High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

Area of Science:

• Immunology
• Biotechnology
• Bioengineering

Background:

• The antibody repertoire of plasma cells and plasmablasts is a valuable source for discovering therapeutic and research antibodies.
• Existing methods for antibody discovery can be limited in throughput and scope, necessitating advanced screening techniques.

Purpose of the Study:

• To present a novel method for high-throughput, single-cell screening of IgG-secreting primary cells.
• To characterize antibody binding to both soluble and membrane-bound antigens using a single platform.
• To enable efficient discovery of therapeutic antibodies by analyzing antibody repertoire diversity, clonal expansion, and somatic hypermutation.

Main Methods:

• Development and application of CelliGO, a droplet microfluidics system.
• Integration of high-throughput screening for IgG activity using fluorescence-based in-droplet single-cell bioassays.
• Incorporation of in-droplet single-cell barcoded reverse transcription for sequencing paired antibody V genes.

Main Results:

• Analysis of IgG repertoire diversity, clonal expansion, and somatic hypermutation in immunized mice yielded 100-1,000 IgG sequences per mouse.
• Generation of 77 recombinant antibodies, with 93% recognizing the soluble antigen and 14% recognizing the membrane antigen.
• Successful recovery of ~450-900 IgG sequences from ~2,200 activated human memory B cells, demonstrating platform versatility.

Conclusions:

• The CelliGO platform provides a versatile and efficient method for high-throughput, single-cell antibody discovery.
• This technology facilitates the characterization of antibody binding to diverse antigens and analysis of immune responses.
• CelliGO holds significant potential for accelerating the discovery of therapeutic and research antibodies from primary B cells.