猪水肿毒素单抗竞争ELISA试剂盒的研制及应用  

Development and Application of McAb-based Competitive ELISA Kit for Shiga-like Toxin IIe

作　　者：张建民[1,2] 吴斌[1] 赵战勤[1] 卢顺[1] 何华[1] 向敏[1] 李利娅 张福涛[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物医学院农业微生物学国家重点实验室,武汉430070 [2]华南农业大学兽医学院,广州510642

出　　处：《畜牧兽医学报》2009年第7期1054-1058,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技支撑计划项目(2006BAD06A18);湖北省十一五公关计划资助项目(2006AA202)

摘　　要：本研究旨在建立猪水肿毒素抗体的快速检测方法。以纯化的猪水肿毒素A亚基基因片段的原核表达产物作为抗原免疫BALB/c小鼠制备单抗,并利用表达蛋白和猪水肿毒素A亚基单抗酶结合物建立了竞争ELISA方法检测猪水肿毒素抗体。经过研究确定抗原包被浓度为0.32μg.mL-1,待检血清最佳稀释度为1∶2,酶标单抗工作浓度为1∶3200,血清抑制率大于40%为阳性。应用单抗竞争ELISA和细胞毒性中和试验同时对60份血清进行猪水肿毒素抗体检测,竞争ELISA的检出率为33.8%,细胞毒性中和试验检出率为30.9%,两者符合率达88.2%。试验结果表明,该ELISA方法特异性强,敏感性高,稳定性和重复性好,操作简便。本方法的建立在实验室诊断的标准化、猪水肿病疫苗免疫效果的评价及流行病学调查方面具有较高的应用价值。The study aimed to establish a rapid and simple assay to detect Shiga-like toxin lie （SLT-IIe）. Monoclonal antibodies （McAbs） against SLT-IIe A subunit were produced by using Escherichia coli （E. coli） expressed recombinant SLT-IIe A subunit protein as antigen to immunize BALB/c mice. A competitive ELISA for detecting antibodies against SLT-IIe was developed on the basis of recombinant protein expressed by E. coli and HRP-labelled McAbs against SLT- IIe A subunit. The optimum conditions of the ELISA were developed as following： the concentration of recombinant for coating ELISA plates was 0.32 ug·mL^-1 ; the best dilution of serum to be tested was 1 ： 2; the working titer for HRP-labelled McAbs was 1 ： 3 200; and the inhibition rate above 40% was selected as the positive judging standard. A total of 60 serum samples were detected in parallel by both competitive ELISA and vitro neutralization assay, 33.8% of them were detected as positive by competitive ELISA, while 30.9 % of them were positive by vitro neutralization assay. The coincidence rate of the two assays was 88.2%. The results showed that the competitive ELISA had the advantages of higher sensitivity, specificity, reproducibility, stability and easy operation. It would be very useful in diagnosis, surveillance of SLT-IIe antibodies and epidemiologieal survey.

关 键 词：猪水肿毒素 单克隆抗体 单抗竞争ELISA 抗体检测 

分 类 号：S852.612[农业科学—基础兽医学] S854.43[农业科学—兽医学]