Establishment Of Indirect ELISA For PEDV IgA And Study On The Relationship Between Specific Antibody IgA And IgG

Porcine epidemic diarrhea virus（PEDV）mainly harms suckling piglets,and the clinical manifestations of sick piglets are vomiting,acute diarrhea,dehydration and high mortality.The epidemic situation of porcine epidemic diarrhea is severe,which has caused huge economic losses to the pig industry.in China.Newborn piglets acquire milk-derived immunity through breast milk is the most effective method to resist PEDV attack and the secreted immunoglobulin A（s IgA）antibody plays a major protective role.Therefore,s IgA level in breast milk is an important indicator for assessing immune status and formulating prevention and control plans.The S protein of porcine epidemic diarrhea virus is the main immunogenic protein and plays a key role in the induction of PEDV neutralizing antibodies.The COE region is the core neutralizing epitope on the S protein and has good reactogenicity.In this study,we expressed and purified the soluble recombinant protein GST-COE using an E.coli expression system,and the recombinant protein was used as an antigen to coat an enzyme-labeled plate.An indirect ELISA method was established to detect IgA antibodies in the milk and serum of vaccine-immunized sows with high sensitivity and specificity.Taking the virus neutralization test（VN）as a reference,and it was verified that the positive samples detected by the indirect ELISA detection method established in this study contained PEDV-specific neutralizing antibodies,indicating that the established method can be applied to clinical detection.The details are as follows:（1）Expression and purification of PEDV recombinant protein GST-COEAccording to the PEDV AJ1102 strain,a pair of PCR amplification primers were designed to amplify the target fragment COE,which was cloned into the prokaryotic expression vector p GEX-KG.The recombinant plasmid p GEX-KG-COE was successfully constructed and transformed into the host strain BL21（DE3）.The soluble protein GST-COE was induced to express,and the recombinant protein was verified by SDS-PAGE and Western blot to be the target protein.（2）Establishment of indirect ELISA detection methodThe recombinant protein GST-COE purified by GSH affinity chromatography was used as coated antigen to establish the indirect ELISA.We optimize each reaction condition and finally determine the best working conditions of the method:the optimal antigen coating concentration in whey detection is 3.125ug/m L,the optimal primary antibody dilution ratio is 1:10,the primary antibody reaction condition is incubation at 37℃ for 60 minutes,the optimal blocking solution is5%BSA,the optimal blocking condition is 37℃ for 150 min,and the optimal dilution ratio of enzyme-labeled secondary antibody 1:20000,the optimal conditions for enzyme-labeled antibody is incubation at 37℃ for 45 minutes and the optimal substrate color development time is 10 minutes in the dark.Optimum conditions used were 6.25μg/m L antigen concentration and 1:10 serum dilution.,the primary antibody reaction condition is incubation at 37℃ for 45 minutes,the optimal blocking solution is 5%BSA,the optimal blocking condition is 37℃ for120 min,and the optimal dilution ratio of the enzyme-labeled secondary antibody is 1:20000,the best conditions for enzyme-labeled antibody is incubation at 37℃ for 45min,and the best substrate color development time is 10min in the dark.Whey and serum samples share a common criterion:it is judged to be positive when the OD₄₅₀of the sample to be tested is greater than 0.295;it is judged to be negative when the OD₄₅₀of the tested sample is less than 0.248,and it is judged to be suspicious in between.（3）Validation of specificity,sensitivity and reproducibility of indirect ELISA detection methodsThe established indirect ELISA method was used to detect Foot and mouth disease virus,Transmissible gastroenteritis virus,Classical swine fever virus,Porcine circovirus 2 and Porcine reproductive and respiratory syndrome virus antibody-positive serum respectively.The results did not cross-react,indicating that the method has good specificity.The sensitivity of this method was tested.When four copies of positive whey and serum were diluted to 1:1280,and their OD₄₅₀values were still higher than 0.295 indicating good sensitivity.Then the coefficient of variation of the inter-assay and intra-assay were both less than 10%indicating good repeatability.（4）Application of indirect ELISA detection methodThe self-built indirect ELISA detection method and the kit produced by Wuhan Keqian Biological Company simultaneously detected 25 clinical whey and50 serum samples,and the coincidence rate reached 100%,indicating that the method can be used for clinical diagnosis.The IgG and IgA contents in 63 PEDV antibody-positive serums were detected simultaneously by the established indirect ELISA method.It was found that the serum IgA and IgG antibodies were positively correlated.Combined with relevant reports,the level of PEDV IgA antibody in milk is positively correlated with the level of PEDV IgG and IgA antibody in serum.Therefore,in the case of difficult milk collection,the level of IgA antibody in milk can be evaluated by detecting serum PEDV IgG and IgA antibody.