Goat anti human iga

Anti-SARS-CoV-2 full-length spike (S) protein and receptor binding domain (RBD) IgG and IgA seropositivity were identified via validated serology enzyme-linked immunosorbent assay (ELISA), as described by Huynh et al., 2021 [14 (link)]. Briefly, NUNC™ Maxisorp 384 well plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with S (5 μg/mL) or RBD (2 μg/mL) antigens in 50 mM carbonate buffer (pH 9.6) overnight at 4 °C, and blocked with 3% skim milk in PBS-0.05% Tween-20. After washing with phosphate-buffered saline (PBS), plates were coated with diluted serum (1:100) for 1 h at room temperature, washed, and incubated with 25 μL alkaline phosphatase-conjugated antibodies goat anti-human IgG (1:2000) or goat anti-human IgA (1:500) (Jackson ImmunoResearch, West Grove, PA, USA). Plates were washed, and antibody levels were quantified by adding 50 μL of substrate buffer (0.27 µM p-nitrophenyl phosphate/diethanolamine buffer, 1 M, pH 9.6) and reading optical density (OD) at 405 nm detection every 1 min, with 490 nm reference on a BioTek 800TS microplate reader (BioTek Instruments Inc., Winooski, WT, USA).

Properdin (Calbiochem); human IgG, IgM and IgA (Sigma); EBNA-1 protein (Tebu-Bio) and neutravidin (Thermo Scientific) were coupled to beads and utilized to determine the optimal assay conditions for measurement of complement activation. For detecting the various antibody isotypes, goat anti-human IgM (µ chain specific) F(ab')₂ –Cy3, goat anti-human IgG (γ chain specific) F(ab')₂ –PE or goat anti-human IgA (α chain specific) F(ab')₂ –DyL549 (Jackson ImmunoResearch Laboratories) antibodies were used. Goat anti-human complement C3 specific F(ab')₂ antibody (MyBioSource) was labeled with R-Phycoerythrin (Lightning-Link-R-Phycoerythrin Conjugation Kit, Innova Biosciences) according to manufacturer's protocol and was used to detect complement activation driven C3 deposition on beads.

As described previously,³⁶ IgM, IgA and IgG levels were determined in supernatants from in vitro B-cell cocultures using an ELISA. Plates were coated with goat anti-human IgM, IgG or IgA (1 mg/mL; Southern Biotech, Uithoorn, The Netherlands). Horseradish peroxidase conjugated rabbit anti-human IgM (Jackson), goat anti-human IgG (Thermo Fisher Scientific) or goat anti-human IgA (Jackson) were used to detect bound antibodies.