Preparation And Application Of Monoclonal Antibody Against Bovine Viral Diarrhea Virus

MDBK cells were infected with Bovine viral diarrhea virus (BVDV) HN-1 strain. The virus was centrifugated and purified. BALB/c mice were immunized with the purified BVDV. Using lymphocyte hybridoma technique,mouse spleen cells were fused with myelomas cells which was named NS0. Four hybridoma ceLL strains which can excrete stably monoclonal antibody were obtained by indirect ELISA and tri-time limiting dilution.The four hybridoma cell strains are named 3A₈,3C₇,3C₁₁ and 3E₉. By evaluating,three strains belong to IgG1 subset, and the other one is IgG2a subset. The monoclonal antibodies secreted by 3A₈,3C₇,3C₁₁ and 3E₉ strains have no cross reactions with BCV and BRV. The monoclonal antibody secreted by 3A₈,3C₇,3E₉ strains have cross reactions with HCV,BDV,but the monoclonal antibody secreted by 3C₁₁ strain has no cross reactions with HCV and BDV. The specificity of monoclonal antibody secreted by 3Cn strain is better. ELISA titers of monoclonal antibodys which are existent in superior schedule of culture of hybridoma cells and ascites of mice are 1:1000 and 1:200000 respectively. After continuous cultivated to 20 generations, the hybridoma cells strains could still excrete antibody stablely. The monoclonal antibody was very important to the investigation of BVDV and the creating of diagnosis method.The hybridoma cell line named 3Cn which couLd secrete McAb stabely was anabiosised. Then the ascites were produced and the IgG was purified by the common method. Applying the rabbit-anti-BVDV IgG and McAb, the double sandwich enzyme-linked immunosorbent assay (ELISA) for detecting bovine viral diarrhea virus (BVDV) was developed. Comparied with Virus Isolation and Neutralization Test, the positive coincidence rates are 86.67% and 93.33% and the nagetive coincidence rates are all 100%.100 samples of whole bloods of bovines were detected by the assay and commercial ELISA kit ,and the positive coincidence rate was 96.15%. The least detecting titer of this assay was 200ng/μL The assay was applied to detecting BVDV in whole blood from 562 bovines of Nanyang,Zhoukou,Anyang regions in Hennan province. The results demonstrated that BVDV-carrier rates of cattle were 16.75%,which suggested that the disease still exists in these areas. The assay has a promising application future.