Rapid single B cell antibody discovery using nanopens and structured light

ABSTRACT

Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today’s fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction–based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.