A FLUORESCENCE POLARIZATION COMPETITION IMMUNOASSAY FOR TYROSINE KINASES

We have recently reported a homogeneous, nonradioactive fluorescence p olarization method to assay protein tyrosine kinase activity. Our orig inal approach can only be used with a peptide substrate and requires l arge amounts of anti-phosphotyrosine antibody. To overcome these probl ems an alternate fluorescence polarization competition immunoassay was designed and evaluated. In this assay, phosphorylated peptide or prot ein produced by kinase reaction will compete with a fluorescent phosph opeptide used as a tracer for immunocomplex formation with phosphotyro sine antibody. In this format kinase activity will result in the loss of the polarization signal. To validate the fluorescence polarization competition immunoassay, Lck activity was compared with a more commonl y used (PO4)-P-32-transfer assay using Lck peptide or enolase as the s ubstrate. In both the assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and the peptide/enolase substrate concentrations with the FP signal inversely proportional to the amount of (PO4)-P-32 transferred to the substrate. Inhibition by staurosporine and the Lck inhibitor methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine was simil ar in these two assays. The advantages of this assay over other kinase assays include use of nonisotopic substrates and a more simple proced ure in which the kinase assay is done in a single tube (well of a micr otiter plate), without separation, precipitation, or washing. This met hod is easily automated for high-throughput drug discovery screening. (C) 1998 Academic Press.