牛分枝杆菌重组MPT83蛋白间接ELISA方法的建立  

Development of an indirect ELISA based on recombinant MPT83 protein of Mycobacterium bovis

作　　者：鲁俊鹏[1] 罗满林[1] 邹潍力[1] 林琳 彭南秀 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]广东省动物防疫监督总所,广东广州510230 [3]广州市畜牧兽医总站,广东广州510050

出　　处：《中国兽医科学》2007年第3期236-240,共5页Chinese Veterinary Science

基　　金：广东省教育厅自然科学研究项目(Z03008);广东农业科技攻关项目(粤农2004)

摘　　要：以纯化的牛分枝杆菌重组MPT83蛋白为包被抗原,建立了检测牛分枝杆菌抗体的间接ELISA方法。确定了间接ELISA各组分的最适反应条件:抗原包被浓度为1μg/mL,酶标二抗稀释度为1∶1600,血清稀释度为1∶60,抗原和血清、血清和二抗均在37℃反应30 min,底物在37℃显色15 min,D655 nm阴性、阳性临界值为0.5。经阻断试验、交叉试验、重复性试验,表明该方法特异性强、重复性好。用该方法对18份结核菌素试验阳性牛血清和36份结核菌素试验阴性牛血清进行检测,结果显示,阳性血清的符合率为27.8%,阴性血清的符合率为91.7%。Using the purified recombinant MPT83 protein of Mycobacteriurn boris to coat microplate, an indirect ELISA assay was developed to detect anti-Mycobacteriurn bovis sera. The optimal concentration of the recombinant MPT83 protein for coating was 1 μg/mL, the working concentration of HRP-labeled goat anti-bovine IgG was 1 ： 1 600, the dilution of serum sample was 1 ： 60, the serum sample and the HRP-labeled goat anti-bovine IgG should be incubated at 37 ℃ for 30 rain, and the substrate for ELISA was incubated at 37 ℃ for 15 min before read. Following the determination of conditions of ELISA,the cutoff value of ELISA was 0.5. The ELISA assay was confirmed to have good reproducibility and specificity through the repeat test,cross reaction assay and blocking test. Eighteen positive serum samples and thirty six negative serum samples by PPD were examined by the established ELISA. The accordance rates of ELISA with PPD to the positive serum samples and the negative serum samples were 27.8% and 91.7%, respectively.

关 键 词：牛分枝杆菌 重组MPT83蛋白 间接ELISA 

分 类 号：S852.618[农业科学—基础兽医学]