猪瘟病毒抗体间接ELISA检测方法的建立及优化

Development and Optimization of Indirect ELISA Detection Method of Classical Swine Fever Virus Antibody

以原核表达、纯化的猪瘟病毒(CSFV)E2主要抗原区蛋白为目标检测物,通过对各反应条件的筛选和优化,建立了检测CSFV抗体的间接ELISA检测方法。结果表明,本研究的最佳抗原包被浓度和最佳血清稀释度分别为1.0μg/mL和1∶200;最佳抗原包被条件和最佳封闭时间均为37℃1h;最佳酶标二抗稀释度和作用时间分别为1∶10 000和37℃45min;阴性临界值判断标准为当D450nm<0.30时猪血清样品为CSFV抗体阴性;批内和批间重复试验变异系数分别为4.8%和6.9%,表明该检测方法具有较高的稳定性;特异性试验结果表明,间接ELISA方法与猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪圆环病毒(PCV)、猪细小病毒(PPV)阳性血清均无交叉反应;应用该间接ELISA方法随机检测80份猪血清样品,与进口阻断ELISA试剂盒相比其阳性符合率为83.58%,阴性符合率为76.92%,总体符合率为80.25%。结果表明本试验建立的间接ELISA方法具有很好的临床应用价值。

In this study,through a series of screening and optimization of reactions condition,an indirect ELISA method was developed for detections the antibodies against classical swine fever virus（CSFV）with the purified recombinant CSFV E2 protein.The results showed that the optimal concentration for coating antigen was 1.0μg/mL and incubated at 37 ℃for 1h.The proper serum sample was diluted by 1∶200and the sealing time was 37 ℃for 1h.Enzyme labeled second antibody was diluted by 1∶10 000 and the reaction time was 45 min incubating at 37℃.The D450nm〈0.30 of sample defined as negative of CSFV antibody in the serum sample.The intrabatch and inter-batch variation coefficients were 4.8% and 6.9%,respectively.It indicated that the method in this study had a high stability.Specific test showed that the indirect ELISA had no crossing reactions with the antibodies against PRV,PRRSV,PCV and PPV.80 serum samples were detected in this study,the positive coincidence rate of indirect ELISA was 83.58%,the negative coincidence rate was 76.92%,and the total coincidence rate was 80.25%compared to the results of import blocking ELISA kit.The results suggested that the established indirect ELISA method in this study had an excellent clinical application.