Establishment of ELISA for detecting porcine epidemic diarrhea virus specific SIgA in mucus

ABSTRACT

The aim of this study is to establish an indirect ELISA technique for detecting the SIgA antibody against porcine epidemic diarrhea virus (PEDV) to evaluate its mucosal immunity. Firstly, the S1D gene (534-789 aa) of PEDV was cloned into the pET-28a(+) vector, and induced in Escherichia coli BL21 (DE3) by IPTG, the product of which was in the form of inclusion bodies. According to Western-blot, the target protein S1D with antigenic activity was 32 ku in molecular weight and could be well detected. Then, the S1D protein was denatured by 8 mol/L urea, purified and gradient as the coating antigen to establish an indirect ELISA for detecting the PEDV specific SIgA antibody in nasal or oral mucus by optimizing conditions. And the optimal antigen coating concentration of ELISA was 2 micro g mL, the working concentrations of nasal mucus was 11 and the optimal blocking solution was 50 g/L skimmed milk, while the working concentrations and optimal blocking solution were 12 and 30 g/L BSA in oral mucus, the working concentrations of the enzyme-labeled antibody was 12 000 in nasal and oral mucus. Finally, 84 samples of oral and nasal mucus from immunized pigs were detected by S1D of ELISA, and the coincidence rate could reach 95.2% compared with purified PEDV of ELISA. In conclusion, the indirect ELISA established in this study provided a quick, simple, sensitive, and specific method to detect PEDV specific SigA for evaluating the level of PEDV mucosal immunity.