Multiplex analysis using membrane-bound sensitizers

• US 20030170915A1
• Filed: 03/04/2003
• Published: 09/11/2003
• Est. Priority Date: 03/05/2002
• Status: Active Grant

• Current Assignee: Aclara Biosciences, Inc (Laboratory Corporation Of America Holdings), Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Chan-Hui, Po-Ying
• Granted Patent: US 6,949,347 B2
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/567 utilising isolate of tissue or organ as binding agent
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing

Abstract

The present invention is directed to methods and compositions for determining the presence, absence, and/or amounts of one or more membrane-associated analytes in a sample. In accordance with the invention, binding compounds derivatized with releasable molecular tags specifically bind to selected membrane-associated analytes, after which the molecular tags are released upon activation of cleavage moieties, or sensitizers, anchored in the same membrane as the membrane-associated analytes. The released molecular tags are then identified by their distinct separation and detection characteristics.

Claims

1. A method for determining the presence or absence of one or more membrane-associated analytes, the method comprising the steps of:

providing a sensitizer-treated membrane comprising the one or more membrane-associated analytes;

providing one or more binding compounds, such that there is at least one binding compound specific for each of the one or more membrane-associated analytes, each binding compound having one or more molecular tags, each molecular tag being attached by a cleavable linkage, and the molecular tags of each binding compound being distinguishable from those of every other binding compound by one or more physical and/or optical characteristics;

combining the sensitizer-treated membrane and the plurality of binding compounds specific for the one or more membrane-associated analytes such that in the presence of a membrane-associated analyte a complex is formed between such membrane-associated analyte and a binding compound specific therefor;

activating the sensitizer-treated membrane so that an active species is produced that cleaves the cleavable linkage of the binding compounds forming such complex so that molecular tags are released; and

separating and identifying the released molecular tags by the one or more physical characteristics to determine the one or more membrane-associated analytes in the sample.

2. The method of claim 1 wherein said step of separating includes electrophoretically separating said released molecular tags, and wherein each of said molecular tags has a molecular weight in the range of from 50 to 1500 daltons.

3. The method of claim 2 wherein said sensitizer-treated membrane is a photosensitizer-treated membrane, wherein said active species is singlet oxygen, and wherein each of said molecular tags attached to said binding compounds are selected from a group defined by the formula:

-L-(M,D)

wherein;

L is a cleavable linkage;

D is a detection moiety; and

M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

4. The method of claim 3 wherein D is a fluorescent label, a chromogenic label, or an electrochemical label.

5. The method of claim 4 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

6. The method of claim 5 wherein D is a fluorescein.

7. The method of claim 6 wherein said fluorescein is selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxyfluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, and 2′

,4′

,5′

,7′

-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein.

8. The method of claim 3 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.

9. The method in accordance with claims 1, 2, 3, 4, 5, 6, 7, or 8 wherein said one or more binding compounds are a plurality of binding compounds in the range of from 2 to 50.

10. The method of claim 9 wherein each of said binding compounds is an antibody binding composition.

11. The method of claim 9 wherein said one or more physical characteristics is electrophoretic mobility.

12. The method of claim 11 wherein said step of separating and identifying include forming an electropherogram in which each of said released molecular tags forms a distinct peak.

13. A composition for determining the presence or absence of one or more membrane-associated analytes, the composition comprising:

a sensitizer-treated membrane containing one or more membrane-associated analytes; and

one or more binding compounds, such that there is at least one binding compound specific for each of the one or more membrane-associated analytes, each binding compound having one or more molecular tags, each molecular tag being attached by a cleavable linkage, and the molecular tags of each binding compound being distinguishable from those of every other binding compound by one or more physical and/or optical characteristics;

wherein at least one binding compound of the plurality is specifically bound to at least one membrane-associated analyte.

14. The composition of claim 13 wherein said sensitizer-treated membrane comprises a cell surface membrane having photosensitizers attached, the photosensitizers each having a lipophilic moiety for anchoring the photosensitizers in the cell surface membrane.

15. The composition of claim 13 wherein said one or more membrane-associated analytes are selected from the group consisting of GPCRs.

16. The composition of claim 13 wherein said one or more binding compounds each comprises an antibody binding composition.

17. The composition of claim 16 wherein said antibody binding composition is a monoclonal antibody or a polyclonal antibody composition.

18. The composition according to claims 13, 14, 15, 16, or 17 wherein said one or more binding compounds is a plurality of said binding compounds in the range of from 2 to 50 and wherein said one or more physical characteristics is electrophoretic mobility.

19. The composition of claim 18 wherein said molecular tags released from different said binding compounds form distinct peaks in an electropherogram.

20. A kit for determining the presence or absence of one or more membrane-associated analytes, the kit comprising:

a lipophilic photosensitizer for generating a photosensitizer-treated membrane having one or more membrane-associated analytes; and

one or more binding compounds, such that there is at least one binding compound specific for each of the one or more membrane-associated analytes, each binding compound having one or more molecular tags, each molecular tag being attached by a cleavable linkage, and the molecular tags of each binding compound being distinguishable from those of every other binding compound by one or more physical and/or optical characteristics.