Cloning antibodies from single cells in pooled sequence libraries by selective PCRF

Abstract

Antibodies function by binding to antigens. Antibodies must be cloned and expressed to determine their binding characteristics, but current methods for high-throughput antibody sequencing yield antibody DNA pooled from many cells and do not readily permit cloning of antibodies from single B cells. We present a strategy for retrieving and cloning antibody DNA from single cells within a pooled library of cells. Our strategy, called selective PCR for antibody retrieval (SPAR), takes advantage of the unique sequence barcodes attached to individual cDNA molecules during sample preparation to enable specific amplification by PCR of antibody heavy- and light-chain cDNA originating from a single cell. We show through computational analysis that most human antibodies sequenced using typical highthroughput methods can be retrieved using SPAR, and experimentally demonstrate retrieval of full-length antibody variable region cDNA from three cells within pools of ∼5,000 cells. SPAR enables rapid low-cost cloning and expression of native human antibodies from pooled single-cell sequence libraries for functional characterization.