Liao, H.X. et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. J. Virol. Methods 158, 171-179

Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, 27710, United States.
Journal of virological methods (Impact Factor: 1.78). 07/2009; 158(1-2):171-9. DOI: 10.1016/j.jviromet.2009.02.014
Source: PubMed

ABSTRACT 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.

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Available from: Ruijun Zhang, Oct 31, 2014
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    • "Transient and recombinant antibody expressions were performed as previously described (Liao et al., 2009, 2011). "
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    Immunity 12/2014; 41(6):909-918. DOI:10.1016/j.immuni.2014.11.014 · 21.56 Impact Factor
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    • "When combined with the downstream transfection of pVITRO1 expression vectors into Human Embryonic Kidney (HEK) 293-F cells, followed by hygromycin B selection, this strategy can yield reproducible generation of tens of milligram quantities of functional recombinant mAb in less than four weeks from cloning of the V- and C-region DNA, through to harvesting of selected cell supernatants. Recombinant mAbs have been produced in HEK 293-F cells2526273738 and although cloning the dual antibody cassette into pVITRO1 for expression in these cells has facilitated appreciable expression yields (Figure 3), the cassette can be transferred to any compatible expression vector consisting of two transcription units for use in alternative systems, if required. "
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    • "On the other hand, the single B cell-based human monoclonal antibody (MoAb) gene PCR cloning method has been developed as a rapid and simple technology without undesirable selection or bias in recent years. Using this technology, several researchers have demonstrated that human immunodeficiency virus (HIV)-infected subjects possessed neutralizing serum antibodies that neutralized the majority of viruses with diverse genetic subtypes, and successfully identified and synthesized a human monoclonal antibody from a single memory-B cell that neutralized over 90% of HIV-1 isolates [18] [19]. "
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