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BOVIGAMTM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0.75μg/mL和1:8000,该方法Cut-off值为47.10%,检测限为0.800μg/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BOVIGAMTM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。
[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 BOVIGAMTM 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 0.75μg/mL and 1:8000,respectively.The established method showed the Cut-off of 47.10%,the limit of detection of 0.800μg/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 BOVIGAMTM 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 preliminari
作者
李昕
夏爱鸿
翟伟
谢宇晴
申峻松
徐正中
陈祥
焦新安
LI Xin;XIA Aihong;ZHAI Wei;XIE Yuqing;SHEN Junsong;XU Zhengzhong;CHEN Xiang;JIAO Xin’an(Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,Jiangsu,China;Key Laboratory of Prevention and Control of Biological Hazard Factors(Animal Origin)for Agrifood Safety and Quality of Ministry of Agriculture and Rural Affairs,Yangzhou University,Yangzhou 225009,Jiangsu,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2024年第5期1701-1712,共12页
Microbiology China
基金
国家重点研发计划(2021YFD1800403)
江苏省农业科技自主创新基金[CX(21)1004]
江苏省科技计划(BE2021331)
高等学校学科创新引智计划(D18007)
江苏高校优势学科建设工程项目(PAPD)。
