Development Of Indirect ELISA Antibody Test Kit For Japanese Encephalitis Virus

Epidemic encephalitis B is a zoonotic, insect-borne viral disease caused by Japanese encephalitis virus. To provide a quick and easy tools and experimental methods for clinical diagnosis of porcine epidemic encephalitis after immunization, antibody detection and epidemiological investigation, the experimental use of the E protein of JE virus antigen was established indirect ELISA for detection of JE virus antibody, and developed on this basis can be used for clinical detection of JE virus antibody indirect ELISA kit.1. The preparation of recombinant E proteinIn this study, recovery in the preserved in the laboratory of the E protein expression strain vector pET28a (+)-E (E. coli BL21) recombinant strain, after the PCR method for identification of bacteria, The SDS-PAGE showed that laboratorysave pET28a (+)-E (E. coli BL21) recombinant strain can still be a lot of expression of E protein. NI ion affinity chromatography purification of the E protein and dialysis, the preparation of a package with better reactogenicity of antigen E protein.2. The establishment of the indirect ELISAPurified complex of the E protein as coating antigen, the best package of antigen concentration, serum optimum dilution, antigen coated condition, a closed fluid type, serum optimum time, HRP secondary antibodythe appropriate duration of action, time optimization of the chromogenic substrate, the establishment of an indirect ELISA for antibody detection of porcine epidemic of Japanese encephalitis virus. The optimization results show that the best package of the antigen concentration of7.1ug/ml serum dilution1:80, antigen coated condition is37℃package1h at4℃overnight, and the choice of blocking solution1%BSA, serum, and HRP anti-reaction time of1h, the substrate reaction time was10min.3. The assembly of the test kit and its quality researchDetection kit materials needed for the establishment of an indirect ELISA, Japanese encephalitis virus antibody test kit was developed. Kit, sensitivity, specificity, repeatability, shelf life studies show that the kit sensitivity on almost commercial kits; on specific aspects of pseudorabies (PR), porcine parvovirus virus (PPV), porcine reproductive and respiratory syndrome (PRRS), swine fever (HC)-positive serum reaction, the test results belong entirely negative; repeatability within a batch and between batches repeat experimental results show that the detection of the kit of the same serum results are about5%, with good reproducibility; kit stored at4℃for4months can still be used to detect.In this study, the clinical diagnosis of Japanese encephalitis virus and immune antibody monitoring provides a fast, simple and less demanding on the equipment and technical personnel for grass-roots used detection methods, and its also laid a certain foundation for the commercialization of the test kit.