Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening.
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 10(8) 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 K:(d) = 1 x 10(-7) M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.
Philogen S.R.L., Piazza La Lizza 7, 53100 Siena, Italy. leonardo.giovannoni@philogen.it
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001667:Binding, Competitive; D003001:Cloning, Molecular; D018604:Epitope Mapping; D005909:Glioblastoma / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007150:Immunohistochemistry; D008546:Melanoma, Experimental / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000276:immunology*; D019151:Peptide Library; D011485:Protein Binding
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library