Assay method for group transfer reactions
AssigneeBellbrook Labs, LLC
InventorsRobert Lowery, Karen Kleman-Leyer, Matt Staeben, Thane Westermeyer
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Abstract
The present invention relates to methods for detecting, quantifying and high throughput screening of donor-products and the catalytic activities generating the donor-products in group-transfer reactions. The invention further provides immunoassays, antibodies and kits that may be used to practice the methods of the invention.
Claims30
An antibody produced against a donor product of an enzymatically catalyzed group transfer reaction, wherein the antibody comprises the ability to preferentially distinguish between a donor-product and a donor in the presence of a high donor concentration;
- wherein the enzyme catalyzing the group transfer reaction is selected from the group consisting of a kinase, a sulfotransferase, a methyltransferase, a glycosyltransferase, an acetyl transferase, a UDP-glucuronosyltransferase, a glutathione transferase, and an ADP-ribosyltransferase.
A homogeneous competitive binding assay method for a donor product of a group transfer reaction, the assay comprising the steps of:
- a) combining the donor-product with a tracer and a macromolecule to provide a mixture, the macromolecule being specific for the donor product, the tracer comprising the donor-product conjugated to a fluorophore, the tracer being able to bind to the macromolecule to produce a detectable change in fluorescence polarization;
- b) measuring the fluorescence polarization of the mixture to obtain a measured fluorescence polarization;
- and
- c) comparing the measured fluorescence polarization with a characterized fluorescence polarization value, the characterized fluorescence polarization value corresponding to a known donor-product concentration.
A method for detecting a donor-product of an enzymatically catalyzed group transfer reaction, the method comprising the steps of:
- a) combining the donor-product with a tracer and a macromolecule to provide a mixture, the macromolecule being specific for the donor product, the tracer comprising the donor-product conjugated to a fluorophore, the tracer being able to bind to the macromolecule to produce a detectable change in fluorescence polarization;
- and
- b) detecting the donor-product of the group transfer reaction by measuring the fluorescence polarization of the mixture, wherein the enzyme catalyzing the group transfer reaction is selected from the group consisting of a kinase, a sulfotransferase, a methyltransferase, a glycosyltransferase, an acetyl transferase, a UDP-glucuronosyltransferase, a glutathione transferase, and an ADP-ribosyltransferase;
- and wherein the donor-product is selected from the group consisting of phosphoadenosine-phosphosulfate (PAP), adenosine diphosphate (ADP), guanosine diphospate (GDP), uridine diphosphate (UDP), cytidine diphosphate (CDP), thymidine diphosphate (TDP), s-adenosylhomocysteine (SAM), nicotinamide, and Coenzyme A (CoA).
A method for detecting a donor-product of an enzymatically catalyzed group transfer reaction, the method comprising the steps of:
- a) combining the donor-product with a tracer and a macromolecule to provide a mixture, the macromolecule being specific for the donor product, the tracer comprising the donor-product conjugated to a fluorophore, the tracer being able to bind to the macromolecule to produce a detectable change in fluorescence polarization;
- and
- b) detecting the donor-product of the group transfer reaction by measuring the fluorescence polarization of the mixture, wherein the enzyme catalyzing the group transfer reaction is a UDP-glucuronosyltransferase;
- and wherein the donor-product is uridine diphosphate (UDP).
An assay kit for characterizing a donor-product of a group transfer reaction, the assay kit comprising:
- a macromolecule and a tracer, each in an amount suitable for at least one homogeneous fluorescence polarization assay for donor-product, wherein the macromolecule is a an antibody or an inactivated enzyme;
- and wherein the macromolecule and the tracer may exist separately or together in a container.
A tracer compound for use in a homogenous competitive binding assay to detect a donor-product of a group transfer reaction;
- wherein the tracer comprises a fluorophore conjugated to a nucleotide.
A tracer compound for use in a homogenous competitive binding assay to detect a donor-product of a group transfer reaction;
- wherein the tracer comprises a fluorophore conjugated to a nucleotide through a position selected from the group consisting of an N6 amino, a C8 or a C2 position of adenine, a C5 or C6 position of uridine, thymidine or cytidine, and a 2′ or 3′ OH position of ribose.
A composition for use in a homogenous competitive binding assay to stabilize an assay signal by preventing breakdown of a donor product;
- wherein the composition is ethylene glycol-bis(-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA).
A composition for use in a homogenous competitive binding assay to quench a group transfer reaction;
- wherein the composition is sodium vanadate.
Dependencies are inferred from the claim text.