Ren Xuanxiu, Zhang Yiwei, Zhang Gan, Yang Shangyu, Yu Feiyang, Cheng Rao, Deng Zengqin, Zhao Haiyan
State Key Laboratory of Virology and Biosafety, College of Life Sciences, Wuhan University, Wuhan 430072, China.
State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430207, China.
Biophys Rep. 2025 Aug 31;11(4):246-257. doi: 10.52601/bpr.2025.240067.
Identifying immunoglobulin (Ig) genes from antigen-specific B cells is crucial for understanding immune responses and generating monoclonal antibodies for diagnostic and therapeutic purposes. Despite single B cell PCR-based mouse antibody development is well established, several practical challenges remain. Here, we present an optimized protocol for the sequencing and cloning of variable regions of antibodies from single antigen-specific mouse B cells, along with high-throughput antibody expression and characterization. This method builds upon existing techniques, incorporating laboratory refinements and detailed troubleshooting insights. By integrating fluorescence-activated cell sorting (FACS) with reverse transcription polymerase chain reaction (RT-PCR) to amplify immunoglobulin heavy and light chain genes, along with a 12-well format for antibody expression, our refined approach enables efficient monoclonal antibody production and functional screening, thereby accelerating the antibody discovery workflow across a range of experimental applications.
从抗原特异性B细胞中鉴定免疫球蛋白（Ig）基因对于理解免疫反应以及生成用于诊断和治疗目的的单克隆抗体至关重要。尽管基于单B细胞PCR的小鼠抗体开发已很成熟，但仍存在一些实际挑战。在此，我们提出了一种优化方案，用于对单个抗原特异性小鼠B细胞抗体可变区进行测序和克隆，以及高通量抗体表达和表征。该方法基于现有技术，融入了实验室改进和详细的故障排除见解。通过将荧光激活细胞分选（FACS）与逆转录聚合酶链反应（RT-PCR）相结合以扩增免疫球蛋白重链和轻链基因，以及采用12孔格式进行抗体表达，我们改进的方法能够实现高效的单克隆抗体生产和功能筛选，从而加速一系列实验应用中的抗体发现工作流程。
