Cloning antibodies from single cells in pooled sequence libraries by selective PCR.

Felix Horns,Stephen R Quake

doi:10.1371/journal.pone.0236477

Felix Horns, Stephen R Quake

Open Access

https://doi.org/10.1371/journal.pone.0236477

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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 high-throughput 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.

Highlights

Antibodies enable immune recognition by binding to target molecules called antigens
Antibody DNA can be produced by gene synthesis [3, 5], but this approach is more costly and time-consuming than
selective PCR for antibody retrieval (SPAR) enables a simple workflow for cloning antibody genes for functional characterization

Summary

Introduction

Antibodies enable immune recognition by binding to target molecules called antigens. Characterization of antibody binding properties, such as specificity and affinity, is essential for understanding the recognition capability of the immune system and discovering antibodies for research and therapeutics. Sequence information alone is not sufficient to predict antibody specificity and affinity. Single-cell approaches enable high-throughput determination of native antibody sequences, but remain inadequate for functional characterization at similar scale. Droplet- and microwell-based single-cell sequencing techniques can identify >10,000 natively paired antibody heavy- and light-chain gene sequences per experiment [1,2,3,4]. Antibody DNA can be produced by gene synthesis [3, 5], but this approach is more costly and time-consuming than

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