High-throughput monoclonal antibody screening from immunized rabbits via droplet microfluidics†

Abstract

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, in vitro 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.