牛结核病抗体胶体金快速检测技术的建立和应用

为了建立一种快速检测牛分支杆菌抗体的新方法用于诊断牛结核病,利用胶体金免疫层析技术原理,用原核诱导表达的牛分支杆菌抗原蛋白MPB83和MPB70分别作为胶体金标记抗原和检测线上的捕获抗原,制备牛结核抗体检测试纸条.结果表明,粒径为40 nm的胶体金制备的试纸条敏感性最高,胶体金最佳标记pH为6.0,MPB83抗原最适标记量为每毫升胶体金6.5 μg,MPB70抗原的最适包被浓度为3.0 mg/mL,抗MPB83蛋白IgG的最佳包被浓度为2.5 mg/mL,交叉试验证明试纸条不与牛的其他非相关疾病的阳性血清反应,具有较高的特异性.比较试验证明其敏感性显著高于韩国进口试纸条.在上述试验条件下生产了一批胶体金试纸条进行临床样品检测,并与细菌分离培养、结核菌素皮内变态反应(TST)和韩国试纸条比较.本试纸条与牛分支杆菌分离培养的符合率为85%,与TST的符合率为79.73%,与韩国试纸条的符合率为98.75%.快速检测牛结核抗体的免疫层析试纸条具有敏感、特异、简便、快速的特点,适用于对牛结核病进行普查和检疫,也可作为TST的辅助诊断方法,在牛结核病根除计划中具有良好的应用前景.

Establishment and Preliminary Application of Immunochromatographic Strips for Rapid Detection of Antibodies against M. bovis

To develop a new method to rapidly detect antibodies against Mycobacterium bovis for diagnosis of bovine tuberculosis(bTB).Strips were prepared with the principle of colloidal gold immunochromatography assay(GICA) with the two recombinant Mycobacterium bovis antigens MPB83 and MPB70.MPB83 was labeled by colloidal gold and MPB70 was used as the test capture reagent to detect the corresponding antibodies.The highest sensitivity was found with strips prepared by 40 nm particles of colloidal gold.The optimal labeling pH of colloidal gold solution was 6.0.The best amount of MPB83 for colloidal gold labelling was 6.5 g per ml.The optimal concentration of capture reagent MPB70 and control coating protein anti-MPB83 IgG was 3.0mg/mL and 2.5mg/mL,respectively.The strips did not react with positive sera of other unrelated bovine diseases and displayed high specificity.Comparison test showed that the strips had a higher sensitivity than a commercial MPB70 test strips from Korea.A batch of colloidal gold test strips were manufactured to detect clinical samples,in parallel with culture of M.bovis,tuberculin skin testing(TST) and test strips from Korea.The agreement ratios were 85%,79.73% and 98.75%,respectively,between this strip with M.bovis isolation,TST,and the Korea MPB70 strip.This novel colloidal gold test strip for bTB antibody detection is sensitive,specific,convenient to be used,rapid to get results.It will be a good method in bTB survey used alone or as a supplementary diagnostic method for TST in bTB eradication program.