Assay method for group transfer reactions

1. An isolated antibody produced against a donor product of an enzymatically catalyzed group transfer reaction, wherein the antibody has the ability to preferentially recognize a donor-product in the presence of a donor molecule, and wherein the antibody binds the donor-product, guanosine diphosphate (GDP) with at least ten-fold higher affinity than to the donor molecule, GTP.

2. The antibody of claim 1 , wherein the antibody is generated using an immunogen made from a nucleotide conjugated to a carrier protein.

3. The antibody of claim 2 , wherein the immunogen includes a linker region between the nucleotide and the carrier protein comprising a chemical formula of NH 2 —X-Z, wherein X is a saturated or unsaturated chain of 2 to 20 carbons and Z is a functional group capable of covalently binding to a protein, and wherein the group includes an NH 3 .

4. The antibody of claim 1 , wherein the antibody binds the GDP, with at least fifty-fold higher affinity than to GTP.

5. The antibody of claim 1 , wherein the antibody binds the GDP with at least one hundred-fold higher affinity than to GTP.

6. The antibody of claim 1 , wherein the antibody binds the GDP with at least one hundred and thirty-fold higher affinity than to GTP.

7. A universal homogenous assay method for directly detecting a donor-product produced in a group transfer reaction in the presence of a donor molecule, the method comprising the steps of:

a) reacting a donor molecule, which is guanosine triphosphate (GTP), with an acceptor in the presence of a catalytically active enzyme to form the donor-product, which is guanosine diphosphate (GDP), such that the GTP is partially consumed;

b) combining the GDP produced in a group transfer reaction with a tracer and the antibody of claim 1 to provide a reaction mixture, the tracer comprising a nucleotide conjugated to a fluorophore, and capable of binding to the antibody to produce a detectable change in fluorescence polarization;

c) measuring the fluorescence polarization of the mixture to obtain a measured fluorescence polarization; and

d) comparing the measured fluorescence polarization with a characterized fluorescence polarization value corresponding to a known GDP concentration to directly detect the GDP produced in the group transfer reaction.

8. A method of using the assay of claim 7 , for screening a chemical library to identify a molecule which is an acceptor substrate for an enzyme catalyzing a group transfer reaction or is capable of inhibiting or activating such an enzyme.

9. A universal homogenous assay method for directly detecting a donor-product produced in a group transfer reaction, the method comprising:

a) reacting a donor molecule which is an guanosine triphosphate (GTP) with an acceptor, in the presence of a group transfer enzyme;

b) forming the donor-product which is an guanosine diphosphate (GDP), such that the GTP is partially consumed;

c) contacting the GDP with a first complex comprising an antibody that specifically recognizes the GDP and a tracer capable of producing an observable, wherein the antibody is the antibody of claim 1 ;

d) competitively displacing the tracer of the first complex by the GDP, to generate a second complex, GDP-antibody complex and a displaced tracer, to directly detect the donor-product in the group transfer reaction; and

e) detecting a change in the observable produced by the tracer in the first complex bound to the antibody and the displaced or free tracer.

10. A universal homogenous assay method for directly detecting a donor-product produced in a group transfer reaction, the method comprising the steps of:

a) providing a reaction mixture having products of the group transfer reaction, a tracer and an antibody, wherein the products of the reaction include the donor-product, which is a guanosine diphosphate (GDP), in the presence of a donor molecule which is a guanosine triphosphate (GTP), wherein the antibody is the antibody of claim 1 , and wherein the tracer comprises a nucleotide conjugated to a fluorophore and is capable of binding to the antibody to produce a detectable change in the fluorescence properties of the tracer;

b) measuring the fluorescence properties of the reaction mixture to obtain a measured value; and

c) comparing the measured fluorescence properties with a characterized fluorescence property value corresponding to a known GDP concentration to directly detect the GDP produced in the group transfer reaction.

11. An assay kit for detecting the donor-product of a group transfer reaction, the assay kit comprising

an isolated antibody produced against a donor product of an enzymatically catalyzed group transfer reaction, wherein the antibody has the ability to preferentially recognize a donor-product in the presence of a donor molecule, and wherein the antibody binds the donor-product, guanosine diphosphate (GDP) with at least ten-fold higher affinity than to the donor molecule, GTP; and a tracer.