SARS-CoV-2 RBD Antibody Detection by ELISA

Antibody was tested by indirect ELISA with the SARS-CoV-2 RBD protein (Sino Biological Inc., China) and peroxidase conjugated goat anti-cat IgG (Sigma-Aldrich, USA). Briefly, ELISA plates were coated overnight at 4°C with RBD protein (1 μg/ml, 100 μl per well). After blocked with PBS containing 5% skim milk for 2 h at 37°C, the plates were added with sera at a dilution of 1: 40. After incubation for 30 min at 37°C, the plates were washed five times with washing buffer (PBS containing 0.05% Tween-20). A 1:20,000 diluted anti-cat IgG was added and incubated for an additional 30 min. After another 5 washes, TMB Substrate (Sigma-Aldrich, USA) was added and incubated for 10 min. Then the reaction was stopped, and optical density (OD) was measured at 450 nm. As the judgment method described previously[16 (link), 17 (link)], those sera were considered positive if the OD values were twice higher than the mean OD of the 39 sera collected between Mar. and May, 2019.