SARS-CoV-2 Spike Protein Antibody Assay

Serum samples were inactivated with γ-irradiation (2 mRad) and analyzed as previously described [28 (link)]. In brief, maxisorp plates (Nunc) were coated with 50 ng spike protein (generated in-house) per well and incubated overnight at 4 °C. After blocking with casein in phosphate buffered saline (PBS) (ThermoFisher) for 1 h at room temperature (RT), serially diluted 2-fold serum samples (duplicate, in blocking buffer) were incubated for 1 h at RT. Spike-specific antibodies were detected with goat anti-hamster IgG Fc (horseradish peroxidase (HRP)-conjugated, Abcam) for 1 hr at RT and visualized with KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120-0047, Milford, MA, USA). The reaction was stopped with KPL stop solution (Seracare) and read at 450 nm. Plates were washed 3 to 5× with PBS-T (0.1% Tween) for each wash. The threshold for positivity was calculated as the average plus 3× the standard deviation of negative control hamster sera.