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Nat Med. 2015 Jan;21(1):86-91. doi: 10.1038/nm.3743. Epub 2014 Dec 15.

In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire.

Author information

1
Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.
2
1] Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA. [2] Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
3
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.
4
Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, USA.
5
1] Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA. [2] Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA. [3] Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA. [4] Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas, USA.

Abstract

High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the >1 × 10(6) B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 10(6) B cells per experiment with demonstrated pairing precision >97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or 'public', VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza.

PMID:
25501908
DOI:
10.1038/nm.3743
[Indexed for MEDLINE]

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