Liu Siguo, Guo Sheping, Wang Chunlai, Shao Meili, Zhang Xiuhua, Guo Yang, Gong Qiang
Division of Bacterial Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
Tuberculosis (Edinb). 2007 May;87(3):212-7. doi: 10.1016/j.tube.2006.07.007. Epub 2006 Oct 4.
Enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine tuberculosis has been widely explored over the years. Three Mycobacterium bovis-specific antigen genes, namely, mpb70, mpb83, and esat-6 were recombined in tandem by spliced overlap extension technology and expressed in Escherichia coli to obtain the fusion protein (rM70-83-E6). Western blot analysis showed that rM70-83-E6 can specifically react with bovine tuberculosis-positive sera but not those from cattle infected with other bovine diseases such as bovine paratuberculosis. An indirect ELISA (iELISA) method was established with rM70-83-E6 as the diagnostic antigen. The diagnostic criteria were determined using 150 serum samples from healthy cattle. Analyses of 85 serum samples from cattle with bovine tuberculosis and 100 serum samples from healthy cattle demonstrated that the sensitivity of the iELISA was 69.4% (59/85) and the specificity was 96.0% (96/100). Moreover, 46 out of 67 purified protein derivative (PPD) skin test-positive samples were also positive by iELISA, giving a positive coincidence of 68.7%, while all 50 PPD skin test-negative samples were negative by iELISA, giving a negative coincidence of 100%. The total coincidence between iELISA and the PPD skin test was 82.1%. This study demonstrated that iELISA using rM70-83-E6 antigen is simple, sensitive and easy to perform and can be used to analysis of a large number of samples for serodiagnosis of bovine tubercuiosis.
多年来，用于诊断牛结核病的酶联免疫吸附测定（ELISA）已得到广泛研究。通过拼接重叠延伸技术将三个牛分枝杆菌特异性抗原基因，即mpb70、mpb83和esat-6串联重组，并在大肠杆菌中表达以获得融合蛋白（rM70-83-E6）。蛋白质印迹分析表明，rM70-83-E6能与牛结核病阳性血清特异性反应，但与感染其他牛病（如牛副结核病）的牛血清不发生反应。以rM70-83-E6作为诊断抗原建立了间接ELISA（iELISA）方法。使用来自健康牛的150份血清样本确定诊断标准。对85份牛结核病牛血清样本和100份健康牛血清样本的分析表明，iELISA的敏感性为69.4%（59/85），特异性为96.0%（96/100）。此外，67份纯化蛋白衍生物（PPD）皮肤试验阳性样本中有46份通过iELISA也呈阳性，阳性符合率为68.7%，而所有50份PPD皮肤试验阴性样本通过iELISA均为阴性，阴性符合率为100%。iELISA与PPD皮肤试验的总符合率为82.1%。本研究表明，使用rM70-83-E6抗原的iELISA方法简单、灵敏、易于操作，可用于大量样本的牛结核病血清学诊断分析。
