摘要
以纯化的env蛋白为包被抗原,建立了检测REV抗体的间接ELISA(ienv-ELISA)方法。ienv-ELISA工作条件优化结果表明,最佳包被液为CB;最佳封闭液浓度为1%BSA-PBS;最佳抗原包被量为8μg/mL;血清最佳稀释度为1∶300;血清最佳作用时间为1 h;二抗的最佳稀释度为1∶5000;二抗最佳作用时间为1 h;底物最佳显色时间为25 min。重复性试验、特异性试验和对比试验结果显示,建立的ienv-ELISA方法有良好的可重复性,标准差均小于15%;与鸡MDV、ALV、NDV、H5N1、H9N1等病毒阳性抗体均无交叉反应;与REV抗体检测试剂盒(以REV全病毒为包被抗原)对比,所建立的ienv-ELISA诊断敏感性、诊断特异性以及准确率分别为90.625%9、3.75%和92.7%。
Using the recombinant and purified env protein as antigen to coat micro-plate of 96 wells,an indirect ELISA assay（ienv-ELISA）was developed to detect REV antibody.The optimum reaction conditions for ienv-ELISA were investigated,and the results showed that optimal coating buffer was CB;optimal confining liquid was 1% BSA-PBS;optimal concentration of antigen was 8 μg/mL;optimal dilution of sera was 1∶300;optimal reaction time of sera was 1 h;optimal dilution of HRP-labled goat-anti chicken IgG was 1∶5000;optimal reaction time of HRP-labled goat-anti chicken IgG was 1 h;optimal reaction time of substrate was 25 min.Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 15%.Cross-reactivity assay showed that this assay was REV-specific.This assay was validated by comparison with a REV-based ELISA.The diagnostic sensitivity（DSN）,specificity（DSP） and accuracy of the ienv-ELISA were 90.625%,93.75% and 92.7% respectively.
出处
《中国兽药杂志》
2011年第3期26-30,共5页
Chinese Journal of Veterinary Drug
基金
国家十一五科技支撑计划项目(2006BAD06A11)
