The Preparation Of Anti-PEDV Monoclonal Antibody And Development Of A Double Antibody Sandwich For Detection Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED),which is caused by porcine epidemic diarrhea virus,is a highly contagious enteric infectious disease.The swine infected with PEDV mainly shows the symptoms as fever,vomiting,dehydration and even badly diarrhea.PEDV has its structure including four structural proteins,spike protein,membrane protein,envelope protein and nucleocapsid protein.In these proteins,nucleocapsid protein is a major one,and highly conserved.Therefore,nucleocapsid protein is signal for detection of PEDV.In the present study,cell-adapted PEDV CV777 preserved in our lab was purified by density gradient centrifugation.Purified PEDV shows a good quality after analysis of SDS-PAGE and Western blot.BALB/c mice were immunized with purified PEDV and sacrificed for preparation of monoclonal antibodies.Three Hybridomas secreting anti-N protein antibodies were obtained,designed as 13C3A10-2,6G and 12E2H8-2 respectively.Indirect immunofluorescence assay shows that three MAbs all can reacted with Vero C1008 call infected with PEDV.And three MAbs also can react with na?ve nucleocapsid protein and recombinant nucleocapsid protein.In the present study,in order to detect PEDV in sample,a double antibody sandwich ELISA was established with MAb 13C3A10-2 and swine serum which is PEDV positive.The former is capture antibody while the latter as bingding antibody.Because of the N protein hidden in the virion,it is important to release N protein by disintegration of PEDV before detection.The experiment taking the process of lysis of influenza virus for reference,completed the release of N protein.To detect PEDV in the samples,the present study established a double antibody sandwich ELISA using MAb and polyclonal antibody.Afterwards,the specific,sensitivity and stability were identified.Taking advantages of the ELISA,65 samples were detected.The results were compared with RT-PCR and show that the agreement of two methods is 94.7%.All results means the ELISA established could be occupied to detect PEDV.