Sars cov 2 spike s1 s2 ecd his recombinant protein

Nunc MaxiSorp™ flat-bottom 96-well plates (ThermoFisher, Ottawa, ON) were coated with 1 µg/mL of either SARS-CoV-2 Spike S1+S2 ECD-His recombinant protein or SARS-CoV-2 Spike RBD-His recombinant protein (Sino Biological, Beijing, China) in PBS and incubated overnight at 4°C. Plates were washed with PBS containing 0.1% Tween-20 (PBS-T) before blocking with 3% (w/v) Bovine Serum Albumin (IgG-Free, Protease-Free) (Jackson Immuno Research, West Grove, PA) in PBS-T for 2 hours at 37°C. The plates were washed again and two-fold serial dilutions of hamster serum, starting from 1:50 up to 1:102400 were added to the wells and incubated for 1 hour at 37°C. Plates were then washed with PBS-T and Peroxidase AffiniPure Goat Anti-Syrian Hamster IgG (H+L) (Jackson Immuno Research, West Grove, PA) was added to each well at 1:4000 and incubated at 37°C for 1h. Plates were washed again with PBS-T and 100 µL of Tetramethylbenzidine (TMB) substrate (Cell Signaling Technology, Danvers, MA) was added to each well. After a two-minute incubation at room temperature, 100 µL of 0.16 M sulfuric acid was added to terminate the reaction and absorbance was measured at 450 nm. Endpoint titers were expressed as the reciprocals of the final detectable dilution with an OD above the cut-off value, which was defined as the average OD of the pcDNA3.1-empty samples plus 3 standard deviations.

Serum from the rabbit of different groups were analyzed by enzyme‐linked immunosorbent assay (ELISA) to determine sera antibody titers. ELISA plate (Corning, USA) was coated with 1 µg/ml SARS-CoV-2 Spike S1 + S2 ECD-His recombinant protein (Sino Biological, China) in Dulbecco’s phosphate-buffered saline (DPBS) (ThermoFisher, USA) for 2 h at room temperature. Plate was washed for 3 times with DPBS + 0.05% Tween 20 (Scharlau, Spain) and then blocked with PBS + 1% BSA (ThermoFisher, USA) + 0.050% Tween-20 for 2 h at 37 °C. The plate was washed for 3 times then incubated with rabbit sera and SARS-CoV-2 Spike antibody (Sino Biological, China) for 2 h at 37 °C. After washing for 3 times, the plate was incubated with HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (ThermoFisher, USA) for 50 min at room temperature. Final washing was done for 3 times and then developed for colorimetric reaction with Pierce TMB substrate (ThermoFisher, USA) for 10 min. The reaction was stopped with 1 N HCl and the plate was read at 450 nm wavelength within 30 min.