Assay Method for Group Transfer Reactions

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.

1 . A method of detecting a donor-product of a group transfer reaction, the method comprising:
 a) reacting an activated form of a donor with an acceptor in the presence of a catalytically active enzyme;   b) forming the donor-product and an acceptor-X;   c) contacting the donor-product with a first complex comprising a detectable tag capable of producing an observable;   d) competitively displacing the detectable tag of the first complex by the donor product to generate a second complex and a displaced detectable tag; and   e) detecting a change in the observable produced by the detectable tag in the first complex and the displaced detectable tag.   
   
   
       2 . The method of  claim 1 , further comprising,
 f) quantifying the observable of step (e).   
   
   
       3 . The method of  claim 1 , wherein,
 a) the activated form of the donor comprises donor-X;   b) the acceptor comprises a substrate for the catalytically active enzyme, wherein the substrate is selected from the group consisting of a polypeptide, a protein, a nucleic acid, a lipid, a carbohydrate and a small molecule substrate;   c) the donor-product comprises a nucleotide or a non-nucleotide, wherein the non-nucleotide is a metabolic intermediate selected from the group consisting of s-adenosylhomocysteine, nicotinamide or coenzyme A;   d) the acceptor-X comprises a reaction product in which X is a covalent adduct; wherein the covalent adduct is selected from the group consisting of a phosphate, a sulfate, a carbohydrate, a naturally occurring amino acid, a synthetically derived amino acid, ADP-ribose, a nucleotide, a methyl, an acetyl, and a glutathione moiety; and wherein the covalent adduct is optionally capable of altering either the function, the stability, or both the function and the stability of the acceptor;   e) the first complex comprises a macromolecule and a detectable tag; and   f) the second complex comprises the macromolecule wherein the detectable tag is competitively displaced by the donor-product resulting in the production of an observable.   
   
   
       4 . The method of  claim 3 , wherein the macromolecule is selected from the group consisting of an antibody, a polypeptide, a protein, a nucleic acid molecule, and an inactivated enzyme that is capable of contacting the donor-product with high affinity. 
   
   
       5 . The method of  claim 4 , wherein the antibody is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. 
   
   
       6 . The method of  claim 4 , wherein the antibody is specific for the donor product, and wherein the level of antibody cross-reacting with the donor-X is less than the level of specificity that the antibody exhibits towards the donor-product. 
   
   
       7 . The method of  claim 1 , wherein the detectable tag is a tracer, wherein the tracer is a fluorescent or a chemiluminiscent molecule conjugated to a nucleotide or a non-nucleotide. 
   
   
       8 . The method of  claim 1 , further comprising detecting a catalytic activity, wherein the catalytic activity generates the donor-product in the group transfer reaction. 
   
   
       9 . The method of  claim 8 , wherein the catalytic activity comprises a chemical catalytic activity, an enzymatic activity, or a combination thereof; wherein the enzymatic activity comprises a sulfotransferase, a kinase, a UDP-glucuronosyltransferase, a methyl transferase, a acetyl transferase, a glutathione transferase, and a ADP-ribosyltransferase. 
   
   
       10 . The method of  claim 1 , wherein the method is an immunoassay. 
   
   
       11 . The method of  claim 10 , wherein the immunoassay is selected from the group consisting of fluorescence polarization immunoassay (FPIA), fluorescence resonance energy transfer (FRET), enzyme linked immunosorbant assay (ELISA), chemiluminescence immunoassay. 
   
   
       12 . The method of  claim 1 , wherein the method is used for screening a chemical library to identify a molecule which is capable of activating or inhibiting a group transfer reaction enzyme. 
   
   
       13 . The method of  claim 12 , wherein the molecule is capable of altering either the function, the stability, or both the function and the stability of the acceptor. 
   
   
       14 . The method of  claim 12 , wherein the molecule is capable of exhibiting a therapeutic effect. 
   
   
       15 . The method of  claim 12 , wherein the library is screened using a high-throughput screening technique comprising a multiwell plate, a microarray or a microfluidic system. 
   
   
       16 . An antibody produced against a donor product of a 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. 
   
   
       17 . The antibody of  claim 16 , wherein the donor-product is selected from the group consisting of a nucleotide or a non-nucleotide. 
   
   
       18 . The antibody of  claim 16 , wherein the antibody is specific for a phosphate portion of a nucleotide, and wherein the antibody has the ability to distinguish between a 5′-phosphate, a 5′-phosphosulfate, a 5′-diphosphate and a 5′-triphosphate. 
   
   
       19 . A homogeneous competitive binding assay 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.   
   
   
       20 . The assay of  claim 19 , wherein the group transfer reaction is catalyzed an enzyme. 
   
   
       21 . The assay of  claim 19 , wherein the enzyme is selected from the group consisting of a kinase, a sulfotransferase, a methyltransferase a UDP-glucuronosyltransferase, a acetyl transferase, a glutathione transferase, and a ADP-ribosyltransferase. 
   
   
       22 . The assay of  claim 19 , wherein the donor-product is selected from the group consisting of phosphoadenosine-phosphosulfate (PAP), adenosine diphosphate (ADP), uridine diphosphate (UDP), s-adenosylhomocysteine, nicotinamide, and Coenzyme A. 
   
   
       23 . The assay of  claim 19 , wherein the fluorophore is selected from the group fluorescein, rhodamine, Texas red and derivatives thereof. 
   
   
       24 . A method of using the assay of  claim 19  to screen a chemical library to identify a molecule which is capable of inhibiting or activating a group transfer reaction enzyme. 
   
   
       25 . An assay kit for characterizing a donor-product from 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 be separate or together in the container.   
   
   
       26 . The assay kit of  claim 25 , further comprising packaging, and instructions for using the antibody and the tracer in the homogeneous fluorescence polarization assay, the antibody being specific for donor-product, the tracer comprising donor-product conjugated to a fluorophore, the tracer being able to bind to the antibody to produce a detectable change in fluorescence polarization. 
   
   
       27 . The assay kit of  claim 26  wherein the fluorophore is selected from the group consisting of fluorescein, rhodamine, Texas red and derivatives thereof.