Assay methods for group transfer reactions

1. A 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 adenosine triphosphate (ATP), with an acceptor in the presence of a catalytically active enzyme to form the donor-product which is adenosine diphosphate (ADP) and an acceptor phosphate, such that the ATP is partially consumed;

b) combining the ADP produced in a group transfer reaction with a tracer and an antibody to provide a reaction mixture, the antibody being specific for the ADP, the tracer comprising the ADP 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 ADP concentration to directly detect the ADP produced in the group transfer reaction.

2. A 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 adenosine triphosphate (ATP) with a polypeptide, in the presence of a kinase;

b) forming the donor-product which is an adenosine diphosphate (ADP) and a phosphorylated polypeptide;

c) contacting the ADP with a first complex comprising an antibody, that specifically recognizes the ADP and a tracer capable of producing an observable;

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

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

3. A 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 reaction is a kinase reaction, wherein the products of the reaction include the donor-product which is an adenosine diphosphate (ADP), in the presence of a donor molecule which is an adenosine triphosnhate (ATP), wherein the antibody is specific for the ADP, and wherein the tracer comprises the ADP conjugated to a fluorophore and is capable of binding to the antibody to produce a detectable change in fluorescence polarization;

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

c) comparing the measured fluorescence polarization with a characterized fluorescence polarization value corresponding to a known ADP concentration to directly detect the ADP produced in the kinase reaction.