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

Peneff, C.; Lefranc, M. P.; Dariavach, P.

J Immunol Methods

2000-03-06 / vol 236 / pages 105-15


In order to obtain single chain Fv fragments (scFv) specific for the protein tyrosine kinase Syk, we screened a human synthetic phage-display library. Two glutathione S-transferase (GST):Syk fusion proteins containing both SH2 domains of Syk were used to perform three rounds of selection of the library. Among the scFv fragments resulting from the third round of selection, the ones specific for the GST portion of the fusion proteins were eliminated by performing enzyme-linked immunosorbent assay tests on GST:Syk versus GST coated plates, and the monoclonal scFv fragments binding only to the GST:Syk coated plates with high affinities were further analysed. We report here the in vitro characterisation of G4G11 and G6G2 anti-Syk scFvs. G4G11 shows the best performance in immunoprecipitation and immunofluorescence experiments, and G6G2 is able to detect Syk in immunoprecipitation, immunofluorescence and on Western blots. Both scFvs are also able to detect the phosphorylated form of Syk, and neither of them binds to Zap-70, the other member of the Syk family of protein tyrosine kinases.

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Humans; Animals; Mice; Phosphorylation; Flow Cytometry; Base Sequence; Precipitin Tests; Cell Line; Blotting, Western; Fluorescent Antibody Technique; Rabbits; ZAP-70 Protein-Tyrosine Kinase; Intracellular Signaling Peptides and Proteins; Cross Reactions; Antibodies, Monoclonal/*immunology; Antibody Specificity; DNA Primers/genetics; Enzyme Precursors/chemistry/genetics/*immunology; Immunoglobulin Fragments/*immunology; Peptide Library; Protein-Tyrosine Kinases/chemistry/genetics/*immunology; Recombinant Fusion Proteins/chemistry/genetics/immunology; src Homology Domains

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