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

关键词:  网状内皮组织增生症  env蛋白  间接ELISA  试剂盒  抗体检测

Key words:  REV  env protein  indirect ELISA  kit  antibody detection

摘要:

用纯化的env蛋白为包被抗原，建立了检测抗REV抗体的间接ELISA（ienv-ELISA）。经对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1h；二抗的最佳稀释度为1：5000；二抗最佳作用时间为1h；底物最佳显色时间为25min。经重复性试验、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%, 93.75% 和92.7%。

Abstract:

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 anti-REV sera. The optimum recation conditions for ienv-ELISA were investigated, and the result showed: optimal coating buffer was CB；optimal confining liquid was 1%BSA-PBS；optimal concentration of antigen was 8μg/ml；optimal dilution of sera was 1：300；optimal reaction time of sera was 1h；optimal dilution of HRP-labled goat anti chicken IgG was 1：5000；optimal reaction time of HRP-labled goat anti chicken IgG was 1h；optimal reaction time of substate was 25min. 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. Some sera samples which came from some chicken farms in Shandong were detected by the ELISA kit.