ELISA for SARS-CoV-2 Receptor Binding Domain Antibodies

Enzyme-linked immunosorbent assay (ELISA) was used to test antibody binding to SARS-CoV-2 receptor binding domain (RBD) (catalog numbers 40592-V08H, Sino Biological US Inc., Wayne, PA, USA) (31 (link), 32 (link)). High-binding 96-half-well microplates (Corning Life Sciences, Tewksbury, MA, USA) were coated with 50ng RBD protein in PBS overnight at 4°C. The next day, plates were washed five times with PBS + 0.5% Tween 20 and treated with blocking buffer (PBS + 0.5% Tween 20 + 2% BSA + 2% normal goat serum [Jackson ImmunoResearch Inc., West Grove, PA, USA]) for two hours. Different dilutions of human plasma in blocking buffer were added and incubated for one hour. Plates were washed and incubated for one hour with HRP-conjugated anti-human IgG antibody (Jackson ImmunoResearch Inc.) (1:10,000 dilution). The process was stopped by adding 50µL of 1N sulfuric acid to 100L TMB substrate (Thermo Fisher Scientific, Rockford, IL, USA). BioTek Synergy 2 plate reader was used to measure OD at 450 nm (BioTek Instruments Inc., Winooski, VT, USA).