Preparation Of Monoclonal Antibodies Against Duck Hepatitis Type 1 Virus

Duck Hepatitis Virus (DHV) is the causative agent of Duck Viral Hepatitis (DVH), an acute and fatal disease of ducklings. Three types of DHV (DHV-1, DHV-2 and DHV-3) showed no cross reaction among them. Traditional DHV-1 is the most widely distributed and can cause more than 90% mortality in ducklings under 3 weeks of age, while DHV-2 and DHV-3 are only reported in China, UK, and the USA with low mortality. DHV-1A is the most popular serotype in China. Which belongs to picornaviridae with single RNA strand and about 20 nm in size. In this study, the purified DHV-1 was used to immunize mice to produce monoclonal antibodies. 10 monoclonal antibodies against DHV-1 were obtained. These monoclonal antibodies could be used to develope a rapid diagnostic method for DHV-1 infection in the future.Chicken embryos were used for virus propagation. Allantoic fluid were collected after 24 h～96 h infection or when chicken embryos died. After freezing and thawing for 3 times, the virus was extracted with chloroform and then concentrated with PEG-6000. The ultracentrifugation was performed for 16 h at 5×104×g. The purified antigen was used to immunize 6-week-old female BALB/c mice. The spleen cells of immunized mice were fused with SP2/0 myeloma cells by PEG-1450. Indirect ELISA was used to screen hybridoma cells and limited dilution method was performed to subclone positive clones for 3 times. 10 monoclonal antibodies cell lines were obtained eventually, named AF8, DB6, AF2, AF3, AD4, AE2, AG1, DB6, BG6, and DH11, respectively. The titers of 10 supernatant hybridoma cells can reach 1:2048. The titer of abdominal fluid can reach 1:409600. The hybridoma cell strains can steadily secrete antibodies after 25 times passages culture and 6 months stored in liquid nitrogen. 8 antibodies are IgG1 subtype and 2 IgM subtype withκlight chain. 6 monoclonal antibodies showed cross-activity to DHV-1C, while all monoclonal antibodies showed no cross-activity to SPF allantoic fluid, avian reovirus, and goose parvovirus.These obtained monoclonal antibodies would help to develope rapid sensitive diagnostic ELISA method for detecting DHV-1 infection, and also can help to study on molecular pathogenesis of DHV-1.