A Fluorescence Polarization Competition Immunoassay for Tyrosine Kinases

Abstract

We have recently reported a homogeneous, nonradioactive fluorescence polarization method to assay protein tyrosine kinase activity. Our original approach can only be used with a peptide substrate and requires large amounts of anti-phosphotyrosine antibody. To overcome these problems an alternate fluorescence polarization competition immunoassay was designed and evaluated. In this assay, phosphorylated peptide or protein produced by kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with phosphotyrosine 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 commonly used32PO4-transfer assay using Lck peptide or enolase as the substrate. 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 of32PO4transferred to the substrate. Inhibition by staurosporine and the Lck inhibitor 4-amino-5-(methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine was similar in these two assays. The advantages of this assay over other kinase assays include use of nonisotopic substrates and a more simple procedure in which the kinase assay is done in a single tube (well of a microtiter plate), without separation, precipitation, or washing. This method is easily automated for high-throughput drug discovery screening.