Membrane receptor reagent and assay

• US 20040033624A1
• Filed: 05/23/2003
• Published: 02/19/2004
• Est. Priority Date: 06/17/2002
• Status: Active Grant

• Current Assignee: Stephen Eliot Zweig
• Original Assignee: Stephen Eliot Zweig
• Inventors: Zweig, Stephen Eliot
• Granted Patent: US 6,790,632 B2
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: G01N 33/5432 Liposomes or microcapsules
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

A membrane receptor reagent and assay is disclosed in which liposomes are bound to an evanescent wave emitting surface. Membrane receptors on the liposome'"'"'s fluid lipid bilayer membrane are labeled with a fluorescent or luminescent moiety. These membrane receptors are free to diffuse randomly throughout the liposome surface, and thus tend to redistribute according to externally applied forces. The evanescent wave-emitting surface additionally contains reagents that reversibly bind to the membrane receptors, tending to bring them closer to region of high evanescent wave intensity. Test analytes that disrupt or promote the association between the membrane receptors and the surface reagents act to change the average distance between the membrane receptors and the evanescent wave emitting surface, resulting in a change in the fluorescent or luminescent signal. This reagent and assay system functions with physiologically important membrane receptors such as GPCR receptors, other 7-tm receptors, drug transport proteins, cytochrome P450 membrane proteins and other clinically important membrane components. The reagent and assay methods may be incorporated into microarrays, capillaries, flow cells and other devices, and used for drug discovery, ADMET, and other biomedically important assays.

Claims

1. :

A reagent for measuring the interaction between test analytes and target membrane components, comprising;

a fluid lipid membrane anchored to a surface that emits energy that varies as a function of distance from the surface;

said membrane containing said target membrane components;

said target membrane components being associated with a moiety that emits a detectable signal upon receiving energy from said energy emitting surface;

said surface additionally containing reagents that reversibly bind to the target membrane components;

wherein test analytes influence the average distance between the target membrane receptors and the energy-emitting surface, resulting in a change in the detectible signal.

2. :

The reagent of claim 1, in which the energy emitted by the surface is chosen from the group of evanescent waves, surface plasmon resonance, or electron transfer, and the detectable signal is chosen from the group of fluorescent, luminescent, electrochemiluminescent, electron transport, or surface plasmon resonance signals.

3. :

The reagent of claim 1, in which the fluid lipid membrane is in the form of a liposome or phospholipid vesicle, and the target membrane components are bound to the liposome or phospholipid vesicle.

4. :

The reagent of claim 1, in which the anchor and reversible target membrane binding reagents are bound to the surface by means that are chosen from the group of protein-ligand binding reagents, electrostatic interactions, hydrophobic interactions, polymeric tethers, and polymeric meshes.

5. :

The reagent of claim 1, in which the target membrane component is chosen from the group of 7-transmembrane proteins, GPCR receptors, toll receptors, ion channels, ABC cassette pumps, hormone receptors, biological response modifier receptors, apoptosis receptors, angiogenesis receptors, neuroreceptors, histocompatibility antigens, coagulation factors, immune response antigens, cytochrome P450 family, and other membrane proteins.

6. :

The reagent of claim 1, used for the purpose of drug discovery, absorption distribution metabolism and excretion (ADME)), toxicology, cellular proliferation, cellular regulation, and medical diagnostic assays.

7. :

The reagent of claim 1, in which a reference energy excitation source, which does not substantially vary as a function of distance from the surface, is used to adjust or otherwise normalize the test signal.

8. :

The reagent of claim 1, in which the energy-emitting surface is part of a microarray, capillary tube, fiber optic fiber, or flow cell device.

9. :

A reagent for measuring the interaction between test-ligands and target membrane proteins, comprising;

a liposome containing a fluid lipid bilayer membrane;

said liposome anchored to a surface that emits evanescent energy;

said liposome membrane containing said target membrane proteins;

said target membrane proteins being associated with a moiety that emits a detectable fluorescent or luminescent signal upon receiving evanescent energy from said surface;

said surface additionally containing tethered reagent-ligands that reversibly bind to the target membrane proteins;

wherein test-ligands influence the average distance between the target membrane proteins and the evanescent energy emitting surface, resulting in a change in the fluorescent or luminescent signal.

10. :

The reagent of claim 9, used for the purpose of drug discovery, absorption distribution metabolism and excretion (ADME)), toxicology, cellular proliferation, cellular regulation, and medical diagnostic assays.

11. :

The reagent of claim 9, in which a control fluorescent excitation source, which does not substantially vary as a function of distance from the surface, is used to adjust or otherwise normalize the test signal.

12. :

The reagent of claim 9, in which the evanescent wave-emitting surface is part of a microarray, capillary tube, fiber optic fiber, or flow cell device.

13. :

A method for measuring the interaction between test analytes, and target membrane components that are bound to a surface by a lipid membrane, comprising;

observing changes in a detectible signal emitted from moieties associated with said target membrane receptors in response to test analytes;

In which the changes in the detectible signal are due to a change in the average distance between the target membrane components and an energy-emitting surface.

14. :

The method of claim 13, in which the target membrane components are part of a liposome.

15. :

The method of claim 13, in which the liposomes are anchored to an energy-emitting surface.

16. :

The method of claim 13, in which the energy-emitting surface emits evanescent waves, and in which the detectible signal is a fluorescent or luminescent signal.

17. :

The method of claim 13, in which the changes in the detectible signal are adjusted by a reference signal that does not substantially vary as a function of the average distance between the target membrane components and the energy emitting surface.

18. :

The method of claim 13, in which the surface is part of a microarray, capillary tube, fiber optical fiber, or flow cell.

19. :

The method of claim 13, used to perform a combined receptor ligand binding assay and ADMET analysis.

20. :

The method of claim 13, used in conjunction with fluidic switch elements that dynamically direct the passage of the test analytes to different assay devices.