Preparation Of Monoclonal Antibodies Against PEDV And The Establishment Of Sandwich ELISA Method

Pig epidemic diarrhea(Porcine epidemic diarrhea,PED)is caused by PEDV and it is a highly contact intestinal infectious diseases as the main characteristics of diarrhea,vomiting,dehydration and high fatality rate for lactation piglets.Different ages and varieties of Pigs are susceptible,PED is the most harmful to lactation piglets and the mortality rate is very high.The purified whole PEDV viruses is used as immunogen in this study,6 weeks old BALB/c mice wereimmunized with it for 4 times,then the spleen cells of immunized mice were fused with SP2/0 myeloma cells 3 days after the last immunization.Two stable secrete monoclonal antibody hybridoma cell lines were prepared by indirect ELISA and limited dilution method after the training,selection and the cloning and they were named 5F4 and 5B3,respectively.The number of chromosomes of two strains hybridoma is between 75 and 120.The results of Western blot and indirect immunofluorescence identifications showed that the two strains of monoclonal antibody can specifically react with PEDV,respectively.The results of indirect ELISA showed that two strains of monoclonal antibodies do not react with TGEV,PRV virus and this suggest that the obtained monoclonal antibody and PEDV have certain specificity,respectively.Combined ELISA test showed that two strains of monoclonal antibodies are identified antigen of PEDV protein on the same site.The preparation of PEDV monoclonal antibody provides an important material base for the method of PED etiology immunological detection with monoclonal antibody as the main detection reagent.By purifying and concentrating the cells culture of PEDV to get the whole virus which was used to immunize white rabbits,and prepared the hyperimmune serum against PEDV.The titer of serum antibody tested by ELISA is 1×10-5.The basis of the establishment of the double antibody sandwich ELISA was that the polyclonal antibody and monoclonal antibody were used as capture antibody and the detecting antibody respectively,and the swine fecal samples were used as the detecting antigen.The appropriate dilution of rabbit anti-PEDV antiserum was 1:800.The positive determining standard of this method was determined as S/N>2.by square test,while OD490nm>0.37.The lowest detection limit of this method was 5×103 67 TCID50/ml.The results showed that only the detection against PEDV was positive,the others were negative by detecting the faeces with PEDV,TGEV and PRV in this method,which indicated that the method has a higher specificity.19 samples of the 48 fecal samples from different pig farms were positive detected by the new established ELISA method,21 samples were positive detected by RT-PCR,and the positive rate were 39.6%(19/48),43.8%(21/48)respectively,there was no significant difference between the two detection methods by X2(P>0.05),it showed that the established double-antibody sandwich ELISA method can be used to detect PEDV.It turns out that antigen capture ELISA method has a good specificity and sensitivity,it can be used for the rapid detection of PEDV.Antigen capture ELISA method can not only provide a more simple,rapid and sensitive detection method for the clinical diagnosis of PEDV,but also provide us an experimental basis to explore a more accurate,more intuitive and easier detection method.