Tethered receptor-ligand reagent and assay

• US 20030108972A1
• Filed: 12/03/2002
• Published: 06/12/2003
• Est. Priority Date: 12/06/2001
• Status: Abandoned Application

• Current Assignee: Stephen Eliot Zweig
• Original Assignee: Stephen Eliot Zweig
• Inventors: Zweig, Stephen Eliot
• Application Number:
  US10/308,411
• Publication Number:
  US 20030108972A1
• Time in Patent Office: Days
• US Class Current: 435/7.92
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

A tethered reagent and assay is disclosed consisting of protein receptors tethered to ligands. The protein receptors can be antibodies, enzymes, hormone receptors, integral membrane proteins, and other proteins. Ligands can be antigens, enzymatic inhibitors, hormone agonists, drugs, and other protein binding ligands. The protein receptors and ligands will each be labeled with moieties capable of detecting changes in the average distance between the protein receptors and the ligand, using detection methods in which there is a sharp fall-off in signal as a function of distance. As a result, a change in the average distance between the two label moieties, such as that caused by protein-ligand binding and dissociation, produces a change in a detectable signal produced by the reagent. Tethering means may consist of flexible polymers, typically composed of a material that is chemically distinct from either the receptor or the ligand, so that the receptors and ligands may freely associate and dissociate via their specific binding sites, but not totally diffuse away from each other. When bound to solid phase surfaces, such reagents are particularly well suited for proteomic microarrays and flow cells. Such reagents may have utility for immunoassays, enzyme assays, ligand binding assays, sepsis assays, drug screening assays, and drug ADMET assays.

Claims

1. :

A reagent for measuring one or more test factors, said reagent comprising;

a ligand receptor protein, and a reagent ligand that can bind to a ligand binding site on said protein;

said protein and said reagent ligand being tightly coupled together by a tether that is chemically distinct from the protein and the reagent ligand;

wherein the test factors alter the ability of the reagent ligand to bind to the ligand binding site on said protein;

and wherein the binding or non binding of the reagent ligand to the ligand binding site on said protein creates a detectable signal.

2. :

The reagent of claim 1, in which the receptor protein is an antibody, and the reagent ligand contains an epitope recognized by the antibody.

3. :

The reagent of claim 1, in which the ligand receptor protein is an enzyme, and the reagent ligand is a substrate or inhibitor of the enzyme.

4. :

The reagent of claim 1, in which the reagent ligand binds to an allosteric site on the ligand receptor protein.

5. :

The reagent of claim 1, in which the test factor competes with the reagent ligand for binding to the ligand receptor on the ligand receptor protein.

6. :

The reagent of claim 1, in which the test factor modifies the conformation of the ligand receptor protein or the reagent ligand.

7. :

The reagent of claim 1, in which the test factor chemically modifies the ligand receptor protein or the reagent ligand.

8. :

The reagent of claim 1, in which the ligand receptor protein is labeled with a first moiety, and the reagent ligand is labeled with a second moiety;

in which either said first moiety or said second moiety emits photons; and

the other moiety absorbs said photons by a resonance transfer mechanism;

9. :

The reagent of claim 1, in which the reagent is bound to a surface of a cellular component, bead, microarray, or flow cell.

10. :

A device for measuring one or more test factors,

said device containing a surface with one or more reagents tightly bound to said surface;

said reagent or reagents comprising;

a ligand receptor protein, and a reagent ligand that can bind to a ligand binding site on said protein;

said protein and said reagent ligand being tightly coupled together by a tether that is chemically distinct from the protein and the reagent ligand;

wherein the test factors alter the ability of the reagent ligand to bind to the ligand binding site on said protein;

and wherein the binding or non binding of the reagent ligand to the ligand binding site on said protein alters the resonance transfer of photons between a first moiety associated with the protein, and a second moiety associated with the reagent ligand, producing a detectable signal;

11. :

The device of claim 10, in which the receptor protein is an antibody, and the reagent ligand contains an epitope recognized by the antibody.

12. :

The device of claim 10, in which the ligand receptor protein is an enzyme, and the reagent ligand is a substrate or inhibitor of the enzyme.

13. :

The device of claim 10, in which the reagent ligand binds to an allosteric site on the ligand receptor protein.

14. :

The device of claim 10, in which the test factor competes with the reagent ligand for binding to the ligand receptor on the ligand receptor protein.

15. :

The device of claim 10, in which the test factor modifies the conformation of the ligand receptor protein or the reagent ligand.

16. :

The device of claim 10, in which the test factor chemically modifies the ligand receptor protein or the reagent ligand.

17. :

The device of claim 10, in which the device is a component of a bead, microarray or flow cell.

18. :

A method of analyzing test factors, comprising;

applying said factors to a flow cell containing one or more reagents, said reagents comprising;

a ligand receptor protein, and a reagent ligand that can bind to a ligand binding site on said protein;

said protein and said reagent ligand being tightly coupled together by a tether that is chemically distinct from the protein and the reagent ligand;

wherein the ability of the test factors to cause changes in the detectable signal emitted by the reagents is observed.

19. :

The method of claim 18, in which multiple test factors are observed by the flow cell in a sequential manner

20. :

The method of claim 18, in which the flow cell is calibrated by one or more levels of control test factors.