Flexible Detection Technology
BMG Labtech’s PHERAstar was used to detect both the luminescent signal generated by the Kinase-Glo assay in the microHTS format described above, and the output of a time-resolved fluorescence immunoassay embodied as Invitrogen’s (www.invitrogen.com) Adapta™ Universal Kinase Assay in an HTS format.
BMG and Invitrogen teamed up to host a workshop entitled “Screening for Lipid Kinase Inhibitors on the PHERAstar using Adapta Universal Kinase Assay.” The workshop featured the adaptation of BMG’s multidetection micoplate reader and Invitrogen’s time-resolved fluorescence resonance energy transfer (TR-FRET) technology.
E.J. Dell, Ph.D., an applications scientist at BMG Labtech, commented that lipid kinases pose a particular challenge to kinase assay developers because “they can be sticky, ubiquitously interacting with different proteins and lipids.” This tendency to stick to multiple effectors compromises accurate detection and readout of phosphorylation reactions making them a difficult and expensive kinase target to study.
Invitrogen and BMG Labtech’s solution was to combine PHERAstar’s simultaneous dual emission detection system with the Adapta assay, which Invitrogen licensed from Bellbrook Labs.
According to Dr. Dell, the Adapta assay quantifies kinase activity indirectly in terms of ADP formation, the by-product of a phosphorylation reaction, instead of measuring kinase activity or ATP depletion, thus allowing more accurate measurements of lipid kinases to be achieved.
“Due to the inherent complexity of the PI3 Kinase family, traditional biochemical approaches used with protein kinase assays are not readily transferable to the lipid kinases,” said Robert Horton, Ph.D., senior scientist at Invitrogen. “The sensitivity of the Adapta assay to ADP formation, and its versatility to accommodate a wide gamut of substrates, enables profiling of small molecule inhibitors across the entire PI3 Kinase family with a single assay format.”
Adapta, being a TR-FRET assay, is extremely sensitive to ADP formation. This, according to Dr. Dell, increases the assay’s suitability to detect low-level kinase activity since most of the signal changes occur within the first 10–20% of ATP to ADP conversion. The assay kit can be used for a wide range of ATP concentrations (1–500 mM), eliminating the need for multiple kit purchases, he added.
The assay itself measures ADP formation during the course of a kinase reaction by providing a tracer amount of ADP labeled with a red acceptor fluorophore and an antiADP antibody tagged with a Europium chelate, that when excited in turn can excite the flurophore, causing light to be emitted in the far-red range.
When no ADP is present, the tracer ADP is complexed with the Europium-labeled antibody producing a high TR-FRET signal, measured at 665 nm and 620 nm (emission wavelengths of the red fluorophore and Europium, respectively).
When ADP is formed, it displaces the tracer amount of ADP thereby causing the TR-FRET signal to decrease. Potential lipid kinase inhibitors could be found if a high TR-FRET signal is maintained because there would be no kinase activity and no ADP formation.
According to Dr. Dell, the PHERAstar multidetection microplate reader can simultaneously detect both emissions at once to yield results. “Other instruments need to read the plate twice, decreasing sensitivity and increasing assay time and intra-assay variability in terms of percent CVs and Z prime values.”
Patricia F. Dimond, Ph.D., is a life science consultant. E-mail: [email protected]