Establishment Of Monoclonal Antibodies Against Porcine Parvovirus Nucleocapsid VP2 Portein And Development Of Double Sandwich ELISA

PPV was cultured by Pk-15 cell and purified by differential centrifugation and sucrose density centrifugation as immunized antigens. Eight-week BALB/c mice were immunized according to Kazuhiro's produre. Prokaryote expression vector of PPV-VP2 gene which was constructed and conserved by our lab was induced by IPTG and expressed protein of VP2 gene, then the expressed protein was measured by SDS-PAGE and the result showed that the VP2 gene can be expressed successfully in E.coli. Purified protein was measured by western-blotting and the result showed that expressed protein could be recognized by the PPV positive serum and can be used in diagnostic antigen. Based on the purified recombinant protein,an indirect ELISA method for detection of PPV antibodies was developed and used to screen hybridoma. Hybridoma were generated by fusion of NS₀ myelomas and Splenocytes from immunized mice. The monoclonal antibody(McAb) was screened by indirect ELISA. Positive colonies were cloned three times by limited dilution. Three cell line named PPV—VP2—CC1, PPV—VP2—BD2, PPV—VP2—CD12 which could secret McAb stably was obtained. Only the isotype of CC1 McAb was identified the result showed IgG₁ and the average number of chromosomes of the hybridoma cell line was 99.The ELISA titer of cell supernatant and ascite were 2⁵ and 2¹¹.CC1 McAb did not crossly react with PRRSV,HCV,HRV,which demonstrated subtype-specificity.The CC1 cell line could steadily secret McAb after subcultured twenty-one times. Mouse ascetic fluid was purified and result showed protein concentration: 2.151mg/mL.The obtainment of this McAb established a foundation of the researches to PPV and quick-diagnosis methods.Based on the rabbit-anti-PPV IgG and CC1 McAb,the double sandwich enzyme-lined immunosorbent assay (ELISA) for detection of Porcine Parvovirus was developed. The best coating concentration of the rabbit-anti-PPV IgG was 4.04μg/mL.The best working concentration of McAb was 6.45μg/mL.The sensitivity of the double sandwich ELISA was 215.5ng/mL. The assay had no cross reaction with common pathogenic microorganisms,and the positive coincidence rate with virus isolation assay was high.The whole operation time only need 4.5h(not including...