Recombinant sars cov 2 s protein

Ninety-six-well microtiter plates (Nunc MaxiSorp; ThermoFisher Scientific) were coated with 100 μL of recombinant SARS-CoV-2 S protein (Wuhan strain) at a concentration of 1 μg/mL in PBS (Gibco) at 4 °C overnight; negative control wells were coated with 1 μg/mL of BSA (Sigma). Plates were blocked for 1.5 h at room temperature with 280 μL of blocking solution (PBS supplemented with 0.05% Tween-20 (Sigma) and 10% FBS (Corning)). Serum from mice and hamsters were diluted serially in blocking solution, starting at 1:100 dilution and incubated for 1.5 h at room temperature. The plates were washed three times with T-PBS (1X PBS supplemented with 0.05% Tween-20), and 100 μL of goat anti-mouse IgG (Southern Biotech Cat #1030–05) diluted 1:2,000 in blocking solution or 100 μL of HRP-conjugated anti-hamster IgG(H+L) antibody (Southern Biotech Cat. #6061–05) diluted 1:500 in blocking solution, was added to all wells and incubated for 1 h at room temperature. Plates were washed 3 times with T-PBS and 3 times with 1X PBS, and 100 μL of 1-step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to all wells. The reaction was stopped after 5 min using 100 μL of 1M HCl, and the plates were analyzed at a wavelength of 490 nm using a microtiter plate reader (BioTek).