Preparation of single B-cells antibodies against Mycobacterium tuberculosis MPT64 and establishment of a rapid detection method
10.7644/j.issn.1674-9960.2025.09.004
- VernacularTitle: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
- Author:
Jingmei KANG
1
;
Wenhua HUANG
;
Yuhao REN
;
Yongqiang JIANG
;
Guofen ZHAO
;
Qingyu LÜ
Author Information
1. 内蒙古农业大学生命科学学院麦类种质创新利用自治区高等学校重点实验室,呼和浩特 010011
- Keywords:
Mycobacterium tuberculosis;
MPT64;
single B cell;
rabbit monoclonal antibody;
immunochromatography method
- From:
Military Medical Sciences
2025;49(9):666-673
- CountryChina
- Language:Chinese
-
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.
