Characterisation and specificity of two single-chain Fv antibodies directed to the protein tyrosine kinase Syk

In order to obtain single chain Fv fragments (scFv) specific for the protei n tyrosine kinase Syk, we screened a human synthetic phage-display library. Two glutathione S-transferase (GST):Syk fission proteins containing both S H2 domains of Syk were used to perform three rounds of selection of the lib rary. Among the scFv fragments resulting from the third round of selection, the ones specific for the GST portion of the fusion proteins were eliminat ed by performing enzyme-linked immunosorbent assay tests on GST:Syk versus GST coated plates, and the monoclonal scFv fragments binding only to the GS T:Syk coated plates with high affinities were further analysed. We report h ere the in vitro characterisation of G4G11 and G6G2 anti-Syk scFvs. G4G11 s hows the best performance in immunoprecipitation and immunofluorescence exp eriments, and G6G2 is able to detect Syk in immunoprecipitation, immunofluo rescence and on Western blots. Both scFvs are also able to detect the phosp horylated form of Syk, and neither of them binds to Zap-70, the other membe r of the Syk family of protein tyrosine kinases. (C) 2000 Elsevier Science B.V. All rights reserved.