Mouse anti human iga

Mouse anti-human IgG₁ (#9054-01), mouse anti-human IgG₂ (#31-7-4), mouse anti-human IgG₄ (#9200-01), and mouse anti-human IgE (#9250-01) were obtained from Southern Biotechnology (Birmingham, AL). Mouse anti-human IgG₃ (#05-3600) was obtained from Invitrogen (Carlsbad, CA) and mouse anti-human IgM (#555856) and mouse anti-human IgA (#555886) were obtained from BD Biosciences (San Jose, CA). Secondary detection reagents including biotin-mouse anti-human kappa light chain (#555790) and biotin-mouse anti-human lambda light chain (#555794) were obtained from BD Biosciences (San Jose, CA). R-Phycoerythrin-conjugated streptavidin (#016-110-084) and R-Phycoerythrin-conjugated donkey F(ab’)₂ anti-mouse IgG (H + L) (#715–116-150) were obtained from Jackson ImmunoResearch (West Grove, PA).

MAb specificity for HIV-1 proteins was evaluated on a panel of HIV-1 gp120/gp140 Env glycoproteins, the linear C1 region, and the third variable-loop (V3) peptide epitopes, and V1-V2 scaffolds were measured by a custom binding antibody multiplex assay, as previously described (6 (link), 60 (link), 61 (link)). Positive controls included HIVIG (provided by the NIH AIDS Reagent Program) and 7B2 IgA MAb titrations and single-point HIV-1 IgG MAb controls CH58_IgG1 (9 (link)), VRC01 IgG (62 (link)), CH106 IgG (63 (link)), 2G12 IgG (64 (link), 65 (link)), 17b IgG (66 (link)), and F105 IgG (67 (link)). Blank beads and HIV-1-negative sera were used as negative controls. HIV-1-specific antibody isotypes were detected with mouse anti-human IgA (BD Pharmingen) at 4 μg/ml, followed by washing and incubation with streptavidin-phycoerythrin (PE) (BD Pharmingen). Antibody measurements were acquired on a Bio-Plex instrument (Bio-Rad) with 21CFR Part 11 compliant software, and the readout was expressed as mean fluorescence intensity (MFI). Each test MAb was assayed at a starting concentration of 100 μg/ml and titrated by 2-fold dilutions. Results were reported as area under the curve (AUC) binding titers.