Detecting receptor oligomerization
View Patent ↗Methods are provided for detecting formation of oligomeric complexes of molecules on the surface of cell membranes. These methods employ pairs of tagged probes and cleaving probes, each of which binds specificly to a cell surface molecule. The tagged probe includes a molecular tag that is linked to a first binding compound through a cleavable linkage, and the cleaving probe includes a second binding agent and a cleavage-inducing moiety that can cleave the linkage when within a defined proximity thereto. Binding of the two probes to cell surface molecules that have formed an oligomeric complex results in release of the molecular tag from the binding compound, providing a measure of formation of the complex.
1. A method for detecting a dimer in a sample, the method comprising: mixing (i) a sample, which contains a dimer comprising a first membrane-associated analyte and a second membrane-associated analyte; (ii) a cleaving probe, which is capable of binding the first membrane-associated analyte and has a cleavage-inducing moiety with an effective proximity; and (iii) one or more binding compounds, at least one of which is capable of binding the second membrane-associated analyte, and each binding compound has one or more molecular tags attached thereto by a cleavable linkage, wherein cleavage of the cleavable linkage(s) within the effective proximity of the cleaving-inducing moiety of the cleaving probe releases the molecular tag(s);
wherein detecting the released molecular tag(s) detects the dimer.
2. The method of claim 1 , wherein the first membrane-associated analyte and the second membrane associated analyte are both cell surface receptors.
3. The method of claim 2 , wherein the first membrane-associated analyte and the second membrane associated analyte are the same receptor type.
4. The method of claim 2 , wherein the first membrane-associated analyte and the second membrane associated analyte are different receptor types.
5. The method of claim 2 , wherein the cell surface receptors are selected from the group consisting of epidermal growth factor receptors and G-protein coupled receptors.
6. The method of claim 5 , wherein the cell surface receptors are selected from the group consisting of Her1, Her2, Her3 and Her4.
7. The method of claim 6 , wherein the dimer is selected from the group consisting of a Her1—Her1 homodimer, a Her1–Her2 heterodimer, a Her1–Her3 heterodimer and a Her2–Her3 heterodimer.
8. The method of claim 1 , wherein the cleaving probe comprises an antibody binding composition.
9. The method of claim 8 , wherein the antibody binding composition comprises a monoclonal antibody.
10. The method of claim 8 , wherein the antibody binding composition binds an antigenic determinant of the first membrane-associated analyte.
11. The method of claim 1 , wherein the cleavage-inducing moiety of the cleaving probe is a sensitizer.
12. The method of claim 11 , further comprising inducing the sensitizer to generate an active species that cleaves the cleavable linkage(s) of the binding compound(s) within the effective proximity.
13. The method of claim 11 , wherein the sensitizer is a photosensitizer.
14. The method of claim 13 , further comprising illuminating the photosensitizer to generate an active species that cleaves the cleavable linkage(s) of the binding compound(s) within the effective proximity.
15. The method of claim 12 or 14 , wherein the active species is singlet oxygen.
16. The method of claim 15 , wherein the cleavable linkage of the binding compound is an oxidation-labile linkage.
17. The method of claim 16 , wherein the cleavable linkage of the binding compound is selected from the group consisting of a thioether, an olefin, a thiazole, and an oxazole.
18. The method of claim 1 , wherein the one or more molecular tags have a separation characteristic.
19. The method of claim 18 , further comprising separating the released molecular tags.
20. The method of claim 19 , wherein the separation characteristic of said one or more molecular tags is electrophoretic mobility and the step of separating comprises electrophoretically separating the released molecular tags in a separation buffer.
21. The method of claim 1 , wherein the one or more molecular tags are capable of generating an electrochemical, fluorescent or chromogenic signal.
22. The method of claim 21 , wherein the one or more molecular tags are capable of generating a fluorescent signal.
23. The method of claim 1 , wherein the one or more binding compounds comprise an antibody binding composition.
24. The method of claim 23 , wherein the antibody binding composition comprises a monoclonal antibody.
25. The method of claim 1 , wherein at least two binding compounds are mixed.
26. The method of claim 25 , wherein the at least two binding compounds comprise at least two different binding compounds which bind different antigenic determinants of the first or second membrane-associated analyte.
27. The method of claim 25 , wherein at least one of the binding compounds binds a phosphorylation site of the first or second membrane-associated analyte.
28. The method of claim 25 , wherein at least two of the molecular tags attached to the at least two binding compounds have different separation characteristics.
29. The method of claim 28 , wherein the method further comprises separating the released molecular tags from the at least two binding compounds.
30. The method of claim 29 , wherein the molecular tags from the at least two binding compounds have different electrophoretic mobility and are separated electrophoretically.
31. The method of claim 30 , further comprising incubating the cleaving probe, the at least two binding compounds and the sample in a binding buffer, and exchanging the binding buffer with a separation buffer, wherein said steps of incubating and exchanging are performed prior to said step of separating.