Human anti s1 antibody

The culture supernatant sample or purified S protein was coated onto each well of the microtiter plate and left overnight at 4°C. After the wells were blocked with 1% BSA for 1 h, dilutions of human anti-S1 antibody (Abcam) were added to the wells and incubated for 3 h at room temperature. After being washed, HRP-conjugated rabbit anti-human IgG antibody (Abcam) was added and incubated for 1 h at room temperature. The bound antibodies were detected using TMB substrate (Sigma-Aldrich). Absorbance was read at 450 nm using a Multimode Plate Reader (Promega, Madison, WI, USA).

Purified proteins were mixed with loading buffer and loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were electrophoresed for 110 min at 80 V and 500 mA and visualized using Coomassie Brilliant Blue G250 staining (Sigma-Aldrich) or transferred to a nitrocellulose blotting membrane (Sigma-Aldrich) at a constant current of 250 mA for 90 min. Next, the membranes were blocked in 0.5% bovine serum albumin (BSA) (Sigma-Aldrich) and incubated with human anti-S1 antibody (Abcam, Cambridge, UK) for 3 h at room temperature. After being washed, the membranes were incubated with horseradish peroxidase (HRP)-linked rabbit anti-human IgG antibody (Abcam) for 1 h at room temperature. Membranes were washed three times with PBST [1× phosphate-buffered saline (PBS), 0.1% Tween^® 20 Detergent], and the protein bands were visualized by enhancing the TMB (3,3′, 5,5′-tetramethylbenzidine) substrate solution (Sigma-Aldrich).