动物结核分枝杆菌复合群间接ELISA诊断方法的建立  

Establishment of an Indirect ELISA Method for Diagnosing Mycobacterium tuberculosis Complex Groups in Animals

作　　者：庄勇 向蒙 冯桂丹 杨显超[2] 夏炉明[2] 肖文轩 张蒙 王建[2] 张鹭 ZHUANG Yong;XIANG Meng;FENG Guidan;YANG Xianchao;XIA Luming;XIAO Wenxuan;ZHANG Meng;WANG Jian;ZHANG Lu(School of Life Sciences,Fudan University,Shanghai 201103,China;Shanghai Animal Disease Prevention and Control Center,Shanghai 201103,China)

机构地区：[1]复旦大学生命科学学院,上海200433 [2]上海市动物疫病预防控制中心,上海201103

出　　处：《中国动物传染病学报》2025年第1期125-133,共9页Chinese Journal of Animal Infectious Diseases

基　　金：沪农科推字(2022)第2-1号;兵团科技计划项目(2022BC005)。

摘　　要：本研究通过前期已构建好一些优势抗原的表达载体库,以生物信息学工具分析候选抗原的理化性质、二级结构、B细胞抗原表位,选择优势抗原,以纯化后的融合抗原ECX作为包被抗原,将HRP标记的Protein G作为酶标的二抗,成功建立了一种用于多动物结核分枝杆菌复合群间接ELISA诊断的方法。该方法适用于实验室样品批量检测,其敏感性为0.912,特异性为0.949。应用本研究建立的动物结核分枝杆菌间接ELISA法和商品化动物结核分枝杆菌夹心ELISA抗体检测试剂盒共检测犬、猫、野生动物、SPF实验动物血清共552份。其中间接ELISA方法共检出6份血清抗体阳性,阳性检出率为1.09%(6/552);商品化夹心法试剂盒共检出8份血清抗体阳性,阳性检出率为1.45%(8/552);两者之间阳性重合数为6份,阳性重合率为85.72%(6/7);阴性重合数为544份,阴性重合率为99.82%(544/545)。结果表明,两种诊断方法的检测结果具有较高的一致性。本方法的建立,与国外商品化试剂盒比较,不但有望实现这类检测产品的国产替代,同时实现了一种方法对不同物种来源血清的快速检测。This study successfully established a method for indirect ELISA diagnosis of multi animal Mycobacterium tuberculosis complex by constructing expression vector libraries of some advantageous antigens in the early stage,analyzing the physicochemical properties,secondary structure,and B cell antigen epitopes of candidate antigens using bioinformatics tools,selecting advantageous antigens,using purified fusion antigen ECX as the coating antigen,and HRP labeled Protein G as the enzyme-linked secondary antibody.This method is suitable for batch testing of laboratory samples,with a sensitivity of 0.912 and specificity of 0.949.Using the established indirect ELISA method for animal Mycobacterium tuberculosis and a commercial animal Mycobacterium tuberculosis sandwich ELISA antibody test kit(INGENASA,Spain),both methods tested 552 serum samples from dogs,cats,wildlife,and SPF laboratory animals.The indirect ELISA method detected 6 positive serum antibodies,with a positivity rate of 1.09%(6/552);the commercial sandwich kit detected 8 positive serum antibodies,with a positivity rate of 1.45%(8/552).The positive concordance between the two methods was 6 samples,with a concordance rate of 85.72%(6/7);the negative concordance was 544 samples,with a concordance rate of 99.82%(544/545).This indicates a high consistency in the results of the two methods.The establishment of this method,compared with foreign commercialized reagent kits,not only has the potential to achieve domestic substitution of such detection products,but also realizes a method for detecting serum from different species sources.

关 键 词：抗原 结核分枝杆菌复合群 ECX 间接ELISA 

分 类 号：S855.2[农业科学—临床兽医学]