Preparation Of Monoclonal Antibody Against Canine Adenovirus Type 1 And Establishment Of The Virus Detection Method

Canine adenovirus type 1 is also named Canine infectious hepatitis virus, the virus can cause an acute septic diseases in dogs.The disease is called canine infectious hepatitis, which occurs mainly in dogs, and also be seen in other canids,in dogs mainly reprsent hepatitis and eye diseases, in foxes mainly manifest encephalitis.In dogs the disease can occur regardless of species, sex, season.but mainly occurs in puppies less than 1 year of age, often cause acute necrotizing hepatitis, often mixed with canine distemper virus in clinical infection, so the illness more serious and complex. The disease occurs almost all over the world, the prevalence in the United States, Denmark, Norway, Britain, Canada, Australia and other countries. In nature, CAV-1 mainly occurs in dogs and foxes, but also infects coyotes, wolves, raccoons, black bears and other wild animals, which is the one of the most damaging diseases in dog industry fur animal breeding industry in China.ln Foreign countries the positive rate of canine infectious hepatitis is 45%～75%; in our country, up to 40% positive rate. Because a great threat has been posed on canine by CAV-1 in our country, CAV-1 prevention and treatment of Epidemiology and Immunization has important theoretical value and practical significance.The following two aspects are inclued in the study: First,this study mainly include two fields.First,CAV-1 monoclonal antibody is prepared;Secend, the double-antibody sandwich enzyme-link immunosorbent(ELISA) detection method is established. This detected method of CAV-1 is simple, specific, stable, convenient, high sensitivity, rapid detection, etc.,and thus to provide the base of the early control of the virus spreading and popular. The three canine adenovirus type 1 monoclonal antibody hybridoma cell lines have been prepared by hybridoma selection, named G3, C2, H2. Three monoclonal antibodies have been identified the subgroup all are IgG2b.By indirect ELISA assay three monoclonal antibodies are identified specific, and only aimes at CAV-1.no aming CDV,CPIV,FCV,FPV. The titers of monoclonal antibody of cell culture supernatant and ascites were 210～212 and 105～107. The positive hybridoma cells are injected in intraperitoneal of mice to obtain ascites, massive ascites are collected After ascites are purified by caprylic acid-ammonium sulfate, SDS-PAGE electrophoresis shows two bands, one band is heavy chain of IgG, about 50.0kDa, another is the light chain of IgG,about 25.0kDa.Three positive hybridomas were passaged 10.20 genrations and cryopreservation of 1,3,6 months, which were detected by indirect ELISA,the titer is same with the initial preparation of the hybridoma,indicating that three stable hybridoma cells which secrete anti-CAV-1 monoclonal antibody have been obtained. The first phase of this experiment successfully obtained three hybridoma cells,which steadily secret anti-CAV-1 monoclonal antibody, which is a very important foundation for establishing ELISA detection methods.Second,according to different epitopes are recognized H2 and G3 McAb,and they were respectively used for coating ELISA microtiter plates and the preparation of conjugates.The double-antibody sandwich ELISA detection methods is established by H2 McAb and G3 McAb labeled with HRP. By optimizing various reaction conditions, the double-antibody sandwich ELISA detected method were determined:the optimal dilution of CAV-1 antigen is 1:80; the best packed dilution of H2 McAb is 1:4000; the best closed reagents is 2% BSA and the best closing time is in 37℃, 1h; the best dilution of G3 McAb labeled with HRP is 1:1000 and the best reacting time is in 37℃,1h; the best reacting time of substrate in 37℃,10～15min. The double-antibody sandwich ELISA method was initially established to detect CAV-1 antigen,laying a significant foundation for further developingly diagnostic test kits.