Claims
- 1. A method for determining the relative intracellular conformational states of a protein, comprising:
contacting a cell with a first antibody, said first antibody specific for a first conformation of said protein, and a second antibody, said second antibody specific for at least one other conformation of said protein, said first and second antibodies being distinguishably labeled; detecting the binding of each of said antibodies concurrently by said cell; and determining the relative binding thereof.
- 2. The method of claim 1, further comprising the antecedent step of fixing said cell.
- 3. The method of claim 1, further comprising the step, before detection, of permeabilizing said cell.
- 4. The method of claim 1, wherein said labels are fluorophores and said fluorophores are distinguishable by a laser cytometer.
- 5. The method of claim 4, wherein said laser cytometer is a flow cytometer, and said fluorophores are flow cytometrically distinguishable.
- 6. The method of claim 4, wherein at least one of said antibodies is conjugated directly to a fluorophore.
- 7. The method of claim 4, wherein both of said antibodies are conjugated directly to a fluorophore.
- 8. The method of any one of claims 4-7, wherein said fluorophores are selected from the group consisting of: FITC, PE, PerCP, APC, PE-CY5 tandem fluorophore and PerCP-CY5.5 tandem fluorophore.
- 9. The method of claim 4, wherein one of said fluorophores is FITC.
- 10. The method of claim 4, wherein one of said fluorophores is PE.
- 11. The method of claim 4, wherein one of said fluorophores is PerCP.
- 12. The method of claim 4, wherein one of said fluorophores is PE-CY5 tandem fluorophore.
- 13. The method of claim 4, wherein one of said fluorophores is PerCP-CY5.5 tandem fluorophore.
- 14. The method of claim 1, wherein said protein is pRB.
- 15. The method of claim 14, wherein said first antibody is specific for a conformation assumed by the hypophosphorylated form of pRB.
- 16. The method of claim 15, wherein said second antibody is specific for all functional conformations of pRB.
- 17. The method of any one of claims 14-16, wherein said cell is a human cell.
- 18. The method of claim 1 or claim 14, further comprising the step of contacting said cell with a fluorescent nucleic acid stain, and then detecting the binding to DNA of said stain concurrently with detecting the binding to said protein of said first and second antibody.
- 19. The method of claim 18, wherein said fluorescent nucleic acid stain is selected from the group consisting of DAPI, PI, Hoechst 33258, Hoechst 33342, ethidium bromide, ethidium homodimer-1, hexidium iodide, SYTOX Green, acridine orange, 7-aminoactinomycin D, or dimeric cyanine dye.
- 20. The method of claim 19, wherein said fluorescent nucleic acid stain is selected from the group consisting of DAPI and PI.
- 21. The method of claim 1, further comprising contacting said cell with a third antibody, said third antibody specific for a second protein and distinguishable from each of said first and second antibodies, and then detecting the concurrent binding of each of said antibodies to said cell.
- 22. The method of claim 14, further comprising contacting said cell with a third antibody, said third antibody specific for a second protein and distinguishable from each of said first and second antibodies, and then detecting the concurrent binding of each of said antibodies to said cell.
- 23. The method of claim 22, wherein said second protein is a cyclin.
- 24. The method of claim 23, wherein said cyclin is selected from the group consisting of: cyclin A, cyclin B1, cyclin C, cyclin D1, cyclin D2, cyclin D3, cyclin E, cyclin F, cyclin G, cyclin H, cyclin I, and cyclin K.
- 25. The method of claim 24, wherein said cyclin is cyclin D3.
- 26. The method of claim 22, wherein said second protein is a cyclin dependent kinase.
- 27. The method of claim 26, wherein said cyclin dependent kinase is selected from the group consisting of: Cdk1, Cdk2, Cdk3, Cdk4, Cdk5, Cdk6, Cdk7 and Cdk8.
- 28. The method of claim 27, wherein said cyclin dependent kinase is Cdk4.
- 29. The method of claim 27, wherein said cyclin dependent kinase is Cdk6.
- 30. The method of claim 22, wherein said second protein is a cyclin dependent kinase inhibitor.
- 31. The method of claim 30, wherein said cyclin dependent kinase inhibitor is selected from the group consisting of: p15INK4b, p16INK4a, p18INK4, p19INK4c, p21WAF1/CIP1, and p27Kip1.
- 32. The method of claim 31, wherein said cyclin dependent kinase inhibitor is p16INK4a.
- 33. A method of assaying compounds for antiproliferative activity, comprising:
contacting a sample of proliferating cells with said compound in vitro; contacting said cells with a first antibody specific for a conformation assumed by the hypophosphorylated form of pRB and a second antibody, said second antibody specific for at least one other conformation of pRB and flow cytometrically distinguishable from said first antibody; flow cytometrically detecting the binding of each of said antibodies concurrently by said cell; wherein an increased ratio of hypophosphorylated pRB to total pRB indicates antiproliferative activity of said compound.
- 34. A method of assessing the in vivo antiproliferative effect of a compound, comprising:
contacting a sample of cells obtained from a subject after the in vivo administration of said compound with a first antibody, said first antibody specific for a conformation assumed by the hypophosphorylated form of pRB and a second antibody, said second antibody specific for at least one other conformation of pRB and flow cytometrically distinguishable from said first antibody; and flow cytometrically detecting the binding of each of said antibodies concurrently by said cell; wherein an increased ratio of hypophosphorylated pRB to total pRB, as compared to the ratio obtained from cells identically assayed that were obtained prior to administration of said agent, indicative of in vivo antiproliferative effect.
- 35. A method of assessing the proliferative potential of a heterogeneous population of cells, comprising:
contacting said cells with a first antibody specific for a conformation assumed by the hypophosphorylated form of pRB and a second antibody, said second antibody specific for at least one other conformation of pRB and flow cytometrically distinguishable from said first antibody; flow cytometrically detecting the binding of each of said antibodies concurrently by said cell; wherein cells with a decreased ratio of hypophosphorylated pRB to total pRB are determined to have increased proliferative potential.
- 36. A kit for detecting the phosphorylation status of pRB in individual cells, comprising:
a first antibody, said first antibody specific for a first phosphorylation state of pRB; and a second antibody, said second antibody specific for at least one other phosphorylation state of pRB; wherein said first and second antibodies are distinguishable from one another.
- 37. The kit of claim 36, wherein each of said antibodies is conjugated to a fluorophore, and said fluorophores are flow cytometrically distinguishable from one another.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional application No. 60/075,908, filed Feb. 25, 1998, the disclosure of which is incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This work was supported in part by Grant CA R01 28704 from the National Cancer Institute, National Institutes of Health. The government has certain rights in this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60075908 |
Feb 1998 |
US |