Establishment Of Sandwich ELISA Method And Diagnostic Kit Preparation For The Detection Of Porcine Epidemic Japanese B Encephalitis

Porcine epidemic encephalitis, which is caused by Japanese encephalitis virus (JEV) is a kind of insect-borne viral disease. After the infection, the viral loading and time of porcine are the highest and longest among other kinds of animals, therefore porcine is regarded as the main host for the storage and transmission. In this study, we have established the double-antibody sandwich ELISA method and developed its kit for the detection of the virus, which could be used for the analysis of the time, place and the environment of the viral infection in pigs, this method has offered an effective method in mastering the distribution of encephalitis in China and building the models to provide the incidence trends of the virus in pigs.In this study,the E protein fragment of Japanese encephalitis virus (SA14-14-2)was obtained by PCR amplification and cloned to the vector PET28a to construct the expression vector PET28a-E,which was expressed under the IPTG induction and purified by Ni-NTA to get JEV E protein antigen, and based on the above results we have established a JEV E proteinbased indirect ELISA method. After the immunization of the virus(P3) on the New Zealand white rabbits we have obtained the rabbit polyclonal antibodies (1:16000 titer), which was stored at -80°C, after purification using octanoic acid-ammonium sulfate and hydronium exchange of DEAE. Four 7-week old female Balb/c mice were immunized with the polyclonal antibodies,spleen cells of the mice with the highest titers were fused with the bone marrow cells and the hybridoma cells were screened by indirect ELISA, two strains with stable secreted monoclonal antibodies, named 6A8 strain and 3E9, ascites titers of 3E9 (1:1.2×105) was higher than that of 6A8 (1:8.2×104). Immunohistochemical analysis showed that the immune specificity of 3E9 was higer, therefore, 3E9 strain was selected as the specific antibody to establish double-antibody sandwich ELISA method, the rabbit multiclonal antibody was choosen as the capture antibody, which is easy for the virus to stick and the mouse monoclonal antibody as the detection anti- bodies,which can improve the specificity of the test results. The multiclonal antibody was diluted to 1:8000, and the monoclonal antibody was diluted to 0.5μg/mL, accordingly, construction of the kit.In this study, we have build the indirect ELISA method based on clone and expression of epidemic Japanese encephalitis B virus E protein gene and constructed polyclonal rabbit anti-JE antibody and mouse monoclonal antibody and developed the double-antibody ELISA, its sensitivity, specificity and reproducibility reach experimental requirements. Preparation of the kit, then test for the actual samples and experimental samples.It has offered an effective method for warning platform of the porcine epidemic encephalitis B.