Molecular modification assays involving lipids

• US 20050227294A1
• Filed: 06/06/2005
• Published: 10/13/2005
• Est. Priority Date: 09/15/1997
• Status: Abandoned Application

• Current Assignee: MDS Analytical Technologies US Incorporated (Danaher Corporation)
• Original Assignee: Molecular Devices
• Inventors: Sportsman, J., Gaudet, Elizabeth
• Application Number:
  US11/146,553
• Publication Number:
  US 20050227294A1
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: G01N 2800/044 Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

Assays for detecting molecular modifications, such as phosphorylation, dephosphorylation, and/or cleavage, among others, of lipids, lipid fragments, and/or lipid precursors. These assays may be used to detect the presence and/or activity of enzymes and/or other agents, such as drugs, involved in facilitating, inhibiting, and/or otherwise regulating such modifications.

Claims

1. A method of detecting modification of a lipid or lipid precursor in a sample, comprising:

contacting the sample with a binding partner that binds specifically to a modified form or a nonmodified form of the lipid or lipid precursor, but not both, the binding partner including a metal required for specific binding to the one form; and

detecting a response indicative of the extent of binding between the binding partner and the one form.

2. The method of claim 1, wherein the detected response is indicative of a decrease in the nonmodified form of the lipid.

3. The method of claim 1, wherein the detected response is indicative of the presence of the nonmodified form of the lipid.

4. The method of claim 1, wherein the detected response includes a luminescence polarization response.

5. The method of claim 1, wherein the detected response includes a luminescence resonance energy transfer response.

6. The method of claim 1, wherein the detected response includes at least one of a change in the intensity, polarization, energy transfer, lifetime, and excitation or emission wavelength distribution of a detected luminescence.

7. The method of claim 1, wherein the modified and nonmodified forms are related by an enzymatic modification.

8. The method of claim 7, the enzymatic modification being mediated by an enzyme, further comprising adding a candidate modulator of activity of the enzyme to the sample, prior to the step of detecting a response.

9. The method of claim 7, wherein the enzymatic modification is cleavage.

10. The method of claim 9, wherein the enzymatic modification cleaves the lipid or the lipid precursor between a phosphate moiety and a luminescent moiety.

11. The method of claim 7, wherein the enzymatic modification is phosphorylation.

12. The method of claim 1, wherein the extent of binding between the binding partner and the one form is related to one or more functional groups of the one form.

13. The method of claim 12, wherein the functional group is ionic at physiological pH.

14. The method of claim 12, wherein the functional group is selected from phosphate, sulfonate, and carboxylate.

15. The method of claim 1, wherein the binding partner includes a cationic metal ion.

16. The method of claim 15, wherein the binding partner includes a gallium ion.

17. The method of claim 15, wherein the binding partner includes a particle or macromolecule associated with the metal ion.

18. The method of claim 1, wherein the method is performed with a lipid, and wherein the lipid is a phosphoinositide.

19. The method of claim 1, wherein the method if performed with a lipid precursor, and wherein the lipid precursor includes inositol.

20. The method of claim 1, wherein the step of contacting also contacts the binding partner with a luminescent reporter to which the binding partner can bind, and wherein the step of detecting includes a step of measuring competition, if any, between the one form and the luminescent reporter for binding to the binding partner.

21. The method of claim 1, wherein the step of contacting is performed in the presence of a detergent that increases the response relative to no detergent.

22. A kit configured to perform the method of claim 1.