牛布氏杆菌特异性抗体间接ELISA检测方法的建立及应用

目的建立牛布氏杆菌特异性抗体间接ELISA检测方法,并进行初步应用。方法用大肠杆菌表达的牛布氏杆菌重组BP26蛋白作为检测抗原,用棋盘滴定法确定包被抗原和待检血清的最佳工作浓度,对各种反应条件进行优化,建立间接ELISA方法,并进行验证。采用建立的间接ELISA方法与传统的试管凝集试验同时检测116份血清样品,评价二者的符合率。结果间接ELISA的最佳反应条件为:抗原包被量5μg/孔;待检血清稀释倍数1∶200,作用时间60min;酶标二抗作用时间60min;封闭液为5%BSA,作用时间45min。S/P值≥0.363者判为阳性,S/P值≤0.291者判为阴性,介于两者之间判为可疑。该方法精密性良好,特异性较强,敏感性较高,与传统的试管凝集试验检测结果的符合率为93.3%。结论已成功建立了检测牛布氏杆菌特异性抗体的间接ELISA方法,为布氏杆菌病的流行病学调查提供了一种简便的血清学诊断方法。

Development and Application of Indirect ELISA for Specific Antibody against Bovine Brucella

Objective To develop and preliminarily apply an indirect ELISA for specific antibody against bovine brucella. Methods The recombinant BP26 protein of bovine Brucella expressed in E. coli was served as detection antigen, the optimal working concentrations of coating antigen and serum samples to be tested were determined by chessboard titration, and the reaction condition was optimized, based on which an indirect ELISA method was developed and verified. A 116 serum samples were determined by the developed indirect ELISA and traditional tube agglutination test separately to evaluate the coincidence rate of the two methods. Results The optimal amount of antigen for coating was 5 μg per well, and the optimal dilution and incubation time of serum sample to be tested were 1:200 and 60 min respectively. The optimal incubation time of enzyme-labeled second antibody was 60 min. The BSA at a concentration of 5% was served as blocking agent, and its incubation time was 45 min. If the S / P value was not less than 0. 363, the result was judged as positive; if the S / P value was not more than 0. 291, the result was judged as negative; if the S / P value was more than 0. 291 but less than 0. 363, the result was judged as doubtful. The method showed high precision, specificity and sensitivity. The coincidence rate of determination results by the developed indirect ELISA and by traditional tube agglutination test was 93. 3%. Conclusion The indirect ELISA for specific antibody against bovine Brucella was developed, which provided a simple serological diagnostic method for epidemiological investigation on brucellosis.