TRIVALENT METAL MEDIATED HOMOGENEOUS LUMINESCENT PROXIMITY ASSAY.

摘要

An in vitro protein kinase assay technology that (1) exhibits a high assaysignal to background ratio (S/B) and range (S-B); (2) is homogenous; (3) is non-radioactive;and (4) does not require a phospho-specific antibody. Said assay involves complexinga trivalent metal ion (e.g. Ga3+, Fe3+, Al3+,In3+, Ru3+, Sc3+, Y3+) to the surfaceof amplified luminescent proximity assay acceptor or donor beads, e.g., viaa suitable linker such as nitrilotriacetic acid (NTA; also referred to as carboxymethyl-lysine),iminodiacetic acid (IDA), or an appropriately substituted N-containing heterocycle,for example a triazoheterocycle, for example a triazocyclononaneononane,such as 1-propylamino-4-acetato-1,4,7-triazacyclononane. A protein (orconstituent part) or other kinase substrate is bound to the surface of the otherof an amplified luminescent proximity assay acceptor or donor bead and, if phosphorylated,brought into proximity with the trivalent metal ion-complexed acceptor beadto generate a luminescent signal. Presence of a kinase inhibitor inhibits phosphorylationand therefore signal generation and, in this way, is detectable. As the inventiondescribed herein recognizes the presence or absence of phosphate groups on aprotein, (or constituent part), or other biological macromolecule (e.g.,mono, di, or trinucleotides, cyclic nucleotides or phosphate substituted inositols),it is broadly applicable to any phosphorlylation or dephosphorylation reactionenzymes and provides a highly robust and flexible assay format for protein kinasesand other enzyme classes, including lipid kinases, phosphatases, phosphodiesterasesand others.