Preparation Of Monoclonal Antibody Against DHV And Development Of ELISA For Detecting The Virus

In this study, the susceptible duck embryos and primary duck embryo liver cells were used to culture new type DHV(N-DHV) and type 1 DHV(DHV-1) for preparing the viral culture.The primary purified antigens were prepared by release of the viruses using the method of repeated freezing and thawing, defating of the culture by chloroform and being filtered by 0.22 um filter to remove macro-molecular impurities and bacteria. The viruses concentrated by dialysation were purified by Sephorase CL-4B gel filtration.After that,the purified virus antigens were used for immunizing mice to prepare spleen cells, immunizing rabbits to gain polyclonal antibodies and being applied to an indirect ELISA test.The immunogens prepared by emulsification of the purified virus antigens with Freund’s complete adjuvant or Freund’s incomplete adjuvant were used to immunize BALB/c mice through appropriate Immunization programs. The immunized mice with high antibody titers were attained in order to lay a basis on preparing immunized spleen cells.Mean while, an indirect ELISA for screening positive cell clones was established by detecting mouse antibody titers using purified antigen.Spleen cells of immunized mice were fused with SP2/0 myeloma cells by using conventional techniques. After primary screening and re-examination by the established ELISA using DHV-1,N-DHV and healthy duck embryo liquid, 5 hybridoma cell cultures secreting the antibody against DHV-1 and N-DHV were screened out. The ascites antibodies were prepared by subcloning the hybridoma cells for five times using limited dilution method, expanding cell culture and injecting CD-1 mice with the culture. Two stable hybridomas that secreted antibodies against DHV-1,N-DHV were obtained and were named Ya4C7 and Ga3B6, respectively. The titer of Mc Ab secreted by cell lines of Ya4C7 against the viruses of 2 serotypes is 1:40 and the titers of mice ascites Mc Ab against the N-DHV and DHV-1 are more than 1:100,000 and 1:3200, respectively. The titer of Mc Ab secreted by cell lines of Ga3B6 against the viruses of 2 serotypes is 1:20 and the titers of mice ascites Mc Ab against the N-DHV and DHV-1 are more than 1:50,000 and 1:6400, respectively. The affinity force of Ya4C7 Mc Abs to two antigens is stronger than Ga3B6’s. Two Mc Abs belonged to Ig G1 subclass. No reactions between Ya4C7 Mc Ab and the other common pathogens of ducks were observed.The ascites Mc Ab Ig G and the home-made high immune Ig G in rabbit sera were purified by using caprylic acid-ammonium sulfate method. The results of protein content determination using SDS-PAGE electrophoresis and potency determination showed that the Ig G’s purity and activity were significantly increased. By using purified Mouse Mc Ab Ig G to coat microtiter plates, rabbit polyclonal Ig G to response to the viruses and goat anti-rabbit Ig G-peroxidase to combine with rabbit Ig G,a modified antigen captured ELISA(AC-ELISA) method to detect DHV-1 and N-DHV was estalished. The minimum protein contents of DHV-1 and N-DHV detected using this method are 12.6ng/ml and 10.7ng/ml, respectively. This method is negative to detect DPV and other ducks’ pathogens.