军事医学2025，Vol.49Issue(9)：666-673,8.DOI:10.7644/j.issn.1674-9960.2025.09.004
结核分枝杆菌MPT64单个B细胞抗体制备及快速检测方法的建立
Preparation of single B-cells antibodies against Mycobacterium tuberculosis MPT64 and establishment of a rapid detection method
摘要
Abstract
Objective To establish a rapid immunological detection method for MPT64 protein based on red microspheres and select highly-sensitive and highly-specific antibody pairs.Methods A His-tagged prokaryotic expression vector was constructed for expression of MPT64 protein that was used to immunize New Zealand white rabbits after purification and validation.Peripheral blood mononuclear cells(PBMCs)were isolated from the rabbits,and antigen-specific B cells expressing antibodies were sorted using single B-cell flow cytometry.mRNA in B cells was reverse-transcribed into cDNA,and paired antibody heavy-and light-chain sequences were amplified via nested PCR.Expression vectors were constructed,and recombinant antibodies were produced in Expi293F cells.Fluorescent immunochromatography was employed to screen for matched antibody pairs.The selected antibodies were used to establish a rapid detection method based on red microsphere immunochromatography.Results Ten high-affinity monoclonal antibodies against MPT64 were generated.Two antibody pairs were selected for MPT64 immunodetection that reached a sensitivity of 0.0125 ng/mL.Conclusion High-affinity rabbit monoclonal antibodies against MPT64 are obtained via single B-cell technology,and a rapid red microspheres-based immunodetection method is established,enabling highly sensitive detection of Mycobacterium tuberculosis MPT64 protein.关键词
结核分枝杆菌/MPT64/单个B细胞/兔单克隆抗体/层析法Key words
Mycobacterium tuberculosis/MPT64/single B cell/rabbit monoclonal antibody/immunochromatography method分类
医药卫生引用本文复制引用
康景美,黄文华,任宇豪,姜永强,赵国芬,律清宇..结核分枝杆菌MPT64单个B细胞抗体制备及快速检测方法的建立[J].军事医学,2025,49(9):666-673,8.基金项目
病原微生物生物安全全国重点实验室自主研究面上课题(SKLPBS2212) （SKLPBS2212）
