SARS-CoV-2 Spike Protein Seropositivity Assay

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).