Trivalent metal mediated homogeneous luminescent proximity assay

Abstract

An in vitro protein kinase assay technology that (1) exhibits a high assay signal 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 involves complexing a trivalent metal ion (e.g. Ga<3+>, Fe<3+>, Al<3+>, In<3+>, Ru<3+>, Sc<3+>, Y<3+>) to the surface of amplified luminescent proximity assay acceptor or donor beads, e.g., via a 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 (or constituent part) or other kinase substrate is bound to the surface of the other of an amplified luminescent proximity assay acceptor or donor bead and, if phosphorylated, brought into proximity with the trivalent metal ion-complexed acceptor bead to generate a luminescent signal. Presence of a kinase inhibitor inhibits phosphorylation and therefore signal generation and, in this way, is detectable. As the invention described herein recognizes the presence or absence of phosphate groups on a protein, (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 reaction enzymes and provides a highly robust and flexible assay format for protein kinases and other enzyme classes, including lipid kinases, phosphatases, phosphodiesterases and others.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Patent Application No. 60 / 610,799, filed September 17, 2004, entitled "Trivalent Metal-Mediated Homogeneous Luminescent Proximity Assay," which is incorporated herein by reference in its entirety and for all Purpose. Background of the invention [0003] The present invention relates to materials and methods for conducting biological assays, in particular kinase assays. [0004] Phosphorylation of various intracellular proteins and lipid substrates plays an important role in many cellular processes such as growth, proliferation, apoptosis, differentiation and cell cycle progression. Kinases, responsible for phosphorylating enzymes in cells (including other kinases), kinases and lipids were identified as major targets for possible therapeutic intervention. Broadly, kinases are classified as tyrosine (ie, phosphorylation occurs on tyrosine amino acid residues) or serine...

AI Technical Summary

Problems solved by technology

Also, because the intrinsic polarization of macromolecules is very high (i.e., the background B increases significantly), when the substrate is a protein or a large protein fragment, the S / B becomes too small to be used

Assays with low signal-to-noise ratios are more likely to produce false positive results, which can significantly affect the overall validity and reproducibility of the HTS procedure

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