Protocol abstract
Nature Protocols 4, - 372 - 384 (2009)
Published online: 26 February 2009 | doi:10.1038/nprot.2009.3
Subject Categories: Isolation, purification and separation | Cell and tissue culture
Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen
Kenneth Smith1, Lori Garman1, Jens Wrammert2, Nai-Ying Zheng3, J Donald Capra1, Rafi Ahmed2 & Patrick C Wilson3,4
Abstract
We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.
- Department of Clinical Immunology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, Oklahoma 73104, USA.
- Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
- Section of Rheumatology, The Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
- The Committee on Immunology, University of Chicago, Chicago, Illinois 60637, USA.
Correspondence to: Patrick C Wilson3,4 e-mail: wilsonp@uchicago.edu
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