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Nucleic Acids Research 2005 33(1):e10; doi:10.1093/nar/gni010
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Published online 14 January 2005

© 2005, the authors Nucleic Acids Research, Vol. 33 No. 1 © Oxford University Press 2005; all rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use permissions, please contact journals.permissions{at}oupjournals.org.

Methods Online

Covalent antibody display—an in vitro antibody-DNA library selection system

Herald Reiersen*, Inger Løbersli, Geir Å. Løset, Else Hvattum, Bjørg Simonsen, John E. Stacy, Duncan McGregor1, Kevin FitzGerald1, Martin Welschof, Ole H. Brekke and Ole J. Marvik

Affitech AS, Oslo Research Park Gaustadalleen 21, N-0349 OSLO, Norway 1 Isogenica Ltd Barbraham CB2 4AT, UK

*To whom correspondence should be addressed. Tel: +47 22958877; Fax: +47 22958358; Email: herald{at}affitech.com

Received September 13, 2004. Revised December 15, 2004. Accepted December 15, 2004.

The endonuclease P2A initiates the DNA replication of the bacteriophage P2 by making a covalent bond with its own phosphate backbone. This enzyme has now been exploited as a new in vitro display tool for antibody fragments. We have constructed genetic fusions of P2A with single-chain antibodies (scFvs). Linear DNA of these fusion proteins were processed in an in vitro coupled transcription–translation mixture of Escherichia coli S30 lysate. Complexes of scFv–P2A fusion proteins covalently bound to their own DNA were isolated after panning on immobilized antigen, and the enriched DNAs were recovered by PCR and prepared for the subsequent cycles of panning. We have demonstrated the enrichment of scFvs from spiked libraries and the specific selection of different anti-tetanus toxoid scFvs from a V-gene library with 50 million different members prepared from human lymphocytes. This covalent antibody display technology offers a complete in vitro selection system based exclusively on DNA–protein complexes.

Correspondence may also be addressed to Martin Welschof. Email: m.welschof{at}affitech.com


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