为建立一种敏感、特异、快速的猪繁殖与呼吸综合征病毒(PRRSV)抗体检测方法,本研究利用原核表达技术表达了PRRSV N蛋白,亲和层析纯化后作为包被抗原,通过对各反应条件优化选择,建立了PRRSV血清抗体的间接ELISA检测方法,并进行了交叉反应和重复性试验,以及同类成品试剂盒间的应用效果对比试验.最终确定了抗原包被最佳浓度为2 μg/mL,血清最佳稀释度为1∶100,血清及酶标二抗孵育时间均为30 min,显色时间为10 min,检测猪瘟病毒、猪圆环病毒、猪伪狂犬病病毒等5种常见猪病病原的阳性血清均为阴性;该ELISA检测方法批内和批间重复性的变异系数均小于10%;与商品化ELISA试剂盒效果比较显示符合率为94.7%.本研究建立的间接ELISA方法将为猪群感染野毒PRRSV后的快速诊断及流行病学调查提供一种简便易行、快速高效的血清学抗体检测方法.%To establish a sensitive,specific and efficient method of antibody detection for porcine reproductive and respiratory syndrome virus (PRRSV),PRRSV N protein was expressed through prokaryotic expression system and purified by affinity chromatography to act as a coating antigen.Then an indirect ELISA detection method for serum PRRSV antibody was finally set up after the optimization of reaction conditions.Besides,the research also involved cross reaction,repeated experiments,and comparison with other ELISA kits.It was determined that the optimum concentration of coating antigen was 2 μg/mL and that the dilution ratio for serum was 1∶100,with 30 min of incubation and 10 min of chromogenic reaction.With this method,positive serum samples of five common swine pathogens,including classical swine fever virus (CSFV),porcine circovirus (PCV) and porcine pseudorabies virus (PRV) and so on,were tested,and the results were negative.Both intra-assay and inter-assay coefficients of variation were below 10%,and the comparison with commercial ELISA kits indicated that its accuracy was 94.7%.So this indirect ELISA,which had been established in this research,could provide a rapid diagnosis for swine infected by wild PRRSV and applied in epidemiological investigation,as a convenient and efficient serological antibody detection method.
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