Volume 971 of the series Methods in Molecular Biology pp 93-111


Expression Cloning of Human B Cell Immunoglobulins

  • Hedda WardemannAffiliated withResearch Group Molecular Immunology, Max Planck Institute for Infection Biology Email author 
  • , Juliane KoferAffiliated withResearch Group Molecular Immunology, Max Planck Institute for Infection Biology

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The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.

Key words

Antibody Expression cloning Immunoglobulin Repertoire Single cell RT-PCR