Establishment And Application Of IgG And IgA Antibody Detection Method For Porcine Epidemic Diarrhea Virus (PEDV)

Porcine epidemic diarrhea (PED),caused by porcine epidemic diarrhea virus (PEDV) is a kind of high pathogenic contact intestinal infectious disease,which resulted in a high rate of mortality in suckling pigs. Obtaining maternal antibodies from milk is the most important way for the sucking pigs to resist PEDV. The secretion of IgA in breast milk can protect the intestinal mucosa of piglets, and it can protect the suckling sows against PEDV infection. The current commercial sera antibody detecting kits for PEDV are based on detection of neutralizing antibodies or IgG in serum samples. Therefore, it is significant to study the ELISA detection method of IgA antibodies in breast milk for the prevention and control of PED infection in piglets.In this study, PEDV-N protein gene was cloned to the pCold I vector and expressed by prokaryotic expression system. Two indirect ELISA methods based on the recombinant protein was developed to monitor IgG and IgA levels in colostrum or serum samples, named as IgG-ELISA and IgA-ELISA. The methods established in this study was used to detect the serum antibody in pigs challenged with PEDV. The results showed that in serum samples from the experimentally infected pigs, anti-IgG and anti-IgA PEDV antibodies were detected as early as 7 days post-infection, the anti-IgG antibody levels reached the peak 16 days post-infection, the anti-IgA antibody levels rised more slowly compared with the anti-IgG antibody and it reached the peak 32 days post-infection. The anti-IgG and anti-IgA to PEDV persisted for more than 120 days post-infection in most of the experimentally infected pigs.IgG-ELISA was compared with the serum neutralization (SN) test by testing 84 serum samples from the experimentally infected pigs, and the negative coincidence rate was 70.6%, the positive rate was 91%, the total coincidence rate was 86.9%. When compared with the PEDV anti-IgG test kit made by Canada Biovet company by testing 77 field serum samples, the negative coincidence rate was 64.5%, the positive rate was 66.7%, the total coincidence rate was 64.9%, and the further detection on the inconsistent serum samples by SN test showed that the total coincidence rate of IgG-ELISA was 71.4%, and the test kit of Biovet company was only 28.6%. IgA-ELISA was compared with PEDV anti-IgA test kit made by Anigen company by testing 60 milk samples from the sows vaccinated with PEDV, the positive rate of IgA-ELISA was 100%, and the coincidence rate was 98.3%. But the negative samples used for comparison detection were too little, so that there is necessary for further validation. The experimental results showed that the IgG-ELISA and IgA-ELISA had a good sensitivity and specificity, which have laid the foundation for the further development of clinical test kits.In addition, this study found that the fluctuation law of anti-IgG of PEDV was consistent with the neutralizing antibody. Anti-IgG and anti-IgA levels in serum or milk of sows showed a positive correlation with the antibody levels in the serum of the suckling pigs by testing the samples from the sows vaccinated with PEDV and its piglets.