Unknown

Dataset Information

0

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


ABSTRACT: 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.

SUBMITTER: Ding R 

PROVIDER: S-EPMC9055518 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

altmetric image

Publications


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 seque  ...[more]

Similar Datasets

| S-EPMC8020118 | biostudies-literature
| S-EPMC3690483 | biostudies-literature
| S-EPMC4999286 | biostudies-literature
2017-10-06 | GSE104525 | GEO
| S-EPMC9014740 | biostudies-literature
| S-EPMC3360709 | biostudies-other
| S-EPMC8117785 | biostudies-literature
| S-EPMC9027373 | biostudies-literature
| S-EPMC4128248 | biostudies-literature
| S-EPMC3258639 | biostudies-literature