High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies.
Journal Article (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
- Denny, Thomas Norton
- Gao, Feng
- Haynes, Barton Ford
- Liao, Hua-Xin
- Moody, Michael Anthony
- Walter Jr., Emmanuel Benjamin
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
- PMC2805188
Electronic International Standard Serial Number (EISSN)
- 1879-0984
Digital Object Identifier (DOI)
- 10.1016/j.jviromet.2009.02.014
Language
- eng
Conference Location
- Netherlands