牛传染性鼻气管炎病毒重组gD蛋白间接ELISA方法的建立及应用

Development and application of an indirect ELISA for detection of antibodies against infectious bovine rhinotracheitis virus using recombinant glycoprotein D

用大肠杆菌表达的牛传染性鼻气管炎病毒(IBRV)重组gD蛋白纯化后作为包被抗原,建立了检测牛传染性鼻气管炎病毒抗体的间接ELISA方法。交叉反应试验表明,该重组抗原与其它常见的5种牛病阳性血清不发生交叉反应;阻断反应试验表明,IBRV病毒悬液能在很大程度上阻断重组抗原与阳性血清的反应,而对阴性血清没有明显影响。在重复性试验中,批内重复的变异系数小于5%,批间重复的变异系数小于15%。与中和试验相比较,符合率、敏感性和特异性分别为84.1%、85.0%和83.4%。应用该诊断方法和本实验室已建立的以IBRV全病毒作为包被抗原的ELISA诊断方法,同时检测了采集于国内11个省份的2012份血清样本,IBRVgD-ELISA检测的平均阳性率为46.0%(926/2012),而且相对于IBRV全病毒ELISA诊断方法的符合率、敏感性和特异性分别为91.9%、94.2%和90.2%。本研究所建立的IBRVgD-ELISA具有良好的敏感性和特异性,为国内IBR流行病学调查提供了一种快速、简便的血清学诊断方法。

An indirect ELISA was developed to detect antibody against IBRV using purified recombinant gD protein of infectious bovine rhinotracheitis virus （IBRV） expressed in E.coli as coating antigen, designated as gD-ELISA. The recombinant gD protein antigen was highly specific and had no cross-reaction with the positive sere of other bovine diseases. The assay had about 85% consistency with neutralization test. Test on 2012 serum samples collected from eleven provinces in China showed that the gD-ELISA detected 46.0 % （926/2012） positive. Comparison to whole virus ELISA, the agreement rate, sensitivity and specificity of gD-ELISA had 91.9 %, 94.2 % and 90.2 % consistency.