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

2009; Elsevier BV; Volume: 158; Issue: 1-2 Linguagem: Inglês

10.1016/j.jviromet.2009.02.014

1879-0984

Hua‐Xin Liao, Marc C. Levesque, A Nagel, Ashlyn Dixon, Ruijun Zhang, Emmanuel B. Walter, Robert Parks, John F. Whitesides, Dawn J. Marshall, Kwan-Ki Hwang, Yi Yang, Xi Chen, Feng Gao, Supriya Munshaw, Thomas B. Kepler, Thomas N. Denny, M. Anthony Moody, Barton F. Haynes,

Immune Cell Function and Interaction

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 (VH and VL) 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 VH and VL 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 VH or VL genes. The utility of these Ig gene expression cassettes was established using synthetic VH or VL genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using VH and VL 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.