Rapid isolation of antigen-specific B-cells using droplet microfluidics

Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusio...

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Bibliographic Details
Published in:RSC advances 2020-07, Vol.1 (45), p.276-2713
Main Authors: Ding, Ruihua, Hung, Kuo-Chan, Mitra, Anindita, Ung, Lloyd W, Lightwood, Daniel, Tu, Ran, Starkie, Dale, Cai, Liheng, Mazutis, Linas, Chong, Shaorong, Weitz, David A, Heyman, John A
Format: Article
Language:English
Subjects:
Antibodies
Antigens
Binding
Chains
Chemistry
Droplets
Microfluidics
Monoclonal antibodies
Protein synthesis
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Summary:Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusion inefficiency and display bias, respectively. Here we present a platform, featuring droplet microfluidics and a bead-based binding assay, to rapidly identify and verify antigen-binding antibody sequences from primary cells. We used a defined mixture of hybridoma cells to characterize the system, sorting droplets at up to 100 Hz and isolating desired hybridoma cells, comprising 0.1% of the input, with a false positive rate of less than 1%. We then applied the system to once-frozen primary B-cells to isolate rare cells secreting target-binding antibody. We performed RT-PCR on individual sorted cells to recover the correctly paired heavy- and light-chain antibody sequences, and we used rapid cell-free protein synthesis to generate single-chain variable fragment-format (scFv) antibodies from fourteen of the sorted cells. Twelve of these showed antigen-specific binding by ELISA. Our platform facilitates screening animal B-cell repertoires within days at low cost, increasing both rate and range of discovering antigen-specific antibodies from living organisms. Further, these techniques can be adapted to isolate cells based on virtually any secreted product. We use a droplet-microfluidics-based platform to rapidly identify and isolate individual primary cells that secrete desired antibodies. We then retrieve the antibody-encoding sequences and create recombinant antibodies that bind the target protein.
ISSN:2046-2069
2046-2069
DOI:10.1039/d0ra04328a