Clinical assay to predict therapeutic outcomes based on kinase or phostphatase activity.
Researchers at Emory University have developed assays to selectively measure the activity of a specific phosphate modifying enzyme within a heterogeneous samples. Unlike existing detection methods, which rely upon the incorporation or removal of radiolabelled phosphate, this novel assay measures the binding of a TAMRA labeled substrate to fluorescein-coated beads via Fluoresence Resonance Energy Transfer (FRET). This eliminates the need for multiple reaction steps, and permits rapid determination of specific activity in a highly reproducible manner. Our inventors have demonstrated that FRET-based phosphatase/kinase measurements are not hampered by the presence of additional phosphate modifying enzymes; simple, well-characterized modifications to the reaction conditions are sufficient to preferentially detect activity of a chosen enzyme.
While it is recognized that the activity of various kinases/phosphatases can be linked to the outcome of a number of clinical pathologies, variable baseline levels in the population make it difficult to effectively measure enzymatic activity. Our inventors have solved this by identifying factors that stimulate phosphate modifying enzymes, and then comparing baseline activity levels to those of enzymes stimulated with activating factors. This method has enabled our inventors to measure the efficacy of immunosuppressants on activity of the phosphatase Calcineurin in a clinical setting. This superior means of detecting specific differences in activity of phosphate modifying enzymes therefore has the potential to significantly advance therapeutic monitoring in clinical settings.
Publication: Roberts B, et al. (2008). Cell Calcium, 43: 515-19.