Preparation And Tentative Application Of Monoclonal Antibody Against DHV-YZ Strain

Duck viral hepatitis (DVH) caused by duck hepatitis virus (DHV) is a highly mortal disease and spreads rapidly in three-week-old ducklings. Multiplication of three DHV-Ⅰ strains in chicken/duck embryo, chicken embryo fibroblast cells (CEF) and chicken embryo liver cells (CELC) were investigated and DHV antigen of high purity was made in this research. It can be successfully used to carry out indirect ELISA and to immunizing BALB/c mice for producing monoclonal antibodies. After a series of fusion and detecting, four monoclonal antibody lines against DHV-YZ were prepared and they have been applied tentatively.Multiplications of three DHV-Ⅰ strains in three different host systems were compared. When multiplying in chicken embryo and duck embryo, three DHV strains can all cause typical DVH symptoms, such as embryo haemorrhage, dropsy, liver haemorrhage, local necrosis even mottled liver. Embryo inoculated DHV-YZ strain died first and that inoculated DHV-ZJS strain died last, and their ELD50/0.2ml are 104.68, 104.25 and 103.25 . After reducing newborn bovine serum in maintaining solution, three DHV strains can all multiply in CELC but only DHV-ZJS and DHV-NJ can adapt in CEF. The cells begin to turn round, die and fall off at 48h in CEF and within 24h in CELC after inoculating. The result showed that CELC is more sensitive to DHV than CEF and the TCID50/0.2ml of DHV-ZJS and DHV-NJ in CEF are 103.41 and 103.90. DHV strains multiplied in different host systems can all cause typical symptoms in 1-day-old ducklings. The DHV multiplied in chicken embryo and duck embryo were purified with freezing and melting in turn, chloroform distill, filter by membrane, inverse dialyzing and chromatography with Sephadex G-200. It can work well for BALB/c mice immunizing and I-ELISA coating.The optimum work concentration of the indirect ELISA for detecting and screening positive hybridoma cells was established by research again and again. Polystyrene microtitration plates were coated with the purified DHV antigen at the amount of 0.5μg/well. The working concentration of positive and negative serum was 1:6400. All the incubation steps above were done at 37℃ for 60min. In addition SP2/0 cells supernatant and 82 negative hybridoma cells supernatant were detected under this work condition. Based on P/N≥2.1 and P≥N+3sd , the positive standard was determined at 0.193. We got 12 positive hybridoma cell lines at first after detecting and screening, but only 4 hybridoma cell lines were left after 3 sub-cloning. They were named respectively 2B5, 2E8, 5E6 and 6C11. ELISA titer and speciality detecting test showed that titer of 2B5 and 2E8 is the highest, and can reach 102,400. Titer of 5E6 is the lowest, only 6,400. The titer of abdominal fluid is 200~500 times that of hybridoma cells supernatant averagely. The specialities of 4 monoclone antibodies are very good, but virus neutralization characters are feeble.Two methods of purifying immunoglobulin G in duck serum were compared in this research, which included ammonium sulfate precipitation and caprylic acid - ammonium sulfate precipitation. The result of protein concentration analysis, activity detection and sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) demonstnated that protein content of IgG purified by ammonium sulfate is higher than that of caprylic acid - ammonium sulfate, but the purity and activity of IgG purified by caprylic acid - ammonium sulfate is far higher than that of ammonium sulfate. So the method of caprylic acid - ammonium sulfate precipitation is better. Coated with purified IgG, AC-ELISA for detecting DHV was established. The work concentration of IgG and monoclone antibody were 1:80 and 1:100(titer 1:102,400) respectively. Furthermore Dot-ELISA for detecting DHV was also established. The best sample quantity for detecting was 2.723μg/dot, and the least was 1.362μg/dot. Work concentration of monoclone antibody is 1:400(titer 1:102,400). The two methods can all detect DHV in pathogenic materials and their accord rate c...