弓形虫重组MIC3蛋白间接ELISA检测方法的建立  

Establishment of an indirect ELISA method for the detection of antibodies against Toxoplasma gondiibased on recombinant MIC3 protein

作　　者：白昀[1,2] 王海燕[1] 张悦[1] 姚景霆[1] 吴叙苏[1] 刘冬霞[1] 邵国青[1] 

机构地区：[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京天邦生物科技有限公司,江苏南京211102

出　　处：《中国兽医科学》2013年第7期738-743,共6页Chinese Veterinary Science

基　　金：江苏省科技支撑计划(社会发展)项目(BE2010758)

摘　　要：为检测弓形虫特异性抗体,以纯化的重组MIC3蛋白作为包被抗原,用羊抗猪IgG-HRP作为酶标二抗,分别对抗原浓度、血清稀释度、酶标二抗稀释度以及作用时间等进行了优化,初步建立了检测弓形虫特异性抗体的间接ELISA方法。结果显示,该方法检测猪球虫、猪肺炎支原体、猪圆环病毒2型、副猪嗜血杆菌等阳性血清均为阴性,表明该方法具有良好的特异性。组内和组间重复性试验的变异系数均小于8%,表明该方法具有较好的重复性。应用所建立的ELISA方法和商品化试剂盒A(IHA)、B(ELISA)同时检测从南京某猪场采集的猪血清样品166份,符合率分别可达94.58%和95.78%。该方法为我国进行弓形虫病的诊断与流行病学调查提供了一种技术手段,并为试剂盒的研制与开发奠定了前期工作基础。To direct ELISA was as the second anti explore a method for detecting the antibody against Toxoplasma gondii of swine,an in established using recombined MIC3 of T. gondii as coating antigen and goat anti-pig IgG body. The concentrations of coating antigen, the dilutions of sera samples and second antibody as well as the working time were optimized. As a result,a stable and specific indirect ELISA was de- veloped. The assay showed that there were no cross-reactions of the MIC3 protein with the positive sera of swine coecidium,Mycoplasma hyopneumoniae, porcine circovirus type 2, Haemophilus parasuis and so on. And it was highly reproducible with a less than 8% coefficient of variation in intra-batch and inter-batch tests. Total 166 samples of swine sera in Nanjing area were detected by this indirect ELISA method and an other two commercialization kits,A（IHA） and B（ELISA）. The result showed that the coincidence rate were 94.58% and 95.78% respectively. The results of this study provided a method for the diagnosis and epidemiological investigation for toxoplasmosis, and would help to promote the subsequent research and de velopment of the commercial kit.

关 键 词：刚地弓形虫 MIC3蛋白 酶联免疫吸附试验 抗体检测 

分 类 号：S852.723[农业科学—基础兽医学]