MULTIPLEX ASSAY METHODS AND COMPOSITIONS

• US 20100304403A1
• Filed: 02/22/2010
• Published: 12/02/2010
• Est. Priority Date: 02/20/2009
• Status: Active Grant

• Current Assignee: Revvity Health Sciences Canada Incorporated
• Original Assignee: PerkinElmer BioSignal, Inc. (Revvity, Inc.)
• Inventors: Arcand, Mathieu, Bosse, Roger, Roby, Philippe
• Granted Patent: US 8,486,719 B2
• Time in Patent Office: Days
• US Class Current: 435/7.2
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Luminescence assays and compositions for assay of biomolecular interaction and activity and detection of modulators of biomolecular interaction and activity are provided. Technology described herein has utility in a variety of assay formats and types, for example, simultaneous monitoring multiple parameters which affect interaction and activity of biological molecules. Compositions and methods are provided herein which include a first solid-phase support associated with a first specific binding agent and a photosensitizer; a second solid-phase support associated with a second specific binding agent and a first emission system; and a third solid-phase support associated with a third specific binding agent and a second emission system.

Claims

1. An assay method, comprising:

A) providing an assay mixture, comprising, in combination;

a first solid-phase support comprising a first specific binding agent and a photosensitizer, the photosensitizer light excitable to activate oxygen to produce singlet oxygen,

a second solid-phase support comprising a second specific binding agent and a first emission system comprising a first energy acceptor activated by singlet oxygen, the first emission system activated by singlet oxygen to emit a first light signal characterized by a first emission spectrum, and

a third solid-phase support comprising a third specific binding agent and a second emission system comprising a second energy acceptor activated by singlet oxygen, the second emission system activated by singlet oxygen to emit a second light signal characterized by a second emission spectrum, wherein the first light signal and second light signal are distinguishable,

B) irradiating the assay mixture with a light source, activating the photosensitizer to produce singlet oxygen; and

C) detecting the first light signal and the second light signal, wherein detecting the first light signal is indicative of proximity of a first target bound to the first specific binding agent and a second target bound to the second specific binding agent and wherein detecting the second light signal is indicative of proximity of the first target bound to the first specific binding agent and third target bound to the third selective binding agent.

2. The assay method of claim 1, wherein the first solid-phase support, second solid-phase support and third solid-phase support are particles.

3. The assay method of claim 1, wherein the first solid-phase support, second solid-phase support and third solid-phase support are particles.

4. The assay method of claim 1, wherein the first solid-phase support is a cell membrane and the second solid-phase support and third solid-phase support are particles.

5. The assay method of claim 1, wherein the first emission spectrum is characterized by an emission maximum in the range of 450-600 nm and the second emission spectrum is characterized by an emission maximum in the range of 590-670 nm.

6. The assay method of claim 1, wherein the first emission spectrum is characterized by an emission maximum in the range of 545-580 nm and the second emission spectrum is characterized by an emission maximum in the range of 600-620 nm.

7. The assay method of claim 1, wherein the photosensitizer comprises phthalocyanine.

8. The assay method of claim 1, wherein the first energy acceptor and second energy acceptor activated by singlet oxygen comprises thioxene.

9. The assay method of claim 8, wherein the first emission system further comprises an anthracene.

10. The assay method of claim 8, wherein the first emission system further comprises rubrene.

11. The assay method of claim 8, wherein the second emission system further comprises a lanthanide chelate.

12. The assay method of claim 11, wherein the lanthanide chelate is a europium chelate.

13. The assay method of claim 1, wherein the first specific binding agent is selective for a first interacting member and the second specific binding agent is selective for a second interacting member, wherein specific interaction of the first interacting member and the second interacting member brings the photosensitizer and the first emission system into proximity, such that the irradiating generates the first light signal indicative of specific interaction of the first interacting member and the second interacting member, and wherein the third specific binding agent is selective for the first or second interacting member wherein binding of the third specific binding agent with the first interacting member or the second interacting member brings the photosensitizer and the second emission system into proximity, such that the irradiating generates the second light signal indicative of binding of the third specific binding agent and the first or second interacting member.

14. The assay method of claim 13, wherein the first interacting member is an enzyme and the second interacting member is a substrate for the enzyme and wherein the third specific binding agent selectively interacts with enzyme-modified substrate.

15. The assay method of claim 14, wherein the enzyme is a kinase, the substrate is a substrate of the kinase and the third specific binding agent selectively binds to the substrate phosphorylated by the kinase.

16. The assay method of claim 1, wherein the first specific binding agent is selective for a modifiable target and the second and third selective binding agents are selective for modifications of the modifiable target.

17. The assay method of claim 16, wherein the modifiable target is an enzyme substrate for the enzyme, the second specific binding agent is selective for a first enzyme-modified substrate site and the third specific binding agent is selective for a second enzyme-modified substrate site.

18. The assay method of claim 1, further comprising including a test compound in the assay mixture and comparing the first light signal and the second light signal in the absence of the test compound with the first light signal and the second light signal in the presence of the test compound.

19. A composition, comprising:

a first solid-phase support comprising a first specific binding agent and a photosensitizer, the photosensitizer light excitable to activate oxygen to produce singlet oxygen;

a second solid-phase support comprising a second specific binding agent and a first emission system comprising an energy acceptor activated by singlet oxygen, the first emission system activated by singlet oxygen to emit a first light signal characterized by a first emission spectrum; and

a third solid-phase support comprising a third specific binding agent and a second emission system comprising an energy acceptor activated by singlet oxygen, the second emission system activated by singlet oxygen to emit a second light signal characterized by a second emission spectrum, wherein the first light signal and second light signal are distinguishable.

20. An assay method, comprising:

A) providing an assay mixture, comprising, in combination;

a first solid-phase support comprising a photosensitizer, the photosensitizer light excitable to activate oxygen to produce singlet oxygen, the first solid-phase support further comprising a first member of a selective binding pair selected from the group consisting of;

enzyme/known or putative enzyme substrate;

receptor/known or putative ligand;

antibody/antigen; and

nucleic acid/complementary nucleic acid,

a second solid-phase support comprising a second specific binding agent and a first emission system comprising a first energy acceptor activated by singlet oxygen, the first emission system activated by singlet oxygen to emit a first light signal characterized by a first emission spectrum, the second solid-phase support further comprising a second member of the selective binding pair selected from the group consisting of;

enzyme/enzyme substrate;

receptor/ligand;

antibody/antigen; and

nucleic acid/complementary nucleic acid, and

a third solid-phase support comprising a third specific binding agent and a second emission system comprising a second energy acceptor activated by singlet oxygen, the second emission system activated by singlet oxygen to emit a second light signal characterized by a second emission spectrum, wherein the first light signal and second light signal are distinguishable, wherein the specific binding agent selectively binds to a modification of the first or second member of the selective binding pair;

B) irradiating the assay mixture with a light source, activating the photosensitizer to produce singlet oxygen; and

C) detecting the first light signal and the second light signal, wherein detecting the first light signal is indicative of binding of the first member of the selective binding pair with the second member of the selective binding pair and wherein detecting the second light signal is indicative of selective binding of the specific binding agent to a modification of the first or second member of the selective binding pair.

21. The method of claim 21, wherein the first member of the selective binding pair is a kinase, the second member of the selective binding pair is a known or putative substrate for the kinase, and the specific binding agent is an antibody which selectively binds to a phosphorylated or non-phosphorylated form of the known or putative substrate.

22. The method of claim 21, further comprising including a test compound in the assay mixture and comparing the first light signal and the second light signal in the absence of the test compound with the first light signal and the second light signal in the presence of the test compound.

23. An assay method, comprising:

A) providing an assay mixture, comprising, in combination;

a cell having a cell membrane, the cell comprising a first specific binding agent and a photosensitizer associated with the cell membrane, the photosensitizer light excitable to activate oxygen to produce singlet oxygen,

a first solid-phase support comprising a second specific binding agent and a first emission system comprising a first energy acceptor activated by singlet oxygen, the first emission system activated by singlet oxygen to emit a first light signal characterized by a first emission spectrum;

B) irradiating the assay mixture with a light source, activating the photosensitizer to produce singlet oxygen; and

C) detecting the first light signal, wherein detecting the first light signal is indicative of proximity of a first target bound to the first specific binding agent and a second target bound to the second specific binding agent.