SD. Statistical significance was calculated by using repeated measure ANOVA test, *** P < 0.001, **** P < 0.0001. Urine glucose levels of cInsulin‐immunized (red, n = 3 for d0, d26; n = 5 for d14) and CI mice (gray, n = 3) were monitored at indicated days post‐immunization. Upper panel showing visualization of glucose standard (top lane) and representative images of tested animals (middle and bottom lanes), respectively. Dots represent individual mice, mean ± SD. Statistical significance was calculated by using repeated measure ANOVA test, * P < 0.05. Water intake of cInsulin‐immunized ( n = 5) and CI mice (gray, n = 5) monitored from d21 to d26, mean ± SD. Flow cytometric analysis of the pancreas of cInsulin‐immunized (red) and CI mice (gray) at day 26. Upper panel showing pancreatic macrophages (CD11b + Ly6G ‐ ), neutrophils (Ly6G + CD11b+), and B cells (CD19 + ) pre‐gated on living cells (FVD ‐ ). Lower panel showing histograms for insulin binding (left) and streptavidin (SAV)‐binding (right). Representative of two independent experiments with n = 5/group. F: Serum pancreatic lipase titers of mice immunized with cInsulin ( n = 5) or control immunization ( n = 4) measured by ELISA. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann–Whitney U‐test, ** P < 0.01. Serum pancreatic lipase titers of mice immunized with cInsulin ( n = 5) or control immunization ( n = 4) measured by ELISA. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann–Whitney U‐test, ** P < 0.01. Quantification of total (red) and insulin‐specific (salmon) IgG after serum IgG purification of cInsulin‐immunized mice via ELISA with n = 3/group (coating: anti‐IgG). Mean ± SD, statistical significance was calculated by using Mann–Whitney U‐test, * P < 0.05. Coomassie‐stained SDS–PAGE showing purified serum IgG of cInsulin‐immunized (red) and CI mice (gray) under reducing (+β‐ME), and non‐reducing conditions. Representative of two independent experiments. Blood glucose levels of intravenously (i.v.) injected wild‐type mice at indicated hours post‐injection. 20 µg of purified serum IgG from cInsulin‐immunized mice (red, n = 6) or CI mice (gray, n = 5) was used for injection. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, *** P < 0.001, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standard.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Purification, Staining, SDS Page, Injection

Journal: The EMBO Journal

Article Title: Memory IgM protects endogenous insulin from autoimmune destruction

doi: 10.15252/embj.2020107621

Figure Lengend Snippet: A Serum anti‐insulin‐immunoglobulin titers of mice immunized with InsA peptide (red: 0:1, n = 10 and green: 100:1, n = 5) and control immunization (CI: CpG only, gray, n = 3) measured by ELISA at indicated days (coating: insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal–Wallis test, * P < 0.05, *** P < 0.001. B Ratios of IgG to IgM derived from ELISA values plotted against molar ratios of antigens for n = 5/group. Mean ± SD, statistical significance was calculated by using Kruskal–Wallis test, * P < 0.05, *** P < 0.001. C Western blot analysis of insulin‐specific serum IgG derived from InsA peptide‐immunized mice. Top panel (green): 100:1 serum, lower panel (red): 0:1 serum (sInsA:cInsA). Serum of mice was used as primary antibody and detection was done with anti‐mouse‐IgG‐HRP. Black filled arrow: Proinsulin (12 kD), Black non‐filled arrow: insulin (6 kD), β‐actin (42 kD, loading control). Representative of two independent experiments. D ELISpot of InsA peptide‐immunized (red, n = 11) and CI mice (gray, n = 8) showing insulin‐specific IgG‐producing splenocytes on day 14. Representative wells are shown (top lane). Mean ± SD, statistical significance was calculated by using Mann–Whitney U‐test, **** P < 0.0001. E, F Blood glucose levels of InsA peptide‐immunized (red: 0:1, n = 5 for E and n = 10 for F; green: 100:1, n = 5 and n = 4 for F, d30, d32) and CI (gray, n = 5 for E and n = 10 for F) mice were assessed at indicated days. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal–Wallis test (E) and repeated measure ANOVA test (F), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. G Urine glucose levels of InsA‐peptide‐immunized (red: n = 10 for d28, d32 and n = 8 for d30; green: n = 5 for d28, n = 4 for d30, n = 3 for d32) and CI (gray, n = 3) mice were monitored at indicated days post‐immunization. Dots represent individual mice. For 100:1: representative data are shown for two independent experiments with total n = 10. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, *** P < 0.001. H Table comparing human to murine insulin‐A‐chain amino acid sequences. Underlined: sequence used as peptide. Source data are available online for this figure.

Article Snippet: Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standard.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot, Enzyme-linked Immunospot, MANN-WHITNEY, Sequencing

Journal: The EMBO Journal

Article Title: Memory IgM protects endogenous insulin from autoimmune destruction

doi: 10.15252/embj.2020107621

Figure Lengend Snippet: Ratios of IgG to IgM derived from ELISA values plotted on a two‐dimensional graph against blood glucose levels (left panel) and urine glucose levels (right panel) for n = 5/group. Central bands: median, boxes: interquartile range, whiskers: max./min., statistical significance was calculated by using Mann–Whitney U‐test, ** P < 0.01. Serum anti‐insulin‐IgM titers of mice immunized with InsA peptide with a γ/µ ratio < 0.1 (black, n = 5) and CI (gray, n = 3) mice measured by ELISA at indicated days (coating: insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, * P < 0.05. Blood glucose levels of InsA‐peptide‐immunized mice (γ/µ < 0.1, black, n = 5) and CI (gray, n = 6) mice were assessed at indicated days post‐immunization with a commercial blood glucose monitor device. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal–Wallis test, all comparisons were not significant. Insulin‐specific IgM affinity maturation of InsA‐peptide‐immunized mice at indicated days was measured by ELISA. Ratios of IgM binding to InsA(1) and InsA(4) (referring to molar antigen density) were calculated and plotted as relative units ( n = 5 for d7, d28; n = 3 for d78). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ** P < 0.01. Serum dilutions: 1:25. Coomassie‐stained SDS–PAGE showing purified serum IgM of complex InsA‐peptide (cInsA)‐immunized and CI mice under reducing (+β‐ME), and non‐reducing conditions. Representative of two independent experiments. Anti‐insulin affinity of PR‐IgM (blue), primary IgM (red), and isotype control (black) measured by bio‐layer interferometry. IgM binding to insulin was acquired in pm and used to calculate the dissociation constant shown in the graph (K d = 1/K a ). Graph is showing antigen‐antibody association phase. Data are representative for three independent experiments. Blood glucose levels of intravenously injected mice with purified IgM from control immunization (CI, n = 5), complex InsA peptide (cInsA) immunization day 7 (total IgM d7, n = 4) and day 85 (total IgM d85, n = 5). Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test showing comparison of red to black line, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standard.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Binding Assay, Staining, SDS Page, Purification, Injection

Journal: The EMBO Journal

Article Title: Memory IgM protects endogenous insulin from autoimmune destruction

doi: 10.15252/embj.2020107621

Figure Lengend Snippet: A Schematic illustration of immunization schedule with complex InsA peptides (cInsA) intraperitoneally and insulin‐specific protective IgM (PR‐IgM) in 48 h cycles intravenously (i.v.). *monitoring: diabetes symptoms were only observed within cInsA only group. B Blood and urine glucose levels of mice immunized with complex InsA peptides (cInsA) (red, n = 5) and cInsA plus intravenously injected (i.v.) pIgM (salmon, n = 5) at day 7. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann–Whitney U‐test, * P < 0.05. C Blood glucose levels of mice intravenously injected with total IgG pulldown of cInsA immunized mice (red, n = 4), or total IgG (cInsA) together with anti‐insulin PR‐IgM (black, n = 4). Measurements were done 5 h post‐intravenous injection. Data were collected from two independent experiments. Mean ± SD, statistical significance was calculated by using Mann–Whitney U‐test, ** P < 0.01. D, F (D) Serum (dilution: 1:50) and (F) insulin‐specific IgM (concentration: 500 ng/ml) tested for dsDNA (left panel, coating: calf thymus dsDNA) and insulin (right panel, coating: insulin) reactivity measured by ELISA. Mice were immunized with cInsA and serum was collected on day 7 (D: n = 8, left; n = 5, right; F: n = 4) and day 85 (D: n = 4, left; n = 5, right, F: n = 4) post‐immunization. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann–Whitney U‐test, ** P < 0.01, **** P < 0.0001. E, G Anti‐nuclear‐IgM (ANA) of control‐immunized (CI, n = 3) and complex InsA‐peptide(cInsA)‐immunized mice on day 7 ( n = 3) and day 85 ( n = 3) analyzed by indirect immunofluorescence. We used (E) total serum (dilutions: 1:20) or (G) insulin‐specific IgM (concentration: 500 ng/ml) purified from serum at indicated days on commercial HEp‐2 slides. Scale bar: 10 µm. Green fluorescence indicates IgM bound to nuclear structures. Data are representative for three independent experiments with isotype control: n = 3, day 7: n = 3, day 85: n = 3. H Coomassie‐stained SDS–PAGE showing primary (cInsA d7) and memory (cInsA d85) insulin‐specific purified IgM pre‐incubated with insulin and calf thymus dsDNA. Samples were loaded onto the gel after size exclusion with a cut‐off at 10,000 kD (fractions referring to >/< 10 4 kD). IgM heavy chain (HC): 69 kD, IgM light chain (LC): 25 kD, J‐chain: 15 kD. Data shown are representative of three independent experiments. I Blood glucose levels of mice intravenously injected with either IgM isotype ctrl ( n = 6), anti‐insulin PR‐IgM (d85, n = 5), anti‐insulin primary IgM (d7, n = 4) after insulin‐specific pulldown. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test showing comparison of red and black line, *** P < 0.001, **** P < 0.0001.

Article Snippet: Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standard.

Techniques: Injection, MANN-WHITNEY, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Purification, Fluorescence, Staining, SDS Page, Incubation