Protein Protocols Protein Protocols

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Screening of Phage Displayed Antibody Libraries
Heinz Dörsam, Michael Braunagel, Christian Kleist,
Daniel Moynet, and Martin Welschof
1. Introduction
Human monoclonal antibodies are more suitable than monoclonal antibodies of animal
origin for clinical applications because of lower hypersensitivity reactions, less
formation of circulating immune complexes, and lower anti-immunoglobulin
responses. The classical production of human monoclonal antibodies via the hybridoma
technique or Epstein–Barr virus (EBV) transformation is limited by the instability
of cell lines, low antibody production, and the problems of immunizing humans
with certain antigens (1,2). A promising alternative is the production of human recombinant
antibodies (3). Recombinant DNA technology has made it possible to clone
human antibody genes in vectors and to generate antibody expression libraries (4–7).
One approach has been to amplify and recombine the IgG repertoire of an “immunized”
donor. This has been used to isolate several antibodies that were related to diseases
(8–10). To obtain more universal antibody libraries the naive IgM repertoire of several
“unimmunized” donors were pooled (11–13). The complexity of the combinatorial
libraries has been further increased by creating the so-called “semisynthetic” antibody
libraries (14–16).
To prepare antibody DNA from peripheral lymphocytes, spleen lymphocytes, or
B-cell lines, mRNA is first isolated by standard methods. After preparation of the first
strand of cDNA the Fv- or Fab-encoding regions are amplified using the polymerase
chain reaction (PCR) and a set of primers homologous to the variable region of the
heavy (µ,γ) and light chains (κ,λ) (17–21). The PCR products are randomly combined
in an appropriate expression vector. For extra stability, the VL and VH domains of Fv
fragments are often joined with a peptide linker (22,23). The larger Fab fragment contains
the VL–CL and VH–CH1 segments linked by disulfide bonds. To facilitate the
screening of these scFv- or Fab-antibody libraries, phagemid pIII display vectors are
commonly used (24–26). These vectors contain a phage intergenic region to provide a
packaging signal. The expression of the pIII-antibody fusion protein is regulated by a
bacterial promoter under the control of a lac operator.
To display the antibody fragments on the phage surface, the phagemid must be packaged
with proteins supplied by helper phages. First, Escherichia coli is transformed
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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