牛分枝杆菌CFP-10单克隆抗体的制备及其竞争ELISA方法的建立与应用

Preparation of a monoclonal antibody against Mycobacterium bovis CFP-10 and establishment and application of a competitive ELISA for bovine tuberculosis detection

摘要:

【背景】 牛结核病是一种重要的人兽共患传染病，主要由牛分枝杆菌引起，阻碍养殖业发展，并在世界范围内造成巨大经济负担。【目的】 制备牛结核分枝杆菌CFP-10蛋白单克隆抗体，建立牛结核病竞争ELISA方法并对其进行初步应用与评估。【方法】 以rHis-CFP-10蛋白作为免疫原，采用小鼠杂交瘤融合技术获得稳定分泌抗体的杂交瘤细胞株，选用杂交瘤细胞株制备单克隆抗体，并对其纯化后进行辣根过氧化物酶标记。基于CFP-10单克隆抗体建立牛结核病竞争ELISA方法，对155头奶牛采用商品化BOVIGAM^TM试剂盒与建立的竞争ELISA检测方法进行同步检测，验证该方法适用性。【结果】 获得一株稳定分泌CFP-10特异性抗体的阳性杂交瘤细胞株8E6，获得酶标抗体HRP-8E6并建立竞争ELISA方法。棋盘法确定包被抗原CFP-10-ESAT-6融合蛋白的工作浓度和酶标抗体HRP-8E6的稀释倍数分别为0.75 μg/mL和1:8 000，该方法Cut-off值为47.10%，检测限为0.800 μg/mL；其重复性良好，批间与批内试验变异系数均小于10%；敏感性为54.55%，特异性为100.00%。临床检测显示，与BOVIGAM^TM试剂盒相比，阳性符合率为78.57%，阴性符合率为82.35%，总符合率为80.65%。【结论】 制备一株CFP-10特异性抗体，并使用特异性高的CFP-10-ESAT-6融合蛋白建立竞争ELISA检测方法，该方法准确、可靠，可应用于牛结核病的检测。

Abstract:

[Background] Bovine tuberculosis is a zoonotic infectious disease mainly caused by Mycobacterium bovis, which hinders the development of breeding industry and causes huge economic burden worldwide. [Objective] To prepare monoclonal antibodies against Mycobacterium tuberculosis CFP-10 protein and establish a competitive ELISA for the detection of bovine tuberculosis. [Methods] With rHis-CFP-10 protein as the immunogen, the mouse hybridoma fusion technique was employed to establish stable hybridoma cells that secreted antibodies. A monoclonal antibody was obtained from the hybridoma cell line 8E6 and subsequently conjugated with horseradish peroxidase after purification. A competitive ELISA for the detection of bovine tuberculosis was established based on the anti-CFP-10 monoclonal antibody. To assess the applicability of the established method, we employed the commercial BOVIGAM^TM kit and the established competitive ELISA assay to detect 155 dairy cows. [Results] A hybridoma cell line 8E6 stably secreting specific antibodies against CFP-10 was obtained. A competitive ELISA method was established based on the HRP-8E6 antibody. The working concentration of the coating antigen CFP-10-ESAT-6 and the dilution factor of HRP-8E6 were 0.75 μg/mL and 1:8 000, respectively. The established method showed the Cut-off of 47.10%, the limit of detection of 0.800 μg/mL, and good reproducibility (inter and intra-batch coefficients of variance being below 10%). The established method showed the sensitivity of 54.55% and the specificity of 100.00% in the detection of the serum samples from cattle with bovine tuberculosis and healthy cattle. In the clinical trial, the established method showed a positive coincidence rate of 78.57%, a negative coincidence rate of 82.35%, and a total coincidence rate of 80.65% compared with the BOVIGAM^TM kit. [Conclusion] An anti-CFP-10 antibody was successfully prepared and a competitive ELISA method was established using a CFP-10-ESAT-6 fusion protein with high specificity. The method is accurate and reliable and can be preliminarily applied to the detection of bovine tuberculosis.