期刊
RSC ADVANCES
卷 10, 期 45, 页码 27006-27013出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/d0ra04328a
关键词
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资金
- UCB Pharma (Berkshire, UK)
- NIH SBIR [1 R43 AI082861-01, 5R43GM113420-02]
- Harvard University Blavatnik Biomedical Accelerator award
- Massachusetts Life Sciences Center (MLSC) Cooperative Research Matching Grant
- NIH SBIR grant [5R43GM113420-02]
- European Social Fund [09.3.3-LMT-K-712-01-0056]
- DARPA award [HR0011-11-C-0093]
- Bill and Melinda Gates Foundation [OPP1126222]
- UCB Pharma, Inc. [A19019]
- Total S.A. [A21376]
- Capsum award [A28393]
- Cytonome award [A21447]
- Solvay Pharmaceuticals [A19372-A20360]
- NCI Cancer Center support grant [2P30CA006516-48]
- Bill and Melinda Gates Foundation [OPP1126222] Funding Source: Bill and Melinda Gates Foundation
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.
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