Development And Preliminary Application Of Monoclonal Antibodies Against Duck Tembusu Virus

In2010April, for the first time, a disease which could cause the reduction of duck egg was reported in the eastern part of China, then with the rapid spread of this disease, it has become a common and frequently occurring disease, resulting in significant economic loss to our country’s duck raising industry. The disease mainly causes the reduction of duck egg, or various neurological symptoms for the ducking, and as it could infect all kinds of ducks except the Cairna moschata,it can make severe harm to the ducking industry.With the in-depth study of the etiology, the pathogen of this disease was identified as a kinf of virus, Duck Tembusu Virus. The virus belongs to the Ntaya virus (NTAV) group within the genus Flavivirus, family Flaviviridae. Because there is no commercially available vaccine and specific medicine to prevent the disease, It is particularly important to establish an effective detection method for controlling the disease. Currently, the method of detecting the pathogen has been established mainly PCR, RT-PCR, etc., but not for large-scale clinical testing. In this study, by using whole virus immunization, anti-prepared duck Tembusu virus monoclonal antibody, and the establishment of a method for detecting the virus double antibody sandwich ELISA to detect clinically helpful.1. Development of monoclonal antibody against Duck tembusu virusA panel of monoclonal antibodies (MAb) against duck tembusu virus(DTMUV) were developed by fusions between SP2/0and spleen cells from Balb/c mice immunized with duck tembusu virus which was purified. Five hybridomas cell strains stably secreted MAb against DTMUV were named as T-4C10G7, T-6A9B3, T-3E4E7and T-3F5Fl.The subclass of acquired McAbs were identified to be IgG and IgM. T-4C10G7, T-6A9B3and T-3F5F1are positive with ELISA, T-3E4E7, T-3F5F1are positive with IFA. This provides a possibility for development of IFA for DTMUV detection. These MAb showed high titer and specific to DTMUV with indirect immunofluorescence assay, indirect ELISA assay. Four MAb showed good stability of secreting antibodies of hybridoma cell lines stored liquid nitrogen about3months. The development of five hybridoma strains laid a solid foundation for the establishment of a double antibodies sandwich ELISA to detect DTMUV. 2. Establishment of a double antibody sandwich ELISAA double antibody sandwich ELISA method has been established using MAb T-6A9B3and T-4C10G7against DTMUV with T-6A9B3as capture antibodies, T-4C10G7as enzyme labeled antibody. The method has been optimized in the concentration of capture or HRP labeled antibody and incubation time of antigen or HRP labeled antibody. The working concentration of capture antibody and detect antibody were3.5μg/mL and0.25μg/mL. And the o ptimized incubation time of enzyme-labeled secondary antibody with detect antigen are30min.It is determined that the method has no cross-reactivity with other virus and it can be used for clinical application.