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Nucleic Acids Research, 2001, Vol. 29, No. 5 e27
© 2001 Oxford University Press

Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening

Leonardo Giovannoni*, Francesca Viti1, Luciano Zardi2 and Dario Neri1

Philogen S.R.L., Piazza La Lizza 7, 53100 Siena, Italy, 1Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland and 2Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, 16132 Genova, Italy

We describe here a method, based on iterative colony filter screening, for the rapid isolation of binding specificities from a large synthetic repertoire of human antibody fragments in single-chain Fv configuration. Escherichia coli cells, expressing the library of antibody fragments, are grown on a porous master filter, in contact with a second filter coated with the antigen, onto which antibodies secreted by the bacteria are able to diffuse. Detection of antigen binding on the second filter allows the recovery of a number of E.coli cells, including those expressing the binding specificity of interest, which can be submitted to a second round of screening for the isolation of specific monoclonal antibodies. We tested the methodology using as antigen the ED-B domain of fibronectin, a marker of angiogenesis. From an antibody library of 7 x 108 clones, we recovered a number of specifically-binding antibodies of different aminoacid sequence. The antibody clone showing the strongest enzyme-linked immunosorbent assay signal (ME4C) was further characterised. Its epitope on the ED-B domain was mapped using the SPOT synthesis method, which uses a set of decapeptides spanning the antigen sequence synthesised and anchored on cellulose. ME4C binds to the ED-B domain with a dissociation constant Kd = 1 x 10–7 M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.

* To whom correspondence should be addressed. Tel: +39 0577 234922; Fax: +39 0577 234903; Email: leonardo.giovannoni{at}philogen.it Present address: Francesca Viti, Consorzio Siena Ricerche, Via Fiorentina 1, 53100 Siena, Italy


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