Liao, HX, 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 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


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.

Download full-text


Available from: Ruijun Zhang, Oct 31, 2014
  • Source
    • "Transient and recombinant antibody expressions were performed as previously described (Liao et al., 2009, 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognition prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. These data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode. Copyright © 2014 Elsevier Inc. All rights reserved.
    Full-text · Article · Dec 2014 · Immunity
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.
    Full-text · Article · Jul 2014 · Scientific Reports
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Antibody direct cloning from single B cells is simple and efficient and has been successful in antibody identification of infectious diseases. However, although a recent whole-exome sequencing revealed abundant heterogeneic mutation accumulation in cancers, identification and synthesis of autoantibodies against specific cancer-associated antigens is still difficult in cancer patients owing to the very small number of B cells producing autoantibodies. In the present study, to identify autoantibodies targeting tumor antigens, we measured the titer of autoantibodies in high-grade glioma patients’ plasma and identified two patients with elevated autoantibodies to a few transmembrane proteins. Specific B cells producing autoantibody against vascular endothelial growth factor receptor (VEGFR) 2 were immunostained with labeled protein and anti-human IgG antibody, and then collected by a single cell sorter. Finally, 22 antibody genes were successfully identified using direct IgG cloning from single B cell mRNA, and two antibody clones were found to have significant VEGFR2-specific binding affinity. The current direct human IgG gene cloning technique for identifying human antibodies derived from IgG-memory B cells avoids time-consuming procedures such as phage display-based antibody-library screening, and therefore may be applicable to identifying human autoantibodies in a variety of disorders including cancers even when antibody elevation is not detected because of a very small number of memory B cells.
    Full-text · Article · May 2014 · Immunology letters
Show more