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

Publication ,  Journal Article
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 ...
Published in: J Virol Methods
June 2009

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.

Duke Scholars

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Published In

J Virol Methods

DOI

EISSN

1879-0984

Publication Date

June 2009

Volume

158

Issue

1-2

Start / End Page

171 / 179

Location

Netherlands

Related Subject Headings

  • Virology
  • Molecular Biology
  • Humans
  • Herpesvirus 4, Human
  • HIV-1
  • Genes, Immunoglobulin
  • Gene Expression
  • DNA
  • B-Lymphocytes
  • Antibodies, Viral
 

Citation

APA
Chicago
ICMJE
MLA
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Liao, H.-X., Levesque, M. C., Nagel, A., Dixon, A., Zhang, R., Walter, E., … Haynes, B. F. (2009). High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. J Virol Methods, 158(1–2), 171–179. https://doi.org/10.1016/j.jviromet.2009.02.014
Liao, Hua-Xin, Marc C. Levesque, Ashleigh Nagel, Ashlyn Dixon, Ruijun Zhang, Emmanuel Walter, Robert Parks, et al. “High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies.J Virol Methods 158, no. 1–2 (June 2009): 171–79. https://doi.org/10.1016/j.jviromet.2009.02.014.
Liao H-X, Levesque MC, Nagel A, Dixon A, Zhang R, Walter E, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. J Virol Methods. 2009 Jun;158(1–2):171–9.
Liao, Hua-Xin, et al. “High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies.J Virol Methods, vol. 158, no. 1–2, June 2009, pp. 171–79. Pubmed, doi:10.1016/j.jviromet.2009.02.014.
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. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. J Virol Methods. 2009 Jun;158(1–2):171–179.
Journal cover image

Published In

J Virol Methods

DOI

EISSN

1879-0984

Publication Date

June 2009

Volume

158

Issue

1-2

Start / End Page

171 / 179

Location

Netherlands

Related Subject Headings

  • Virology
  • Molecular Biology
  • Humans
  • Herpesvirus 4, Human
  • HIV-1
  • Genes, Immunoglobulin
  • Gene Expression
  • DNA
  • B-Lymphocytes
  • Antibodies, Viral