为建立快速检测鸭坦布苏病毒(DTV)的血清学方法,本研究利用纯化的DTV奉贤株(FX2010)作为包被抗原,建立了检测DTV血清抗体的间接ELISA方法,并且对各种检测条件进行了优化.优化后确定的抗原最适包被浓度为1.675 μg/孔,抗原最佳包被条件为37℃放置2h后,4℃下过夜,血清的最佳稀释度为1:200,酶标抗体最适稀释度为1∶2 000.在优化条件下,阴阳性临界值判定标准为0.432.用建立的间接ELISA方法对禽流感病毒、新城疫病毒、网状内皮增生病病毒、I型鸭肝炎病毒、呼肠孤病毒、禽白血病病毒阳性血清进行了检测,均无交叉反应,表明该方法具有良好的特异性.批内和批间重复试验的最大变异系数分别为2.9％和3.9％,显示该方法具有很好的稳定性.用间接ELISA方法对140份疑似鸭坦布苏病血清样品进行检测,有108份样品呈现阳性,而琼扩试验只有32份呈阳性结果,而且用该方法检测的阳性样品包括了琼扩试验的阳性样品,证明该方法具有较高的敏感性和特异性.本研究快速检测DTV抗体间接ELISA的建立为该病的诊断和流行病学调查提供了新的方法.%An indirect ELISA was established for rapid detection of antibodies against duck Tembusu virus (DTV) using purified DTV (FX2010 isolate) as coating antigen. The reaction conditions were optimized, including 1.675 jig/well coating antigen of purified the virus, 1:200 dilution of testing serum and 1 -2 000 dilution of HRP conjugated anti-duck IgG with cut off-value of 0.432 (OD450nm). The specific tests showed that there were no cross-reaction to the anti-sera against avian influenza virus, Newcastle disease virus, reticuloendotheliosis virus, duck hepatitis virus-I, reovirus and avian leukemia virus, which indicated that the ELISA was specific to anti-sera against DTV. The intra- and inter-assay demonstrated that the coefficient of maximum variation was 2.9% and 3.9% respectively. A total of 140 serum samples from affected ducks were tested and 108 samples were positive detected by the indirect ELISA, while only 32 samples showed positive by agar immunodifrusion test. The results revealed the indirect ELISA could be used for laboratory diagnosis and sero-survey for duck tembus virus infection.
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