Development Of An Indirect ELISA Method For Detecting Porcine Epidemic Diarrhea Virus N Protein And Polypeptide

Porcine epidemic diarrhea（PED）is an acute contact infectious disease in pigs caused by Porcine epidemic diarrhea virus（PEDV）,which is characterized by diarrhea,vomiting,dehydration and weight loss.Although PED can occur in pigs of all ages,it is most harmful to suckling piglets.Before 2010,the disease was not a concern.At the end of 2010,the PEDV mutant strain causing a serious PED killed more than 1 million piglets in just one year in China,causing huge economic losses to China’s pig industry.In clinical practice,the rapid and effective differential diagnosis method is important for the prevention and control of clinical diseases and the monitoring of vaccine immunity effect.To develop an effective method for detecting PEDV antibodies,the indirect ELISA method for detecting PEDV antibodies was established in this study using prokaryotic expression of N protein as the coated antigen.On the basis of previous studies,an indirect ELISA method was established for the differential detection of antibody of PEDV SH and other strain using synthetic polypeptides.The research contents are as follows:1.Establishment of an indirect ELISA method based on N protein of PEDV expressed in prokaryotic expression systemThe N protein recombinant plasmid was transformed into bacteria Rosetta and induced by IPTG.PEDV N protein was purified by Nickel column.After quality test,an indirect ELISA method was established to detect PEDV antibody using N protein as the coated antigen,and the reaction conditions of the ELISA were optimized.To evaluate the method,269 clinical pig sera were detected by the indirect immunofluorescence assay（IFA）and this ELISA.According to the results of these sera,the ROC curve was drawn to analyze the accuracy,sensitivity and specificity of the indirect ELISA method,and the optimal threshold value was calculated.The results were as follows:SDS-PAGE showed that the protein was expressed and purified and the concentration was up to 500μg/m L,which could be used as the coating antigen of indirect ELISA.When NEST detachable plate was used as ELISA coating plate,the antigen was diluted to 0.5μg/m L,coated at 4℃for 14h,and sealed at 37℃with 5%skim milk for 2h.The serum was diluted 1:200 and incubated at37℃for 2h.SPA-HRP was diluted at 1:10,000 and incubated at 37℃for 30min.The substrate TMB was screened from light at room temperature for 15min,and the 2M H₂SO₄was terminated.The ROC curve was used to analyze 269 clinical pig sera detected by IFA and this method.The results showed that the ELISA antibody detection method had good diagnostic value（AUC was 0.887）,the sensitivity was 94%,the specificity was 75%,and the optimal cut-off value was 0.528.2.Establishment an indirect peptide ELISA to detect the antibody to PEDV SH2016strain and other strainsIn our previous study,PEDV SH2016 strain deleting an antigen epitope of N protein was found.To differentially detect the antibody to SH2016 strain and other strains,the i ELISA was developed.According the genes of CV777 and the mutant strain,the two small peptide located at the deletion site of PEDV SH2016 was synthesized and used as coating antigen of indirect ELISA.The processes of the i ELISA was as above.The results were as follows:When the Nest removable ELISA plate was used as the ELISA coating plate,the antigen was diluted to 0.5μg/m L,coated at 37℃for 2h and sealed with 5%skim milk at37℃for 3h;The serum was diluted at 1:50,and incubated at 37℃for 30min.SPA-HRP-was diluted at 1:2000 and incubated at 37℃for 45min.The substrate TMB was incubated37℃for 8min without light.The results of ROC curve showed that the IELISA antibody detection method using two small peptides had good diagnostic value（AUC was 0.849 and0.875,respectively）,the sensitivity was 92%and 96%,the specificity was 61%and 68%,and the optimal cut-off value was 0.578 and 0.518.