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Isolation of human monoclonal antibodies from peripheral blood B cells

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Abstract

Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity.

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Figure 1: Gating strategy for a memory B cell population.
Figure 2: IgG concentrations after 13 d of culture.
Figure 3: Workflow for the isolation of human monoclonal antibodies.

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References

  1. Wilson, P.C. & Andrews, S.F. Tools to therapeutically harness the human antibody response. Nat. Rev. Immunol. 12, 709–719 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).

    Article  CAS  PubMed  Google Scholar 

  3. Plaisant, P. et al. Human monoclonal recombinant Fabs specific for HCV antigens obtained by repertoire cloning in phage display combinatorial vectors. Res. Virol. 148, 165–169 (1997).

    Article  CAS  PubMed  Google Scholar 

  4. Mao, S. et al. Phage-display library selection of high-affinity human single-chain antibodies to tumor-associated carbohydrate antigens sialyl Lewisx and Lewisx. Proc. Natl. Acad. Sci. USA 96, 6953–6958 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Li, J. et al. Human antibodies for immunotherapy development generated via a human B cell hybridoma technology. Proc. Natl. Acad. Sci. USA 103, 3557–3562 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Traggiai, E. et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat. Med. 10, 871–875 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Aman, P., Ehlin-Henriksson, B. & Klein, G. Epstein-Barr virus susceptibility of normal human B lymphocyte populations. J. Exp. Med. 159, 208–220 (1984).

    Article  CAS  PubMed  Google Scholar 

  8. Bonsignori, M. et al. Analysis of a clonal lineage of HIV-1 envelope V2/V3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors. J. Virol. 85, 9998–10009 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tiller, T. et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J. Immunol Methods 329, 112–124 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Scheid, J.F. et al. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science 333, 1633–1637 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Smith, K. et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat. Protoc. 4, 372–384 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wu, X. et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329, 856–861 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Scheid, J.F. et al. Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals. Nature 458, 636–640 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Walker, L.M. et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326, 285–289 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Corti, D. et al. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science 333, 850–856 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Jourdan, M. et al. An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization. Blood 114, 5173–5181 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Ettinger, R. et al. IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells. J. Immunol. 175, 7867–7879 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Huang, J. et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature 491, 406–412 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kwong, P.D. & Mascola, J.R. Human antibodies that neutralize HIV-1: identification, structures, and B cell ontogenies. Immunity 37, 412–425 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kershaw, M.H. et al. Immunization against endogenous retroviral tumor-associated antigens. Cancer Res. 61, 7920–7924 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Georgiev, I.S. et al. Delineating antibody recognition in polyclonal sera from patterns of HIV-1-isolate neutralization. Science 340, 751–756 (2013).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This project has been funded in part with federal funds from the Intramural Research Programs of the NIAID. The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

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Author notes

  1. Jinghe Huang, Nicole A Doria-Rose and Nancy S Longo: These authors contributed equally to this work.

Authors and Affiliations

  1. HIV-Specific Immunity Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases (NIAID), US National Institutes of Health (NIH), Bethesda, Maryland, USA

    Jinghe Huang, Leo Laub, Byong H Kang, Stephen A Migueles & Mark Connors

  2. Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, USA

    Nicole A Doria-Rose, Nancy S Longo, Chien-Li Lin, Ellen Turk, Robert T Bailer & John R Mascola

Authors
  1. Jinghe Huang
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  2. Nicole A Doria-Rose
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  3. Nancy S Longo
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  4. Leo Laub
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  5. Chien-Li Lin
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  6. Ellen Turk
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  7. Byong H Kang
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  8. Stephen A Migueles
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  9. Robert T Bailer
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  10. John R Mascola
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  11. Mark Connors
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Contributions

M.C., L.L., J.R.M., N.A.D.-R. and N.S.L. developed and optimized the B cell culture protocol. J.H., L.L. and B.H.K. performed B cell sorting and isolated potent and broadly neutralizing antibodies. S.A.M. led the clinical care of the patients. C.-L.L., E.T. and R.T.B. screened the B cell culture supernatants for neutralization activity.

Corresponding author

Correspondence to Mark Connors.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

ELISA plate layout. (PDF 217 kb)

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Huang, J., Doria-Rose, N., Longo, N. et al. Isolation of human monoclonal antibodies from peripheral blood B cells. Nat Protoc 8, 1907–1915 (2013). https://doi.org/10.1038/nprot.2013.117

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