Goat anti human serum iga

ELISA plates (MaxiSorp 96-well immunoplates; Thermo Fisher Scientific) were coated with the F(ab′)₂ fragment of 5 µg/mL goat anti-human serum IgA (50 µL/well, Jackson ImmunoResearch Labs, West Grove, PA) in PBS and then incubated at room temperature for 1 h. The plates were then blocked with 1% BSA in PBS and incubated with 50 µL deglycosylated IgA1 (5 µg/mL) at 4°C overnight. Subsequently, the plates were washed with PBS-T and incubated with each biotinylated lectin: Helix pomatia agglutinin (HPA, 5 µg/mL; EY Laboratories, San Mateo, CA), Vicia villosa agglutinin (VVA, 5 µg/mL; Vector Laboratories), peanut agglutinin (PNA, 2 µg/mL; Vector Laboratories, Burlingame, CA), and WFA (5 µg/mL; Vector Laboratories) at room temperature for 2 h. After washing, the plates were incubated with HRP-conjugated streptavidin at room temperature for 1 h. A 3,3′,5,5′-tetramethylbenzidine substrate solution (50 µL/well, Thermo Fisher Scientific) was added to the plates followed by incubation at room temperature until signal development. The reaction was stopped with 2 M sulfuric acid (50 µL), and the optical density was measured at 450 nm with a Wallac 1420 ARVO SX Multilabel Counter (Perkin Elmer).

HIV-1 envelope epitope mapping was evaluated by peptide microarray analysis as previously described (58 (link), 59 (link)), with modifications. Briefly, the peptide arrays contain libraries of 15-mer peptides, overlapping by 12 amino acids, spanning the whole HIV-1 gp120 Env of subtype A, B, C, and D and circulating recombinant form (CRF) AE and AG consensus sequences, as well as the group M consensus sequence, captured on customized microarrays. Three identical subarrays, each containing the full peptide library, were printed on each slide. Array slides were blocked for 1 h to reduce nonspecific binding, followed by a 2-hour incubation with 20 μg/ml IgA MAb for testing or 10 μg/ml of 7B2_IgA2 Dim (Catalent; included as positive control). The slides were subsequently incubated for 45 min with goat anti-human serum IgA (α-chain specific; Jackson ImmunoResearch) that was conjugated with DyLight 649 using a microscale antibody labeling kit (Thermo Scientific) per the manufacturer's instructions. Array slides were scanned at a wavelength of 635 nm with a GenePix 4300 scanner. Images were analyzed using GenePix software to obtain binding intensity values (median intensity of triplicates spots for each peptide).