An enhanced assay to measure phosphorylation activity for therapeutic monitoring of calcineurin activity in transplant patients as well as a number of other enzyme activity monitoring in medicine and research.
- Entire assay can be completed in a single plate.
- Fast, easy to use, non-radioactive method suitable for high-throughput assays.
- Enzyme specific substrates can be used to tailor assay for a number of applications.
This technology is an enhancement to our previous method to measure phosphatase activity. Our modification describes an ?in-solution? method that allows for the entire assay to be completed in a single plate. The principle of this in-solution assay is based on fluorescence resonance energy transfer. The plates contain fluorescent micro-beads of titanium oxide and the phosphorylated peptide substrate is synthesized with a commonly used fluorophore. The phosphorylated peptide substrate will bind to the micro-beads while the de-phosphorylated peptide substrate will not. The green fluorescent signal transfers energy to the red fluorophore signal to produce a unique emission. The emitted signal can be detected and compared to a standard curve in order to determine the amount of phosphorylated substrate in each reaction of the assay. Our current technology is a modification of a previously described improvement of phosphatase activity assays. Previously, we developed an assay that utilized fluorescently labeled peptide substrates and titanium oxide-coated plates to separate phosphorylated from non-phosphorylated substrates. While a significant advancement over previous radioactive methods for determining enzymatic activity, the assay required samples to be moved to multiple plates. Our improvement to the assay allows the entire procedure to be completed in a single plate. Additionally this assay contains micro-beads of titanium oxide instead of a coating of a titanium oxide resin. Overall, this technology continues to provide an assay to monitor calcineurin activity as well as improve the assay as an in-solution, or one plate, technique.
Proof of principle studies were completed to validate the assay.