High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies.

Published

Journal Article

Defining human B cell repertoires to viral pathogens is critical for design of vaccines that induce broadly protective antibodies to infections such as HIV-1 and influenza. Single B cell sorting and cloning of immunoglobulin (Ig) heavy- and light-chain variable regions (V(H) and V(L)) is a powerful technology for defining anti-viral B cell repertoires. However, the Ig-cloning step is time-consuming and prevents high-throughput analysis of the B cell repertoire. Novel linear Ig heavy- and light-chain gene expression cassettes were designed to express Ig V(H) and V(L) genes isolated from sorted single B cells as IgG1 antibody without a cloning step. The cassettes contain all essential elements for transcriptional and translational regulation, including CMV promoter, Ig leader sequences, constant region of IgG1 heavy- or Ig light-chain, poly(A) tail and substitutable V(H) or V(L) genes. The utility of these Ig gene expression cassettes was established using synthetic V(H) or V(L) genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using V(H) and V(L) genes isolated from cloned EBV-transformed antibody-producing cell lines. Finally, this strategy was successfully used for rapid production of recombinant influenza mAbs from sorted single human plasmablasts after influenza vaccination. These Ig gene expression cassettes constitute a highly efficient strategy for rapid expression of Ig genes for high-throughput screening and analysis without cloning.

Full Text

Duke Authors

Cited Authors

  • Liao, H-X; Levesque, MC; Nagel, A; Dixon, A; Zhang, R; Walter, E; Parks, R; Whitesides, J; Marshall, DJ; Hwang, K-K; Yang, Y; Chen, X; Gao, F; Munshaw, S; Kepler, TB; Denny, T; Moody, MA; Haynes, BF

Published Date

  • June 2009

Published In

Volume / Issue

  • 158 / 1-2

Start / End Page

  • 171 - 179

PubMed ID

  • 19428587

Pubmed Central ID

  • 19428587

Electronic International Standard Serial Number (EISSN)

  • 1879-0984

International Standard Serial Number (ISSN)

  • 0166-0934

Digital Object Identifier (DOI)

  • 10.1016/j.jviromet.2009.02.014

Language

  • eng