鸭东方次睾吸虫病间接ELISA诊断方法的建立  

Establishment of an indirect ELISA method for diagnosis of Metorchis orientalis infection in duck

作　　者：高俊峰[1] 毛瑞锋 孙云异 马晓晓 兰卓 常巧呈[1] 王春仁[1] GAO Jun-feng;MAO Rui-feng;SUN Yun-yi;MA Xiao-xiao;LAN Zhuo;CHANG Qiao-cheng;WANG Chun-ren(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国兽医科学》2021年第8期947-954,共8页Chinese Veterinary Science

基　　金：国家自然科学基金面上项目(31972703);黑龙江省博士后面上资助项目(LBH-Z19191);大学生创新创业训练计划项目(202010223014);福建省家畜传染病防治与生物技术重点实验室开放基金项目(2019KF04)。

摘　　要：为评价候选蛋白烯醇化酶(Enolase)作为东方次睾吸虫ELISA诊断抗原的潜力,重组表达东方次睾吸虫烯醇化酶基因,以纯化好的重组蛋白为抗原,优化各检测条件,建立了鸭东方次睾吸虫病间接ELISA诊断方法并用该方法检测临床样品。结果显示,通过棋盘滴定法,确定了抗原的最佳包被质量浓度为2g/mL和血清最佳稀释度为1∶100,其他条件优化结果为:酶标二抗的最佳稀释度为1∶5000,最佳包被条件为4℃包被过夜,最佳封闭条件为50g/L脱脂奶粉溶液封闭2h,显色液最佳显色时间为10min。通过对25份未感染东方次睾吸虫鸭的阴性血清进行检测与统计分析,确定出该重组蛋白间接ELISA方法的阳性临界值为0.322。经优化反应条件后建立的间接ELISA方法具有特异性强、敏感性高和重复性好等优点。利用本研究中建立的ELISA方法检测120份临床血清,阳性率为14.2%,与粪便虫卵检测结果的符合率为94.74%,表明该烯醇化酶可作为东方次睾吸虫病血清学诊断的良好候选抗原。本研究为进一步研发鸭东方次睾吸虫病的血清学诊断试剂盒奠定了坚实的理论基础。To evaluate the potential applications in diagnosis of enolase for the detection of Metorchis orientalis,the recombinant protein of enolase(r Mo ENO)was expressed,purified and identified.The experimental conditions of indirect ELISA were optimized by r Mo ENO as an antigen,and an indirect ELISA diagnostic method for detection of M.orientalis was established.The results showed that the optimal experimental conditions as below:the coating antigen concentration was 2 g/m L,the serum dilution was 1∶100,the enzyme-labeled secondary antibody dilution was 1∶5000,the coating condition was 4℃ overnight,the blocking condition was 50 g/L skimmed milk for 2 h,and the TMB developing time was10 min.By statistical analysis of 25 negative serum samples,the cut-off value for indirect ELISA was0.322.The specificity,sensitivity and repeatability experimental results showed that the established indirect ELISA method has a good specificity,sensitivity and repeatability.One hundred and twenty duck serum samples were tested by both newly developed indirect ELISA method and traditional feces test method,the results showed that the positive rate of serum samples was 14.2%,and the coincidence rateof the two methods was 94.74%,which indicated that the enolase was a good candidate antigen for serological diagnosis of M.orientalis.This study provides an important reference for development of serological diagnostic kits for duck metorchiasis.

关 键 词：东方次睾吸虫 间接ELISA 烯醇化酶 血清学诊断 

分 类 号：S852.735[农业科学—基础兽医学]