华支睾吸虫间接ELISA检测方法的建立及初步应用

Development and primary application of an indirect ELISA for detecting Clonorchis sinensis

RT-PCR扩增华支睾吸虫(Clonorchis sinensis)的Enolase基因后,进行原核表达,采用SDS-PAGE和Western blot检测表达产物;以纯化后的蛋白为包被抗原建立ELISA方法,并对工作条件进行优化。结果表明,ELISA最佳工作条件为:抗原最佳包被质量浓度为5mg/L,37℃1h再4℃过夜;5%脱脂奶粉,37℃封闭1h;待检血清1∶100稀释37℃孵育1h;酶标二抗1∶5 000稀释,37℃孵育45min;TMB显色作用时间10min;确定的阴阳性血清临界值为0.253。所建立的ELISA方法特异性较好,可检测犬华支睾吸虫病阳性血清,与犬卫氏并殖吸虫病阳性血清、犬蛔虫病阳性血清、犬弓形虫病阳性血清、犬新孢子虫病阳性血清均不发生反应。该方法敏感性为1∶3 200。批间批内重复性试验变异系数均小于10%。在临床应用中,对浙江地区353多份犬血清的检测表明,样品阳性率为1.13%。本研究建立的间接ELISA方法可以用于临床病例的血清学快速检测,为华支睾吸虫的血清流行病学调查提供了有效手段。

The enolase gene of Clonorchis sinensis was amplified by RT-PCT,and then cloned into prokaryotic expression system.SDS-PAGE and Western blot were employed to anlyze the recombinant protein.An indirect ELISA for detecting of Clonorchis sinensis was established on the basis of the purified recombinant protein,the working conditions of the ELISA was optimized afterwards.The optical working conditions were determined：coating antigen at 4℃ overnight after37℃for 1hat a concentration of 5mg/L,5%light nonfat-dried milk as blocking agent at 37℃for1h,testing serum diluted at 1∶100at 37℃for 1h,HRP labeled horse anti-dog（1∶5 000）incubated at 37℃for 45 min,the substrate TMB for ELISA being incubated at 37℃ for 10 min.The statistical analysis shows the threshold of positive and negative sera was 0.253.The ELISA assay could detect Clonorchis sinensis specific and showed no cross-reation with four other positive sera of dog diseases（Paragonimus ringeri,Ascaris alata,Toxoplasma gondii,Neospora caninum）.The sensitivity of the indirect ELISA was 1∶3 200.The coefficient of variation within and between plates was all less than 10% showing agood repeatability of the assay.In clinical application,more than 353 serum samples collected in Zhejiang province,showed 1.13% sera positive.The indirect ELISA method can be used for rapid detection of clinical serology,and provides an effective means for other trematode serum epidemiology investigation.