Optimization Of The Indirect Elisa Detection Method And Vaccine Immunization Study On Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus (PEDV) is a highly contagious gastroenteritis pathogen causing disease characterized by watery diarrhea, vomiting and dehydration. The wide geographical distribution, the acute disease and the high mortality rate in the newborn piglets due to PEDV have brought heavy economic losses to the swine industry. On the basis of the optimized PEDV N protein antibody ELISA detection method, the immune effect of the current commercial vaccines and autogenous vaccine were evaluated to select a more efficient and better vaccine for pigs’immunization on farms.In this study, the purified prokaryotic expressed PEDV N preotein was used coated in the ELISA plates as antigen. The ELISA reaction conditions optimised were:20times diluted N protein coated at37℃for2h and blocked by5%skim milk at4℃overnight. The serum samples used for antibody detection were40times diluted and incubated with the restructured protein at37℃for1h.8,000times diluted HRP-conjugated goat anti-pig IgG was used as secondary antibody and incubated at37℃for lh.TMB substrate was used for coloration at room temperature for10minutes. The cut-off value was OD4500.161. Despite the serious damages of PEDV to swine industry, the efficacy and effecitiveness of of commonly used vaccines were not satisfactory. In this study, randomly selected4030-day-old piglets were divided into8groups and immunized respectively by3kinds of commercially vaccines, two autogenous vaccines and a home-made cell derived vaccine. All the pigs were challeged with PEDV on28day post immunization.The results showed that the antibody level of pigs in each group is not high, indicating the limited efficacy and less effectiveness of the all kinds of tested vaccines. The commercial bivalent vaccine had a better efficacy compared with other vaccines. Moreover, the homemade vaccine showed relative good immune effect when vaccinated by combined immunization. Furthermore,immunization method such as oral administration and houhai point injection induced moderately better immune effect when used alternately.In this study, the level of the PEDV antibody of pigs was tracked by the self-developed ELISA kit. Results showed that the breeding sows immunized with autogenous vaccine could induce relative high level of antibodies but decreased gradually.Meanwhile, maternal antibody level of the piglets reached peak at7days and declined to the lowest level at21days after birth. However, the antibody level of piglets and sows displayed no significant linear relationship between each other.