Development Of Monoclonal Antibodies Against PEDV And Double Antibodies Sandwich ELISA For The Virus Detection

Porcine epidemic diarrhea(PED)is an intestinal infectious disease,which mainly due to porcine are infected Porcine Epidemic Diarrhea Virus(PEDV).The main clinical symptoms of sicked piglets are vomiting,severe diarrhea,dehydration and others.Recently,because the prevalence of the disease in China has expanded significantly and the pathogenicity of pathogens has also increased,PEDV has became one of the severe causes of the death of diarrheal piglets.The laboratory differential diagnosis is particularly necessary due to PEDV clinical symptoms similar with other porcine viral diarrhea disease in clinical manifestation.Three monoclonal antibodies which can secret anti-PEDV antibodies were obtained through the PEDV propagated in Vero cell,proliferated and purified by density gradient centrifugation,immunized BALB/c mice,cell fusion,cloning and multiple screening.They was designed as 43B6-H2,75E2-F3,27C9-C5-D8 respectively,and as the main detection reagent for ELISA.Through the antibody subclass identification method,both antibodies were identified as Ig M subclass by.The titers of hybridoma cells' supernatant were determined by indirect ELISA to be 1:6400,1:3200 and 1:3200,respectively,and Western blot and IFA results showed that all three monoclonal antibodies could specifically recognize PEDV.But,the structural protein size of the reaction is different.43B6-H2 and 75E2-F3 recognize proteins of the same molecular size,between 25-35 KD,and 27C9-C5-D8 recognizes about 55 KD.Proteins,which correspond to PEDV M protein and N protein,respectively.Moreover,the result of the superimposed ELISA assay shown that the antigenic sites of 43B6-H2 and 75E2-F3 were similar,whereas different from that of 27C9-C5-D8.The above experimental results indicated that the specificity of these three monoclonal antibodies was excellent and can provide an important material basis for the diagnosis of PED pathogen.Based on the preparation of PEDV monoclonal antibody,purified rabbit anti-PEDV serum was used as capture antibody,and peroxidase-labeled monoclonal antibody was used as detection reagent.Through optimization of various conditions,double antibody sandwich ELISA was used to detect PEDV.The rabbit anti-PEDV polyclonal antibody was diluted to a concentration of 2.8 ?g/m L,100 ?L was added to each well,and coated at 37? for 1 h,and coated overnight,then add 5% skim milk 200 ?L/well to block overnight.After that,add 200 ?L of virus culture for 90 min in 37?,then add enzyme-labeled monoclonal antibody at a concentration of 7.5 ?g/m L,for 100 ?L per well at 37? for 90 min.This test method does not cross-react with porcine rotavirus(Po RV)and porcine transmissible gastroenteritis(TGE);and the minimum detection limit of virus was 3.125×103TCID50/m L;The the inter-batch variational coefficient of positive samples was 1.22%-3.66% and that of the negative samples was 2.90%-8.72%;The intra-batch variational coefficient of positive samples was 3.76%-7.03% and the intra-batch variational coefficient of the negtavie ones was 3.62%-9.32%,both rates were less than10%;finally,taking advantages of the ELISA method to detect 47 clinical samples,and the result to compared with RT-PCR method and goldimmunochromatographicas assay strip's results show,the positive result of RT-PCR was 21,that of ELISA and GICA was 19 and 14,respectively.The positive detection rate of this method is between RT-PCR and gold standard immunochromatographic test strips.