间接ELISA检测牛分枝杆菌抗体方法的建立及初步应用

针对现行牛结核病检疫方法结核菌素皮内变态反应(TST)的不足,本研究选用牛分枝杆菌特异性抗原MPB83、MPB70及CFP10与ESAT-6融合蛋白(CFP10-ESAT-6)分别建立了检测牛分枝杆菌特异抗体的间接酶联免疫吸附方法(ELISA)。上述3种抗原中一种以上抗原的特异性抗体为阳性时,即可判断为结核检测阳性。以TST为标准,判断ELISA方法的敏感性与特异性。结果证明,ELISA方法具有较好的敏感性(71.4%)。对ELISA检测为强阳性的奶牛进行细菌分离,并对5头结核菌分离阳性奶牛进行TST检测,结果有3头为TST阳性,2头可疑,显示出ELISA方法对严重感染牛的检测较TST具有更高的敏感性。由于TST与ELISA分别以细胞免疫与体液免疫为基础,两种检测方法结合应用可进一步提高牛结核病的检出率。

Development and primary application of indirect ELISA for detection of antibodies against Mycobacterium bovis

In order to overcome the problems associated with tuberculin skin test(TST)in bovine tuberculosis quarantine, three indirect enzyme-linked immunosorbent assays(ELISA)were developed for detection of M.bovis antibodies using specific antigens MPB83,MPB70 and fusion protein of CFP10 and ESAT-6(CFP10-ESAT-6).A sample was considered as M.bovis positive when it was detected positive at least by one of the ELISA assays.ELISA and TST were conducted in parallel,and the specificity and sensitivity of ELISA was nomalised using TST as the standard method.The results showed that ELISA has a sensitivity of 70.4 % in relation to TST test.The strong positive cows determined by ELISA were further succumbed to M.bovis isolation.Among 5 M.bovis positive cows,3 were detected positive by TST with 2 being suspicious.The results indicated that ELISA was more sensitive in detecting the serious infected cows.Because TST is based on cellular immunity while ELISA on humoral immunity,it is likely that M.boris detection rate would be improved by combining both methods.