Generation of Monoclonal Antibodies against Ag85A Antigen of Mycobacterium tuberculosis and Application in a Competitive ELISA for Serodiagnosis of Bovine Tuberculosis.
Generation of Monoclonal Antibodies against Ag85A Antigen of Mycobacterium tuberculosis and Application in a Competitive ELISA for Serodiagnosis of Bovine Tuberculosis.
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抗结核分枝杆菌 Ag85A 抗原单克隆抗体的制备及其在牛结核病血清学诊断的竞争性 ELISA 中的应用
DOI：
10.3389/fvets.2017.00107
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发表时间：
2017
影响因子：
3.2
通讯作者：
Jiao X
中科院分区：
文献类型：
--
作者：
Xu Z;Hu T;Xia A;Li X;Liu Z;Min J;He J;Meng C;Yin Y;Chen X;Jiao X
Ag 85复合物作为结核分枝杆菌（M. tuberculosis，M.结核病）和BCG。该复合物由蛋白质Ag 85 A、Ag 85 B和Ag 85 C组成，其中Ag 85 A被认为在复合物中发挥最大作用。然而，由于缺乏商业化的抗Ag 85 A的单克隆抗体（mAb），仍然阻碍了对该蛋白的生物学和应用研究。在本研究中，我们开发和鉴定了抗Ag 85 A的单克隆抗体，并建立了5个杂交瘤细胞。间接免疫荧光试验表明，两株抗Ag 85 A单抗与Ag 85 B和/或Ag 85 C均无交叉反应。此外，我们使用间接ELISA和Western blot分析表明，在本研究中测试的所有mAb都能够与BCG和rBCG：Ag 85 A中的内源性Ag 85 A蛋白反应。以单克隆抗体3B 8为基础，建立了牛结核病（TB）的竞争ELISA（cELISA），对临床诊断的牛结核病（TB）和健康牛血清进行检测，其敏感性为54.2%（26/48），特异性为83.5%（167/200）。本研究表明，抗Ag 85 A的单克隆抗体为进一步研究Ag 85复合物的功能提供了有用的试剂，并可用于牛结核病的血清学诊断。
The Ag85 complex functions as the main secretory protein of Mycobacterium tuberculosis (M. tuberculosis) and BCG. This complex is composed of the proteins, Ag85A, Ag85B, and Ag85C, with Ag85A thought to play the largest role within the complex. However, the lack of commercially available monoclonal antibodies (mAbs) against Ag85A still hinders the biological and applicative research on this protein. In this study, we developed and identified anti-Ag85A mAbs, and five hybridoma cells were established. Using the indirect immunofluorescence test, we found that two anti-Ag85A mAbs did not cross-react with Ag85B and/or Ag85C. In addition, we showed that all of the mAbs tested in this study are able to react with endogenous Ag85A protein in BCG and rBCG:Ag85A using indirect ELISA and Western blot analyses. A competitive ELISA (cELISA) based on mAb 3B8 was developed, the analyses of clinic serum samples from cattle with bovine tuberculosis (TB) and healthy cattle demonstrated that the sensitivity of the cELISA was 54.2% (26/48) and the specificity was 83.5% (167/200). This study demonstrated that the mAbs against Ag85A will provide useful reagents for further investigation into the function of the Ag85 complex and can be used for serodiagnosis of bovine TB.