Establishment And Application Of Indirect ELISA Method For Detection Of IgA Against Porcine Epidemic Diarrhea Virus

Porcine Epidemic Diarrhea(PED)is a highly contagious intestinal disease caused by Porcine Epidemic Diarrhea Virus(PEDV).The main clinical features are acute watery diarrhea,vomiting,dehydration and weight loss.Pigs of all ages are susceptible to the disease,and the morbidity and mortality of suckling piglets can reach to 80%-100%,causing hμge economic losses to the pig industry.Newborn suckling pigs can cause severe diarrhea symptoms within 1-2 days after infection.Due to the imperfect development of the immune system,newborn suckling piglets cannot quickly produce an immune response after infection.Therefore,maternal antibody is a key way for newborn suckling piglets to obtain passive immune protection.Secreted IgA(sIgA)in breast milk enters the intestinal tract of piglets throμgh the digestive tract and is essential to protect the intestinal mucosa of suckling piglets from PEDV invasion.Therefore,the establishment of detection methods for IgA antibodies in breast milk and serum has certain guiding significance for monitoring the amount of maternal antibodies obtained by piglets.In this study,in order to establish indirect ELISA detection methods for detecting PEDV-specific IgA antibodies,the CH/HNPJ/2017(PJ)and CH/HBXT/2018(XT)which separately located in PEDV GIIb and GIIa genogroup isolated from our laboratory were used.Based on the S1,S2 and N proteins,recombinant plasmids were constructed and transferred into E.coli BL21(DE3),and the expression of the strain was induced with IPTG at a final concentration of 1 mmol/L at 37°C.ELISA and Western-Blotting tests showed that the three proteins(PJ-N,PJ-S1 and XT-S2)expressed had good antigenicity.Three purified proteins were used as antigens to prepare ELISA plates,and three indirect ELISA detection methods were established,named PJ-N-iELISA,PJ-S1-iELISA,XT-S2-iELISA,respectively.Among them,PJ-N-iELISA does not cross-react with FMD and PRRSV positive sera,and the total coincidence rate with indirect immunofluorescence test results was 85.19%;the overall coincidence rate between PJ-N-iELISA、XT-S2-iELISA and virus neutralization test results was 86.36% and 81.82%.CV% of stability tests for PJ-N-iELISA,PJ-S1-iELISA,and XT-S2-iELISA were all less than 11%.PJ-N-iELISA,PJ-S1-iELISA and XT-S2-iELISA were used to detect 177 piglet serum samples,respectively.The positive detection rates were 72.32%、79.10%、76.84%;the negativedetection rates were 27.68%、20.90%、23.16%,respectively.The detection methods established in thisexperiment has good specificity and stability,which can provide effective detection methods forPEDV related research,and is of great significance for the prevention and control of PEDV,the rational use of vaccines and the monitoring of effects.