ISOLATION BY PHAGE DISPLAY OF SINGLE-CHAIN Fv ANTIBODIES AGAINST Escerichia coli K99 FIMBRIAE

Single-chain antibodies (scFv) are small, recombinant proteins in which variable domains from immunoglobulin heavy and light chains are physically linked with a flexible peptide. They are monovalent and lack the heavy chain constant domins for activation of complement and binding to cellular receptors. However scFvs can possess biological activity by binding to and inhibiting the action of their molecular targets. ScFvs can be rapidly isolated from recombinant libraries by phage display. In this study, we have isolated and characterised scFvs against the K99 colonisation factor of an enterotoxigenic strain of Escherichia coli. K99 fimbriae were isolated from B41, a bovine ETEC strain, and purified by ion-exchange chromatography. The material was coated onto plastic immunotubes and two synthetic scFv libraries screened for binders by standard phage display methods. Six scFvs that bound strongly to isolated K99 fimbriae in ELISA were expressed in E. coli (HB2151) and purified by nickel-chelating chromatography. Purified scFvs were then used in immunofluorescence microscopy to study their binding to the surface of B41 bacteria. The recognition of fimbriae was also investigated by electron microscopy using immunogold reagents. It was of particular interest to test if any of the anti-K99 scFvs could inhibit the binding of fimbriae to their mammalian receptors. This was tested in haemagglutination assays. As expected, several of the anti-K99 antibodies have shown the inhibitory activity. Our studies illustrate how recombinant antibody technology can be applied to the study of pathogenesis for infectious disease.

Keywords:

Recombinant antibodies , scFv , Phage display , K99