Non-radioisotopic method for the in vitro measurement of EGF receptor tyrosine kinase

A non-radioisotopic method was developed for the assay of epidermal growth factor receptor (EGFR). A peptide with twenty amino acid residues around Tyr 1173, the major phosphorylation site of EGFR, was cloned as a GST fusion protein and used as substrate. Anti-phosphoty-rosine monoclonal antibody PY99 was used for the determination of the extent of phosphorylation. Both the specificity and the sensitivity were substantially higher than that of the existing method. Km value of the fusion protein is much lower (10 μmol/L) than that of the synthetic peptide (110 μmol/L). The method can be applied to the measurement of the tyrosine kinase activity of c-erb B2 (Neu/HER2).