【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%。更多还原

【Abstract】 Using the purified recombinant MPT83 protein of Mycobacterium bovis to coat microplate,an indirect ELISA assay was developed to detect antiMycobacterium 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min,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.更多还原