Cell encapsulation in microfluidic droplets enables the isolation of thousands of natively-paired antibody coding sequences from blood samples – for example, isolation of monoclonal antibodies or potential therapeutic antibodies from human blood samples, or profiling antibody responses from multiple patients. The resulting natively-fused variable fragment libraries can be directly sequenced (e.g. by Illumina sequencing).
This enables the investigation of diversity and clonality in the antibody repertoire, offering new applications and capabilities.
High throughput microfluidic technology is used to enable the generation of thousands of fused single chain antibodies in a single run. A suspension of lymphocytes is pipetted into the µEncapsulator system. The lymphocytes are captured in droplets at limiting dilution, together with poly(dT) beads and lysis mix, such that each bead captures mRNA from a single cell.
The beads are washed, and added to a Reverse Transcription mix, to make first strand cDNA on the beads. The resulting cDNA beads are re-encapsulated at limiting dilution with PCR mix, with primers designed to amplify and fuse the heavy and light chain variable fragments. The resulting fusion products are ‘natively-paired’ – they contain the heavy and light chain variable fragments from the same cell. The fragments can be fused in-frame and cloned into an expression vector to directly make single chain fused antibodies.
This technique enables rapid high throughput isolation of thousands of monoclonal antibody coding sequences from complex samples, in a reliable manner.
Turchaninova, Maria A., et al. “Pairing of T‐cell receptor chains via emulsion PCR.” European journal of immunology 43.9 (2013): 2507-2515. Read more…
DeKosky, Brandon J., et al. “High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.” Nature biotechnology 31.2 (2013): 166-169. Read more…