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Since April 2010,Tembusu virus (TMUV) which is a contagious pathgen of waterfowls,causing symptoms of high fever,loss of appetite and fall in egg production,has been reported in east of China.A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for Tembusu virus (TMUV) was developed,using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein.BALB/c mice were immunized with purified recombinant E protein expressed in E.coli.Three hybridoma cell lines designated as 1281,10C6 and 2D2,were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein,and were characterized subsequently.High-affinity mAbs 1281 and 2D2 were used as capture and detection antibodies,respectively.The reaction conditions for the DAS-ELISA were optimized for TMUV detection.The cross-reactivity of the DAS-ELISA was determined using TMUV,duck plague virus,avian influenza virus subtype H9,Newcastle disease virus,duck hepatitis A virus type l and duck reovirus samples.A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR.The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%.These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%.The method developed in this study can be used for the diagnosis of TMUV infection of duck origin.