Homogeneous Assay for Tyrosine Kinase: Use of Bacteriophage Antibody Conjugates in an Assay for p56lck Kinase

Abstract

Protein kinases play a critical role in almost every cellular regulatory process and have been identified as key players in diseases such as cancer and immune syndromes (1). For this reason, there has been substantial interest in the development of assays for protein kinases for use as both diagnostics and drug discovery tools (2). Although several assays exist for kinases, the most commonly used involve monitoring transfer of the γ-phosphoryl group from [γ-32P]ATP to a peptide substrate (3). These assays are cumbersome to implement because the unreacted [γ-32P]ATP needs to be separated from the phosphorylated peptide by use of separation techniques such as gel electrophoresis. In drug discovery programs, fluorescence resonance energy transfer (FRET) kinase assays are becoming increasingly popular because they are homogeneous, making them relatively easy to automate (4). In typical FRET assays, the anti-phosphotyrosine antibody and the substrate peptide are labeled with “donor” and “acceptor” fluorophores. On phosphorylation of the peptide, the anti-phosphotyrosine antibody binds to the peptide, and the two fluorophores are brought in close proximity to each other. Excitation of the acceptor fluorophore leads to energy transfer to the donor molecule, which emits fluorescence. The principal disadvantage of FRET-based assays is that they are difficult to configure because the two fluorophores need to be within a closely defined distance of each other. The Dual Phage technology is a new ultrasensitive biological amplification system of broad applicability that uses bacteriophages as …