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Isolation of human monoclonal antibodies from peripheral blood B cells

Nature Protocols volume 8, pages 19071915 (2013) | Download Citation


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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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

    • Jinghe Huang
    • , Nicole A Doria-Rose
    •  & Nancy S Longo

    These authors contributed equally to this work.


  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


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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mark Connors.

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    ELISA plate layout.

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