Sars cov 2 s2 protein

Enzyme-linked immunosorbent assay (ELISA) was employed to screening for the specific binding phage-scFv clones. Briefly, 96-well ELISA plates (Corning, 42,592) were coated with SARS-CoV-2 S2 protein (Sino Biological, 40,590-V08B) and incubated at 4°C overnight, then plates were blocked with 1% bovine serum albumin (BSA) (Sangon, A600332–0100)/PBS (Gibco, 10,010–023) at 37°C for 2 hours (300 μL/well) and washed with PBST for five times. 50 μL supernatant from the single clone antibodies pool and 50 μL 1% BSA/PBS were added into each sample well and incubated at 37°C for 1 hour, blank control wells were supplemented with 1% BSA/PBS at the same time (100 μL/well). After five washes with PBST, a 1:3000 dilution of Horseradish peroxidase (HRP) conjugated anti-human Fab (Sigma, A0293) was added and incubated at 37°C for 1 hour (100 μL/well). Following five washes with PBST, 3,3',5,5'-Tetramethylbenzidine substrate (Thermo Scientific, 34,029) was added (100 μL/well) and incubated at room temperature for 10 min, finally 50 μL 2 M H₂SO₄ was added to each well to stop the reaction. Results were monitored at 450 nm, the cut-off value was set as the mean of the blank controls +2 standard deviation. The well with OD 450 value > cut off value was considered as positive well, thus phage-scFv against S2 was separated.