Journal: Cell Reports Medicine

Article Title: The Fc-effector function of COVID-19 convalescent plasma contributes to SARS-CoV-2 treatment efficacy in mice

doi: 10.1016/j.xcrm.2022.100893

Figure Lengend Snippet:

Article Snippet: Peroxidase AffiniPure Goat Anti-Human IgA + IgG + IgM (H + L) , Jackson ImmunoResearch , 109-035-064.

Techniques: Blocking Assay, Control, Virus, Variant Assay, Recombinant, Modification, Saline, Red Blood Cell Lysis, Lysis, Electron Microscopy, Staining, Luciferase, Multiplex Assay, cDNA Synthesis, Oligo Synthesis, Plasmid Preparation, Expressing, Software, Flow Cytometry, Real-time Polymerase Chain Reaction, Spectrophotometry, Membrane, Stripping Membranes, Isolation, In Vivo Imaging

Journal: bioRxiv

Article Title: Inter-individual and inter-regional variability of breast milk antibody reactivity to bacterial lipopolysaccharides

doi: 10.1101/2024.05.16.594523

Figure Lengend Snippet: IgA, IgM and IgG reactivity to LPS in Kenyan breast milk samples. Breast milk samples were normalized to an IgA concentration of 10 μg/ml and applied to LPS arrays. Antibody binding to LPS was detected using either an anti-human IgA and IgM Cy3 or anti-human IgG AlexaFluor647 labelled antibody. Fluorescence signals were recorded at a PMT-gain of 190V. Data are shown as net mean RFU values of 9-replicate dots.

Article Snippet: The secondary antibodies goat anti-human IgA Cy3 (109-165-011), donkey anti-human IgM Cy3 (709-165-073) and donkey anti-human IgG Alexa Fluor 647 (709-605-098) (all from Jackson ImmunoResearch, West Grove, PA, USA) were diluted 1:10,000 in blocking buffer.

Techniques: Concentration Assay, Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Inter-individual and inter-regional variability of breast milk antibody reactivity to bacterial lipopolysaccharides

doi: 10.1101/2024.05.16.594523

Figure Lengend Snippet: Comparison of IgA reactivity between Kenyan and Swiss breast milk samples. 30 breast milk samples from Kenya (left panel) and 30 samples from Switzerland (right panel) were normalized to an IgA concentration of 10 μ g/ml and analyzed on LPS arrays. Antibody binding to LPS was detected using anti-human IgA secondary antibody coupled to Cy3. Fluorescence signals were recorded at a PMT-gain of 190V. The net mean RFU values of 9-replicate dots are shown. Hierarchical clustering of LPS binding signals was performed using the pheatmap package in R studio.

Article Snippet: The secondary antibodies goat anti-human IgA Cy3 (109-165-011), donkey anti-human IgM Cy3 (709-165-073) and donkey anti-human IgG Alexa Fluor 647 (709-605-098) (all from Jackson ImmunoResearch, West Grove, PA, USA) were diluted 1:10,000 in blocking buffer.

Techniques: Comparison, Concentration Assay, Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Inter-individual and inter-regional variability of breast milk antibody reactivity to bacterial lipopolysaccharides

doi: 10.1101/2024.05.16.594523

Figure Lengend Snippet: Differential recognition of specific LPS O-antigens by Kenyan and Swiss breast milk IgA. IgA binding to LPS O-antigens including the immunogenic epitopes rhamnose (green triangles) as found in (A) S. flexneri 2a; (B) E. coli O13 and (C) C. gillenii O9; the α-Gal (two yellow circles) and Forssman (two yellow squares) antigens as found in (D) E. coli O86; the dideoxy sugars abequose (orange rectangle) and tyvelose (green rectangle) as found in (E) S. enterica Typhimurium and (F) S. enterica Typhi. IgA binding was detected using anti-human IgA Cy3, scanned at a PMT-gain of 190V. The grey dotted line shows TOD. Comparisons of net mean RFU values between Kenyan (n=30) and Swiss (n=30) samples was done using an unpaired Wilcoxon test. P-values <0.001 (***), <0.01 (**) were considered statistically significant. The symbols used to display O-antigens is based on the standard symbolic nomenclature for glycans .

Article Snippet: The secondary antibodies goat anti-human IgA Cy3 (109-165-011), donkey anti-human IgM Cy3 (709-165-073) and donkey anti-human IgG Alexa Fluor 647 (709-605-098) (all from Jackson ImmunoResearch, West Grove, PA, USA) were diluted 1:10,000 in blocking buffer.

Techniques: Binding Assay

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

Article Title: Recombinant human IgA1 and IgA2 autoantibodies to type VII collagen induce subepidermal blistering ex vivo.

doi: 10.4049/jimmunol.1400160

Figure Lengend Snippet: FIGURE 1. Recombinant autoantibodies demonstrated the correct m.w. under reducing and nonreducing conditions, labeled the DEJ and bound to type VII collagen extracted from human dermis. (A) Recombinant autoantibodies were separated by 12% SDS-PAGE under reducing conditions and stained with Coomassie blue. The H chains of the recombinant proteins migrated at 50 (rIgG1), 60 (rIgA1), and 55 kDa (rIgA2), and the k L chains migrated at 25 kDa. The m.w. standard is indicated on the left. (B) Equal amounts of recombinant autoantibodies were separated by 7% SDS-PAGE under nonreducing conditions. The majority of the recombinant autoantibodies were assembled as complete 2H2L heterotetramers. In addition, for rIgA1 and rIgA2, additional bands at higher m.w. were detectable, indicating polymerization and a reduced amount of monomeric rIgA1 or rIgA2. (C) Immunoblotting of recombinant autoantibodies was performed using the same conditions as in (B), as well as detection with peroxidase-conjugated polyclonal anti-human k L chain Ab. The Coomassie staining of rIgA1 and rIgA2 appeared to be weaker overall than that for rIgG1, which may be because of the higher glycosylation content of IgA isotypes. The m.w. standard is indicated on the left. (D) IgA1 and IgA2 recombinant autoantibodies reacted with a 290-kDa band after immunoblotting of dermal extracts, whereas the isotype controls for IgA1 (Iso IgA1) and IgA2 (Iso IgA2) were negative. (E) Human neonatal foreskin cryosections were incubated with the different recombinant autoantibodies and visualized by isotype- and subclass-specific FITC-conjugated Abs. Both rIgA1 and rIgA2 presented a defined reactivity with the DEJ (arrows). As controls, a V-gene matched rIgG1 (31) and serum from a healthy volunteer (NHS) in conjunction with FITC-conjugated polyclonal rabbit anti-human IgG was used. Original magnification 3100.

Article Snippet: For the detection of Abs in cryosections, FITC-conjugated polyclonal goat anti-human IgA (Bio-Rad Laboratories GmbH, Munich, Germany) and polyclonal goat anti-human IgG (Sigma-Aldrich Chemie GmbH, Munich, Germany) were used as secondary Abs.

Techniques: Recombinant, Labeling, SDS Page, Staining, Western Blot, Glycoproteomics, Incubation