Methods and compositions for detecting the activation state of multiple proteins in single cells

• US 20060073474A1
• Filed: 07/10/2002
• Published: 04/06/2006
• Est. Priority Date: 07/10/2001
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Nolan, Garry P., Perez, Omar D.
• Granted Patent: US 7,563,584 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 2333/91205 Phosphotransferases in general
• CPC Class Codes: G01N 2333/96466 Cysteine endopeptidases (3.4.22)
• CPC Class Codes: G01N 2405/00 Assays, e.g. immunoassays or enzyme assays, involving lipids lipopolysaccharides G01N2400/50
• CPC Class Codes: G01N 2440/14 phosphorylation
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The invention provides methods and compositions for simultaneously detecting the activation state of a plurality of proteins in single cells using flow cytometry. The invention further provides methods and compositions of screening for bioactive agents capable of coordinately modulating the activity of a plurality of proteins in single cells. The methods and compositions can be used to determine the protein activation profile of a cell for predicting or diagnosing a disease state, and for monitoring treatment of a disease state.

Claims

1. A method of detecting the activation state of at least a first and a second activatable protein in single cells, said method comprising the steps of:

a) providing a population of cells comprising said first and said second activatable proteins;

b) contacting said population of cells with a plurality of activation state-specific antibodies,

wherein said plurality of activation state-specific antibodies comprise;

i) at least one first activation state-specific antibody that is capable of binding to a corresponding isoform of said first activable protein in said population of cells; and

ii) at least one second activation state-specific antibody that is capable of binding to a corresponding isoform of said second activatable protein in said population of cells; and

c) using flow cytometry to detect said binding of said first and second activation state-specific antibodies in single cells of said population of cells, wherein said binding of said first activation state-specific antibody is indicative of a specific activation state of said first activatable protein, and said binding of said second activation state-specific antibody is indicative of a specific activation state of said second activatable protein.

2. The method according to claim 1, wherein said first activatable protein is a kinase.

3. The method according to claim 1, wherein said first activatable protein is a caspase.

4. The method according to claim 1, wherein said first activatable protein is a first kinase and said second activatable protein is a second kinase.

5. The method according to claim 4, wherein said isoform of said first kinase is a first phosphorylated kinase, and said isoform of said second kinase is a second phosphorylated kinase.

6. The method according to claim 5, wherein said first activation site-specific antibody binds to said first phosphorylated kinase, and said second activation site-specific antibody binds said second phorphorylated kinase.

7. The method according to claim 1, wherein said first activatable protein is a first caspase and said second activatable protein is a second caspase.

8. The method according to claim 7, wherein said isoform of said first caspase is a cleaved product of a first pro-caspase, and said isoform of said second caspase is a cleaved product of a second pro-caspase.

9. The method according to claim 8, wherein said plurality of activation site-specific antibodies comprise a first activation site-specific antibody that binds to said isoform of said first caspase, and a second activation site-specific antibody that binds to said isoform of said second caspase.

23. The method according to any of claims 1-9 and 12-14, wherein said plurality of activation state-specific antibodies comprise at least one antibody selected from a group of antibodies consisting of:

anti-AKT-phospho-Ser473, anti-AKT phospho-Thr308, anti-p44/42 MAPK phospho-Thr202/Tyr204, anti-TYK2 phospho-Tyr1054/1055, anti-p38 MAPK phospho-Thr180/Tyr182, anti-JNK/SAPK phospho-Thr183/Tyr185, anti-phospho-tyrosine, anti-phospho-threonine, anti-PIP2, and anti-PIP3.

24. The method according to any of claims 1-22, wherein step a) further comprises contacting said population of cells with an agent that induces the activation of at least said first activatable protein.

25. The method according to any of claims 1-9, 11, and 13-22, wherein step a) further comprises contacting said population of cells with an agent that induces the activation of at least one of said first and said second activatable proteins.

27. The method according to any of claims 1-9, 11, and 13-22, wherein said method further comprises sorting said single cells based on said activation state of said first activatable protein, and said activation state of said second activatable protein.

28. The method according to any of claims 1-9, and 14-22, wherein said first activation state-specific antibody comprises a first label, wherein said second activation state-specific antibody comprises a second fluorescent label and, wherein said sorting is by fluorescent activated cell sorting (FACS).

29. The method according to any of claims 1-9 and 14-22, wherein said first activation state-specific antibody comprises a first FRET label, wherein said second activation state-specific antibody comprises a second FRET label and, wherein said sorting is by fluorescent activated cell sorting (FACS).

30. The method according to any of claims 1-9 and 14-22, wherein said first activation state-specific antibody comprises a FRET label, wherein said second activation state-specific antibody comprises a label and, wherein said sorting is by fluorescent activated cell sorting (FACS).

31. The method according to any of claim 1-22, wherein step a) further comprises fixing said cells.

32. The method according to any of claims 1-22, wherein said cells are mammalian cells.

10. A method of detecting the activation state of at least a first activatable protein in single cells, said method comprising the steps of:

a) providing a population of cells comprising at least said first activatable protein;

b) contacting said population of cells with a plurality of substrates;

wherein said plurality of substrates comprise at least a first substrate that is capable of being modified by a corresponding isoform of said first activatable protein in said population of cells; and

c) using flow cytometry to detect the modification of said first substrate in single cells of said population of cells, wherein said modification is indicative of a specific activation state of said first activatable protein.

11. The method according to claim 10, wherein said population of cells further comprises a second activatable protein, wherein said plurality of substrates further comprise a second substrate that is capable of being modified by a corresponding isoform of said second activable protein in said population of cells and, wherein step c) further comprises using said flow cytometry to detect the modification of said second substrate in single cells of said population of cells, wherein said modification of said second substrate is indicative of a specific activation state of said second activatable protein.

12. A method of detecting a protein activation state profile of single cells based on the activation state of at least a first activatable protein in said cells, said method comprising the steps of:

a) providing a population of cells comprising at least said first activatable protein;

b) contacting said population of cells with a plurality of substrates;

wherein said plurality of substrates comprise at least a first substrate that is capable of being modified by a corresponding isoform of said first activatable protein in said population of cells;

c) contacting said population of cells with a plurality of activation state-specific antibodies,

wherein said activation state-specific antibodies comprise at least one first activation state-specific antibody that is capable of binding to a corresponding isoform of said first activatable protein in said population of cells; and

d) using flow cytometry to simultaneously detect;

i) said binding of said first activation state-specific antibody in single cells of said population of cells,

wherein said binding of said first activation state-specific antibody is indicative of a specific activation, state of said first activatable protein; and

ii) the modification of said first substrate said single cells,

wherein said modification is indicative of said specific activation state of said first activatable protein.

13. The method according to claim 12, wherein said population of cells further comprises a second activatable protein, wherein said plurality of substrates further comprises a second substrate that is capable of being modified by a corresponding isoform of said second activatable protein in said population of cells, and step d) further comprises using said flow cytometry to detect the modification of said second substrate in said single cells, wherein said modification is indicative of a specific activation state of said said second activatable protein.

14. The method according to claim 13, wherein said plurality of activation state-specific antibodies further comprises a second activation state-specific antibody that is capable of binding to a corresponding isoform of said second activatable protein in said population of cells, and wherein step c) further comprises using said flow cytometry to detect said binding of said second activation state-specific antibody said single cells of said population of cells, wherein said binding of said second activation state-specific antibody is indicative of said specific activation state of said second activatable protein.

15. A method of screening for a bioactive agent capable of modulating the activity of at least a first activatable protein in cells, said method comprising:

a) providing a population of cells, each of said cells comprising at least said a first activable protein, a second activatable protein, and a third activatable protein,

wherein said first activable protein can activate said second activatable protein thereby forming a specific isoform of said second activable protein (isoform-2), and

wherein said second activable protein can activate said third activatable protein thereby forming a specific isoform of said third activatable protein (isoform-3);

b) contacting said cells with a second activation state-specific antibody, a third activation state-specific antibody, and a candidate bioactive agent,

wherein said second activation state-specific antibody is capable of binding to said isoform-2, and wherein said third activation state-specific antibody is capable of binding to said isoform-3;

c) using fluorescent activated cell sorting (FACS) to sort single cells of said population of cells based on the presence of said isoform-2 and said isoform-3; and

d) determining the ratio of said isoform-2 to said isoform-3 in said single cells in the presence and absence of said candidate bioactive agent,

wherein a difference in the ratio of said isoform-2 to said isoform-3 in the presence and absence of said candidate bioactive agent is indicative of the ability of said candidate bioactive agent to modify said activity of said first activable protein.

16. The method according to claim 15, wherein said first activatable protein is activated by an activating agent.

17. The method according to claim 15, wherein said first activatable protein is a caspase.

18. The method according to claim 15, wherein said first activatable protein is a kinase.

19. The method according to claim 18, wherein said kinase is PI3K, wherein said second activatable protein is PIP2, wherein said third activatable protein is PIP3, wherein said isoform-2 is PIP 4,5 bisphosphate and, wherein said isoform-3 is PIP 3,4,5.

20. The method according to claim 19, wherein said PI3K is activated by a growth factor or by activation of a cell surface receptor.

21. The method according to claim 20, wherein said first activatable protein is ICAM-2, wherein said activity is apoptosis, second activatable protein is PIP2, wherein said third activatable protein is PIP3, wherein said isoform-2 is PIP 4,5 bisphosphate and, wherein said isoform-3 is PIP 3,4,5.

22. The method according to claim 21, wherein step a) further comprises clustering said ICAM-2 with ICAM-3.

26. The method according to any of claims 15-22, wherein step a) further comprises contacting said population of cells with an agent that induces the activation of at least one of said first, said second, and said third activatable proteins.