Cui Johnson Q, Song Ruyuan, Song Weihong, Li Ouyang, Yuan Xin, Zhou Hongbo, Zhang Lu, Yao Shuhuai
Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 999077, China.
DPBIO, Inc., 324 S Diamond Bar Blvd, Unit Num 717, Diamond Bar, CA 91765, USA.
Lab Chip. 2025 Jul 8;25(14):3482-3494. doi: 10.1039/d5lc00340g.
The discovery of monoclonal antibodies (mAbs) is critical to advancing therapeutics, diagnostics, and biomedical research. While mouse-derived mAbs dominate current applications, their limitations-short serum half-life, human immunogenicity, and restricted recognition of human-specific antigens-highlight the need for alternative sources. Rabbit-derived mAbs have been gaining significant traction with their superior antigen-binding affinity, broader epitope diversity, and higher yield potential. However, the absence of well-defined surface markers on rabbit B cells has hindered efficient enrichment strategies, limiting the exploration of this valuable antibody repertoire. In this study, we present an integrated workflow that combines magnetic negative selection with high-throughput droplet microfluidics to overcome these barriers. By optimizing a pan B cell enrichment protocol using a tailored antibody cocktail, we achieved a notable boost in IgG secretion and B cell enrichment. Through two complementary droplet-encapsulated assays using particle aggregation for soluble antigens and reporter cells for membrane-bound antigens, we identified target cells capable of secreting high-affinity IgGs. Subsequent sequencing, antibody production and characterization confirmed the high affinity rate of the discovered antibodies, outperforming rates previously reported. The use of droplet microfluidics streamlines the analysis of rabbit IgG repertoires, providing adraw robust platform for rabbit single B cell antibody discovery with promising applications in precision medicines and diagnostics.
单克隆抗体（mAb）的发现对于推进治疗学、诊断学和生物医学研究至关重要。虽然目前的应用主要是小鼠来源的单克隆抗体，但其局限性——血清半衰期短、人免疫原性以及对人特异性抗原的识别受限——凸显了寻找替代来源的必要性。兔源单克隆抗体因其卓越的抗原结合亲和力、更广泛的表位多样性和更高的产量潜力而越来越受到关注。然而，兔B细胞上缺乏明确的表面标志物阻碍了有效的富集策略，限制了对这一宝贵抗体库的探索。在本研究中，我们提出了一种将磁性阴性选择与高通量液滴微流控相结合的综合工作流程，以克服这些障碍。通过使用定制的抗体混合物优化全B细胞富集方案，我们显著提高了IgG分泌和B细胞富集水平。通过两种互补的液滴包封检测方法，一种利用颗粒聚集检测可溶性抗原，另一种利用报告细胞检测膜结合抗原，我们鉴定出了能够分泌高亲和力IgG的靶细胞。随后的测序、抗体生产和表征证实了所发现抗体的高亲和力率，优于先前报道的比率。液滴微流控的使用简化了兔IgG库的分析，为兔单B细胞抗体发现提供了一个强大的平台，在精准医学和诊断中具有广阔的应用前景。
