Sandwich ELISA
Mab 1A9 was diluted with coating buffer (0.1 M NaHCO3, 34 mM Na2CO3) and 0.1 µg of antibody was coated onto individual wells of a Maxisorp flat-bottom plate (Nunc) overnight at 4 °C. The plate was washed three times with PBST before blocking was done using 5% BSA/PBST at 37 °C for 60 min. Dilutions of His-tagged full length SARS-CoV-2 S protein (Sino Biological Inc., catalogue number: 40589-B08V1) and His-tagged H7N7-HA (Sino Biological Inc., catalogue number: 11082-V08B) were added to desired wells and incubated at 37 °C for 90 min followed by three washes with PBST. 100 µL of CR3022 antibody was added at a concentration of 1 µg/mL and incubated at 37 °C for 60 min followed by three PBST washes before HRP-conjugated goat anti-human IgG (Thermo Fisher Scientific) was added for 60 min at 37 °C. Finally, after three PBST washes, TMB (Sigma-Aldrich) was added for 5 min and the reaction was stopped by 2 M sulphuric acid. The OD450nm was determined by a Tecan Infinite M1000 reader. Statistical analyses were performed using an unpaired, one-tailed Student’s t-test with Welch’s correction for unequal variances. p values < 0.05 were considered statistically significant.