Identification of paired heavy and light chains from single B-cells from immunized Malian adults with rapid functional confirmation using iPair-BCR™, NGS, and iScreen™

Abstract

Abstract Information about the cognate pairing of TCR alpha-beta chains and BCR IgH and IgKL chains encoded by individual T and B cells is key to understanding adaptive immune responses and developing therapeutic applications. We have previously reported the development of a sensitive technology that allows the amplification and identification of the paired human TCR alpha and beta chains from single T cells, termed iPair-TCR. Here, we report the extension of this technology to identify paired human BCR IgH and IgKL chains from antigen-specific single B cells. In this proof of concept study, we identified paired-VDJ-receptors from antigen-specific B-cells from nine Pfs230-EPA immunized Malian adults using the iPair-BCR method. Next, we developed a method to rapidly generate Fab fragments and demonstrate the binding of several of these single cell targets to the original Pfs230 antigen. Single cells of interest were identified based upon their repeated frequency on the plate, which indicates a clonal selection. The corresponding heavy and light chains were PCR amplified from selected wells. Using overlap extension PCR, all necessary elements for in vitro transcription and translation and either the CH1 or C-kappa-domain were added to both the 5′ and 3′ ends of the single cell VDJ. After in vitro transcription and translation, four out of five tested Fab fragments demonstrated binding through a colorimetric ELISA assay. The overall process after VDJ identification can be performed in under a week indicating the utility of our technology for rapid identification of antigen-specific BCRs and functional binding characteristics.