mouse anti human iga2 biot  (SouthernBiotech)

Journal: Frontiers in immunology

Article Title: The Alternative Pathway Is Necessary and Sufficient for Complement Activation by Anti-THSD7A Autoantibodies, Which Are Predominantly IgG4 in Membranous Nephropathy.

doi: 10.3389/fimmu.2022.952235

Figure Lengend Snippet: FIGURE 2 | Identification of IgA anti-THSD7A autoAbs. (A) Quantitative analysis of anti-THSD7A IgA in MN sera (circles). The threshold for positivity, depicted by the dotted line, was defined as the mean + 4SD of control sera (tringles). B-C. IgA-positive MN sera (circles) show significantly higher binding to THSD7A of IgA1 (B) but not IgA2 (C), compared to IgA-negative MN sera (squares) or control sera (triangles). The significance of differences among groups was analyzed by one-way ANOVA follow by Bonferroni’s test for multiple comparison (ns, not significant; *p<0.05; **p<0.01).

Article Snippet: Secondary antibodies were horseradish peroxidase-conjugated sheep anti-human IgG1-4 (The Binding Site, Birmingham, UK), goat anti-human IgA, and mouse anti-human IgA1 and IgA2 (Southern Biotech, Birmingham, Alabama).

Techniques: Control, Binding Assay, Comparison

Journal: EMBO Molecular Medicine

Article Title: Alteration of microbiota antibody‐mediated immune selection contributes to dysbiosis in inflammatory bowel diseases

doi: 10.15252/emmm.202115386

Figure Lengend Snippet: A–D ELISA assay of fecal IgA1 (A), IgA2 (B), total IgA (C), and secretory component (D) levels from non‐IBD ( n = 7), CD ( n = 18), and UC ( n = 12) patients' stool. E, F Data were further separated according to disease activity for IgA1 (E) and IgA2 (F). G, H In vitro assay of purified IgA1 (G) and IgA2 (H) reverse‐transcytosis abilities on an inverted model of FAE from Caco2 and Raji cells co‐culture. I, J (I) ELISA assay of IgA1‐Dectin‐1 binding, at a rate of one receptor per 10 IgA; (J) ELISA assay of IgA2‐Dectin‐1 binding, at a rate of one receptor per 10 IgA. K–N Percent of IgA1 (K, L) and IgA2 (M, N) reverse‐transcytosis for weak (K, M) and strong (L, N) Dectin‐1 binding. Data information: Data were analyzed using Kruskall–Wallis multiple comparisons with Dunn's correction, when possible, or a Mann–Whitney test. P ‐values are as follows: (A) ** P = 0.0098; (B) * P = 0.0406; (C) Non‐IBD vs. UC * P = 0.0479, CD vs. UC * P = 0.0166; (F) * P = 0.0167; (G) * P = 0.0441; (H) * P = 0.423; (J) Non‐IBD vs. UC * P = 0.0199, CD vs. UC * P = 0.0243; (K) * P = 0.0221; (L) * P = 0.0486. (G–N) For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non‐IBD: n = 7; CD: n = 8; UC: n = 4; IgA2: Non‐IBD: n = 7; CD: n = 8; UC: n = 5. All patient samples (biological replicates) have been tested in technical duplicates meaning n × 2.

Article Snippet: After three washes with PBST, wells were incubated with biotinylated anti‐human IgA1 (Southern Biotech, 9130–08) or anti‐human IgA2 (Southern Biotech, 9140–08) diluted 1/2,500 and incubated at RT for 90 min. After washing in PBST, streptavidin‐HRP (BD, 554066) was added for 60 min.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, In Vitro, Purification, Co-Culture Assay, Binding Assay, MANN-WHITNEY, Antibody Purification, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Alteration of microbiota antibody‐mediated immune selection contributes to dysbiosis in inflammatory bowel diseases

doi: 10.15252/emmm.202115386

Figure Lengend Snippet: A Full MS spectrum of glycopeptides released from CD IgA1 trypsin digest. Main N340‐glycoforms of the IgA1 glycopeptide [332–353] (LAGKPTHVNVSVVMAEVDGTCY) are annotated using CFG nomenclature. Appendix Table summarizes a list of N‐glycopeptides identified. # is the peptide [43–76] of immunoglobulin kappa constant chain (UniProtKB P01834 ). *Isobaric structures not differentiated by MS/MS experiments (not exhaustive N‐glycans illustrations). (HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR). B O‐glycoforms identified for CD. C O‐glycoforms identified for UC. Data information: *corresponds to IgA1 peptide [264–273] (WLQGSQELPR). # shows contamination by other multiply charge species covering the glycoforms signals. Details related to O‐glycoforms are given in Appendix Table . CD: n = 3; UC: n = 1 (biological replicates). All patient samples have been tested in technical duplicates.

Article Snippet: After three washes with PBST, wells were incubated with biotinylated anti‐human IgA1 (Southern Biotech, 9130–08) or anti‐human IgA2 (Southern Biotech, 9140–08) diluted 1/2,500 and incubated at RT for 90 min. After washing in PBST, streptavidin‐HRP (BD, 554066) was added for 60 min.

Techniques: Glycoproteomics, Tandem Mass Spectroscopy

Journal: EMBO Molecular Medicine

Article Title: Alteration of microbiota antibody‐mediated immune selection contributes to dysbiosis in inflammatory bowel diseases

doi: 10.15252/emmm.202115386

Figure Lengend Snippet: A, B Heatmaps displaying standardized log2‐transformed signal intensities per glycan for IgA1 (A) and IgA2 (B) in non‐IBD, CD and UC groups, plotted by hierarchical clustering of Euclidean distance. C–F Volcano plots of log2‐transformed fold changes for IgA1 anti‐glycan reactivity in CD (C) and UC (D) groups, and IgA2 anti‐glycan reactivity for CD (E) and UC (F) compared to non‐IBD IgA1 and IgA2. For CD, P ‐values of differentially targeted glycan motifs are listed in Appendix Table and those of UC in Appendix Tables and . Significance threshold was placed at P ‐value < 0.05 (indicated by the dotted line in C–F); For IgA1, Non‐IBD: n = 4; CD: n = 4; UC: n = 4. For IgA2, n = 6; CD: n = 6; UC: n = 6. IgA1 and IgA2 were taken matched for the same patient. All patient samples (biological replicates) have been tested in technical duplicates. A list of the glycans from each cluster is provided as Appendix Tables and .

Article Snippet: After three washes with PBST, wells were incubated with biotinylated anti‐human IgA1 (Southern Biotech, 9130–08) or anti‐human IgA2 (Southern Biotech, 9140–08) diluted 1/2,500 and incubated at RT for 90 min. After washing in PBST, streptavidin‐HRP (BD, 554066) was added for 60 min.

Techniques: Transformation Assay, Glycoproteomics

Journal: EMBO Molecular Medicine

Article Title: Alteration of microbiota antibody‐mediated immune selection contributes to dysbiosis in inflammatory bowel diseases

doi: 10.15252/emmm.202115386

Figure Lengend Snippet: A–C Flow cytometry analysis of total IgA1‐bound (A) and IgA2‐bound (B) fecal microbiota. (C) Flow cytometry analysis of stool IgA1 + IgA2 − bacteria (gray dots), IgA1 − IgA2 + bacteria (black dots) and IgA1 + IgA2 + bacteria (empty dots). (A–C) Data were analyzed with a Kruskall–Wallis multiple comparison test. In (C) * P = 0.0226. Non‐IBD: n = 7, CD: n = 18, and UC: n = 12. D Shannon and Chao1 diversity indices of IgA1‐bound microbiota in non‐IBD, CD and UC. E PCA plot based on the Jaccard distance between samples. F Phylum‐level composition of IgA1‐bound microbiota in non‐IBD, CD and UC. G Boxplots of OTUs for which the abundance was significantly different between non‐IBD and CD. H Shannon and Chao1 diversity indices of IgA2‐bound microbiota in non‐IBD, CD and UC. I PCA plot based on the Jaccard distance between samples. J Phylum‐level composition of IgA2‐bound microbiota in non‐IBD, CD and UC. K Boxplots of OTUs whose abundance was significantly different between CD and UC, and between non‐IBD and UC, respectively. OTUs present in less than 25% of samples or with read count lower than 50 were filtered out. Differential abundance was tested using negative binomial model implemented in DESeq2 and p‐values corrected with False Discovery Rate (FDR) procedure. Non‐IBD: n = 4; CD: n = 4; UC: n = 4 (two patients excluded for abundance in both IgA1 and IgA2 analyses). All patient samples have been tested in technical duplicates.

Article Snippet: After three washes with PBST, wells were incubated with biotinylated anti‐human IgA1 (Southern Biotech, 9130–08) or anti‐human IgA2 (Southern Biotech, 9140–08) diluted 1/2,500 and incubated at RT for 90 min. After washing in PBST, streptavidin‐HRP (BD, 554066) was added for 60 min.

Techniques: Flow Cytometry, Bacteria, Comparison

Journal: EMBO Molecular Medicine

Article Title: Alteration of microbiota antibody‐mediated immune selection contributes to dysbiosis in inflammatory bowel diseases

doi: 10.15252/emmm.202115386

Figure Lengend Snippet: A–E Optical density (600 nm) variations during in vitro growth assay of Salmonella enterica Typhimurium (SL1344) co‐cultured with stool‐purified IgA1 (A) or IgA2 (B) from non‐IBD ( n = 4), CD ( n = 6) and UC ( n = 3). Comparison between IgA1 and IgA2 coculture in non‐IBD (C), CD (D), and UC (E). F–I Optical density (600 nm) during in vitro growth assay of Escherichia coli (25,922 strain from ATCC) cocultured with stool‐purified IgA1 (F) or IgA2 (G) from non‐IBD ( n = 2) and CD ( n = 2). All patient samples (biological replicates) have been tested in technical duplicates. Comparison between IgA1 and IgA2 co‐culture in non‐IBD (H), and CD (I). These IgA samples were from the same patients throughout each experiment. Data information: Two‐way ANOVA with Holm–Sidak correction after D'Agostino–Pearson nomality test, P ‐values are as follows: (A) UC curve: 2 h – P = 0.0024, 3 h – P = 0.003; CD curve: 2 and 3 h P < 0.0001 (B) UC curve: 4 h – P = 0.0186, 5 h – P = 0.0393, 6 h – P = 0.039, 8 h – P < 0.0001; CD curve: 4 h – P = 0.0001, 5 h – P = 0.0028, 6 h – P = 0.0024, 8 h – P < 0.0001. (C) 3 h – P = 0.0422, 4 h – P = 0.0422, 6 h – P = 0.0012, 8 h – P < 0.0001. (F) Gray curve: 4 h – P = 0.0004, 5 h – P = 0.0014; Black curve: 4 and 5 h – P < 0.0001. (G) De‐glycosylated CD vs. De‐glycosylated non‐IBD: 4 h – P = 0.0003, 5 h – P < 0.0001. (H) Native vs. de‐glycosylated: 4 and 5 h – P < 0.0001; De‐glycosylated IgA1 vs. De‐glycosylated IgA2: 5 h – P = 0.0012. (H) Native vs. de‐glycosylated: 4 h – P = 0.001 and 5 h – P = 0.006; De‐glycosylated IgA1 vs. De‐glycosylated IgA2: 5 h – P < 0.0001.

Article Snippet: After three washes with PBST, wells were incubated with biotinylated anti‐human IgA1 (Southern Biotech, 9130–08) or anti‐human IgA2 (Southern Biotech, 9140–08) diluted 1/2,500 and incubated at RT for 90 min. After washing in PBST, streptavidin‐HRP (BD, 554066) was added for 60 min.

Techniques: In Vitro, Growth Assay, Cell Culture, Purification, Comparison, Co-Culture Assay

Journal: EMBO Molecular Medicine

Article Title: Alteration of microbiota antibody‐mediated immune selection contributes to dysbiosis in inflammatory bowel diseases

doi: 10.15252/emmm.202115386

Figure Lengend Snippet: A Combined FISH‐IF staining for each probe. B Total fluorescence detected per probe and per biopsy. Erec482 – * P = 0.00233; Ato291 – * P = 0.0268; Bac303 – * P = 0.0196. C Percent of co‐occurred IgA2 and FISH‐bound particles. Erec482 – * P = 0.0073. Data information: Two‐way ANOVA with Holm–Sidak correction after D'Agostino–Pearson nomality test. Non‐IBD: n = 3; Active CD: n = 6; Inactive CD: n = 4. All patient samples (biological replicates) have been tested in technical duplicates.

Article Snippet: After three washes with PBST, wells were incubated with biotinylated anti‐human IgA1 (Southern Biotech, 9130–08) or anti‐human IgA2 (Southern Biotech, 9140–08) diluted 1/2,500 and incubated at RT for 90 min. After washing in PBST, streptavidin‐HRP (BD, 554066) was added for 60 min.

Techniques: Staining, Fluorescence

Journal: EMBO Molecular Medicine

Article Title: Alteration of microbiota antibody‐mediated immune selection contributes to dysbiosis in inflammatory bowel diseases

doi: 10.15252/emmm.202115386

Figure Lengend Snippet: While in the healthy gut, IgA1 have mostly neutralizing and/or bacteriostatic properties, IgA2 undergoes reverse‐transcytosis via Dectin‐1 and Siglec‐5 to deliver antigens to the immune cells of the PP. This mechanism is dependent on glycosylations, notably sialylations for Siglec‐5 recognition. This allows for efficient elimination of pathogens and opportunists. IgA‐mediated entrapment of commensal bacteria into the mucus is also glycan‐dependent, wherein bacteria, IgA and mucin glycosylation are required. In CD (middle panel), luminal binding of commensal by‐ and RT of IgA1, would promote higher antigen load to the lamina propria and establishment of responses against both commensals and opportunists, which would ultimately favor opportunist's growth. Mechanisms of IgA1 retro‐transport remain to be determined. In addition, de‐sialylation of IgA1 limits its effector function, and would affect adequate commensal selection. Tissular IgA2, in turn, is less efficient at neutralizing opportunists, with increased binding to commensals during active disease, which would favor opportunists again. In UC (right panel), absence of RT for IgA2 and overall loss of anti‐glycan reactivity would lead to impaired tolerogenic responses to commensals but also impaired responses against pathogens and opportunists, resulting in opportunist's resurgence within the microbiota.

Article Snippet: After three washes with PBST, wells were incubated with biotinylated anti‐human IgA1 (Southern Biotech, 9130–08) or anti‐human IgA2 (Southern Biotech, 9140–08) diluted 1/2,500 and incubated at RT for 90 min. After washing in PBST, streptavidin‐HRP (BD, 554066) was added for 60 min.

Techniques: Bacteria, Glycoproteomics, Binding Assay, Selection

Journal: Frontiers in Immunology

Article Title: Analysis of the Targets and Glycosylation of Monoclonal IgAs From MGUS and Myeloma Patients

doi: 10.3389/fimmu.2020.00854

Figure Lengend Snippet: Viral targets of purified monoclonal IgAs from myeloma patients, as determined by the multiplexed infectious antigen micro-array (MIAA) revealed using a Dylight TM 680-labeled goat anti-human IgA Fc antibody. For each patient, serum and purified monoclonal (Mc) IgA were incubated in parallel in the MIAA assay; results shown as fluorescent intensity represent either unseparated IgAs (left) or the patient's monoclonal IgA (right). (A) A patient with a Mc IgA that does not react with any pathogen of the MIAA. (B–E) Four patients with Epstein–Barr virus (EBV)-specific Mc IgAs. (F) One patient with a hepatitis C virus (HCV)-specific Mc IgA. EBV nuclear antigen (EBNA-1) signals are shown in dark blue dots, HCV core signals in red dots, and positive thresholds are shown in dotted lines. (A) For patient X11, the serum contained IgAs that recognized Borrelia burgdorferi , EBV EBNA-1, EBV VCA, Helicobacter pylori lysates 1 and 2, HCV NS3, and varicella zoster virus (VZV) ORF26 protein, whereas the purified Mc IgA did not recognize anything on the MIAA array. (B) For patient X01, the serum contained IgAs that recognized a mix of cytomegalovirus (CMV) antigens, EBV EBNA-1, EBV VCA, herpes simplex virus (HSV-1) gG, HCV NS3, and VZV ORF26; the purified Mc IgA recognized EBV EBNA-1 only. (C) For patient X04, the serum contained IgAs that recognized B. burgdorferi , CMV antigens, EBV EBNA-1, EBV VCA, HSV-1 gG, HCV NS3, HCV NS4, VZV gE, and ORF26, whereas the purified Mc IgA recognized EBV EBNA-1 only. (D) For patient X06, the serum contained IgAs that recognized EBV EBNA-1, EBV VCA, and HCV NS3; the purified Mc IgA recognized EBV EBNA-1 only. (E) For patient X09, both IgAs in serum and the purified Mc IgA recognized the EBV EBNA-1 protein only. (F) For patient X12, the serum contained IgAs that recognized EBV EBNA-1, EBV VCA, HSV-1 gG, HSV-1 lysate, HSV-2 lysate, and HCV core, whereas the purified Mc IgA recognized HCV core only. (B–F) The fluorescence values shown for EBV EBNA-1 or HCV core were obtained after subtraction of the non-specific fluorescent background. Thresholds of specific positivity were defined for each viral pathogen or protein (1,400 for EBV EBNA-1, blue threshold; 500 for HCV core, red threshold) ( , , ). Note that dots may be superimposed; horizontal bars represent the means of results obtained for a pathogen, Ag, or lysate. Experiments were performed in triplicates, repeated at least once.

Article Snippet: After washing, 50 μl of biotinylated mouse anti-human IgA1 or IgA2 antibody (0.5 μg/ml; Southern Biotech, Birmingham, AL, USA) was added (2-h incubation, 37°C).

Techniques: Purification, Microarray, Labeling, Incubation, Virus, Fluorescence

Journal: Frontiers in Immunology

Article Title: Analysis of the Targets and Glycosylation of Monoclonal IgAs From MGUS and Myeloma Patients

doi: 10.3389/fimmu.2020.00854

Figure Lengend Snippet: Confirmation of the specificity of recognition of EBV EBNA-1 or HCV core proteins by purified monoclonal IgAs. (A) Dot blotting assays with purified recombinant EBNA-1 were performed in parallel with PBS, as control (CTRL), and with the serum and the purified monoclonal IgA from six patients. As assessed by the MIAA array, both serum and purified monoclonal IgAs of the patient X05 did not recognize EBV EBNA-1, and only unseparated IgAs from the serum of the patient X11 recognized EBNA-1 (negative controls). For patients X01, X04, X06, and X09, both serum and purified monoclonal IgAs recognized EBV EBNA-1, thus confirming the results obtained with the MIAA array. (B) A dot blotting assay with purified recombinant HCV core protein was performed in parallel with PBS, as control (CTRL), and with the serum and purified monoclonal IgA of patients. As assessed by the MIAA array, both the serum and the purified monoclonal IgA of patient X08 did not recognize the HCV core (negative control). For patient X12, both the serum and the purified monoclonal IgA recognized the HCV core, confirming the results obtained with the MIAA array. Experiments were performed at least twice.

Article Snippet: After washing, 50 μl of biotinylated mouse anti-human IgA1 or IgA2 antibody (0.5 μg/ml; Southern Biotech, Birmingham, AL, USA) was added (2-h incubation, 37°C).

Techniques: Purification, Recombinant, Control, Negative Control

Journal: Frontiers in Immunology

Article Title: Analysis of the Targets and Glycosylation of Monoclonal IgAs From MGUS and Myeloma Patients

doi: 10.3389/fimmu.2020.00854

Figure Lengend Snippet: Lysoglucosylceramide (LGL1) is specifically recognized by subsets of purified monoclonal IgAs. LGL1-specific immunoblotting assays were performed as described in the Materials and Methods section ( , , ). Samples of serum (left) or purified monoclonal IgAs (right) were first submitted to agarose gel electrophoresis; then, the gels were blotted onto LGL1-saturated membranes. After blocking for 1 h, membranes were incubated with anti-human IgA horseradish peroxidase (HRP)-conjugated secondary antibody, then washed and revealed by chemiluminescence. The positive control (CTRL+, left) was a sample of serum from a patient known to have LGL1-specific IgAs. Negative controls (CTRL–) were samples of serum without LGL1-reactive IgAs (one from a healthy volunteer, two from patients). The lines of sample deposit are indicated by white arrowheads. The positive signals characteristic of LGL1-reactive Igs are encircled. Patterns of migration may differ for serum and purified monoclonal IgAs because serum may contain both monoclonal and polyclonal LGL1-reactive IgAs.

Article Snippet: After washing, 50 μl of biotinylated mouse anti-human IgA1 or IgA2 antibody (0.5 μg/ml; Southern Biotech, Birmingham, AL, USA) was added (2-h incubation, 37°C).

Techniques: Purification, Western Blot, Agarose Gel Electrophoresis, Blocking Assay, Incubation, Positive Control, Migration

Journal: Frontiers in Immunology

Article Title: Analysis of the Targets and Glycosylation of Monoclonal IgAs From MGUS and Myeloma Patients

doi: 10.3389/fimmu.2020.00854

Figure Lengend Snippet: Hyposialylation of IgAs from monoclonal gammopathy of undetermined significance (MGUS) and myeloma patients. The sialylation level of IgAs in the serum of healthy volunteers (HV, n = 12), MGUS ( n = 6), and myeloma ( n = 22) patients was assessed using an enzyme-linked lectin assay (ELLA) technique, as described in the Materials and Methods section. Unfortunately, we were not able to constitute a control cohort of patients with excessive amounts of non-clonal IgAs. Results are expressed as percentages of sialylated forms of IgAs in serum. (A) Sialylation level of IgAs from HV (green dots) and MGUS and myeloma (MM) patients (red dots); *** p < 0.001, Mann–Whitney U -test. (B) Sialylation level according to age, in HV under or over 60 (green dots), and according to the diagnosis of MGUS (orange squares) or MM (red squares). (C) Sialylation level of IgAs from MM patients with a pathogen-specific monoclonal IgA, as determined by the MIAA (filled red squares, MIAA+) and MM patients with a monoclonal IgA of undetermined specificity (open red squares, MIAA–), compared to MGUS patients (orange squares). Bars indicate means±SEM. (B,C) Significant differences are indicated by stars; * p < 0.05 and ** p < 0.01, Kruskal–Wallis test followed by Dunn's post-hoc test (ns, not significant).

Article Snippet: After washing, 50 μl of biotinylated mouse anti-human IgA1 or IgA2 antibody (0.5 μg/ml; Southern Biotech, Birmingham, AL, USA) was added (2-h incubation, 37°C).

Techniques: Control, MANN-WHITNEY, Biomarker Discovery