REV重组env蛋白间接ELISA诊断试剂盒的研制  

Development of an Indirect ELISA Kit to Detect REV Sera with Recombinant Env Protein

作　　者：李彦[1,2] 牛星 王青娟[1,2] 牛钟相[1,2] 

机构地区：[1]山东农业大学动物医学院,山东泰安271018 [2]山东省动物生物技术与疫病防治重点实验室,山东泰安271018 [3]山东畜牧兽医职业学院,山东潍坊261061

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)

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.

关 键 词：网状内皮组织增生症病毒 env蛋白 间接ELISA 试剂盒 抗体检测 

分 类 号：S854.4[农业科学—临床兽医学]