Spike Protein Antibody ELISA

Serum samples were analyzed as previously described^{50 (link)}. In brief, maxisorp plates (Nunc) were coated with 50 ng Lineage A spike protein (generated in-house) per well. Plates were incubated overnight at 4 °C. Plates were blocked with casein in phosphate buffered saline (PBS) (ThermoFisher) for 1 h at room temperature. Serum was diluted 2-fold in blocking buffer and samples (duplicate) were incubated for 1 h at room temperature. Secondary goat anti-hamster IgG Fc (Cat.No. 5220-0371 Lot. 10492253, Seracare; each lot is tested to assure specificity and lot-to-lot consistency using an in-house ELISA assay. Reference number: 14-22-06 (https://www.seracare.com/AntiHamster-IgG-HL-Antibody-PeroxidaseLabeled-5220-0371/)) spike-specific antibodies were used (diluted at 2500X) for detection and visualized with KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120-0047). The reaction was stopped with KPL stop solution (Seracare) and plates were read at 450 nm. The threshold for positivity was calculated as the average plus 3 x the standard deviation of negative control hamster sera. Endpoint titers were determined.