Preparation And Identification Of Monoclonal Antibodies Against DTMUV NS5 Protein And Establishment Of The Colloidal Gold Test Strip Rapid Detection Method

Since the summer of 2010, a disease caused by a newly virus which characterized by a sudden drop in egg production, slow growth and ovarian haemorrhage on egg-laying duck was outbreak in Jiangsu, Zhejiang and Shandong provinces in succession. The disease spread rapidly to most areas of China which caused great economic losses for the egg-laying duck bleeding. Infected ducks mainly performed feed intake and egg production falling rapidly.with the different infected ages changed, the mortality rates was between 5%~10%. After many domestic laboratories diagnosised and isolated virues, identification of the viral pathogen showed that the pathogen was Duck Tembusu virus(DTMUV), which was a member of the Ntaya virus(NTAV) group, genus Flavivirus, and family Flaviviridae.Research the fuction and structure for the DTMUV protein and establish the rapaid detection method were the effectively way to preventing the occurrence of this disease. Our research estabilished the colloidal gold strip method based on the monoclonal antibody and polyclonal antibody against envelope protein, prepared the monoclonal antibodies against NS5 protein.The theoretical basis and technical support for preventing this disease can be provided by these methods. The research content was divided into following two parts:1. Preparation of monoclonal antibody against NS5 protein of DTMUVPET28a-NS5 vector which was preserved by our lab was transformed respectively into Escherichia coli Rosetta(DE3), the NS5 protein was purified by the urea gradient method.6~8 week BALB/c mices were immunized by the purified protein NS5 of DTMUV, three hybridoma cells named by A4D1, B4B8 and C12D2 were screened by cell fusion technology,indirect ELISA and limited dilution method continually. A4D1 and B4B8 were identified by some related feature. Titers of the both strains in cell culture medium were 1:100, while the tlters of the strains in ascetic fluids were respectively 1:128000 and 1:64000. The result of the indirect ELISA showed that both monoclonal antibodies could react with DTMUV specifically, no cross-reaction was found with other duck pathogens. The result of Western blot and indirect immunofluorescence assay(IFA) test analysis showed that the two McAbs reacted with DTMUV and purified NS5 protein, respectively. The success preparation of themonoclonal antibody against DTMUV NS5 protein could lay the foundation of researching immunological diagnosis and pathogenesis for DTMUV.2. Establishment of the colloidal gold strip rapid detection method for DTMUVThe gold particle in colloidal solution was coupled with the purified monoclonal antibody A12D3 against envelope protein as gold conjugate antibody, while the preparation of purified polyclonal antibody from BALB/c mice against envelope protein served as the capture antibody. The double antibody sandwich-based gold immunochromatographic(IC)strip for the detection of DTMUV was assembled in regular order. The result showed that the IC strip could detect specifically for DTMUV and observed the results in about 10~15 min. 50 clinical suspected samples were simultaneously detected by IC strip and RT-PCR while the results showed 93.8% accuracy between them. The IC strip has high specificity and repeatability rates and can be used for rapid clinical detection of DTMUV.