S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen

Makadiya, Niraj; Brownlie, Robert; van den Hurk, Jan; Berube, Nathalie; Allan, Brenda; Gerdts, Volker; Zakhartchouk, Alexander

Immulon 2 HB 96-well plates (ThermoFisher Scientific) were coated overnight with 0.1 ml/well of 0.5 μg/ml purified recombinant S1 protein. The plates were washed five times in phosphate buffer saline (PBS) containing 0.05 % Tween 20. Sera serially diluted in assay diluent buffer (0.1 M PBST, 0.05 % Tween-20, 1 % fish gelatin) were added to respective wells. After a 2 h incubation, the plates were washed, and 1/3000 diluted HRPconjugated anti-pig IgG antibodies were added. After 1 h incubation, the plates were washed, and developed with 1-Step Ultra TMB-ELISA substrate solution (Ther-moFisher Scientific). The reaction was stopped by adding 30 μL of 2 M sulphuric acid to each well, and optical density values were measured at 450 nm using an ELISA plate reader.