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

• US 7,563,584 B2
• Filed: 07/10/2002
• Issued: 07/21/2009
• Est. Priority Date: 07/10/2001
• Status: Expired due to Fees

• 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: Perez, Omar D., Nolan, Garry P.
• Primary Examiner(s): GABEL, GAILENE
• Application Number:
  US10/193,462
• Publication Number:
  US 20060073474A1
• Time in Patent Office: 2,568 Days
• Field of Search: 435/2, 435/4, 435/6, 435/7.1, 435/7.21, 435/7.23, 435/7.24, 435/7.4, 435/7.92, 435/8, 435/18, 435/23, 435/40.5, 435/375, 435/973, 436/503, 436/519, 436/538, 436/546, 436/10, 436/17, 436/37, 436/56, 436/63, 436/64, 436/172, 436/174, 422/68.1, 422/82.05, 422/73
• US Class Current: 435/7.2
• 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, wherein said first and second activatable proteins are distinct proteins that each have at least an activated isoform, and a non-activated isoform;

b) permeabilizing said population of cells;

c) contacting said permeabilized population of cells with at least two distinguishably labeled activation state-specific antibodies,

wherein a first of said at least two distinguishably labeled activation state-specific antibodies is specific for said activated isoform of said first activatable protein; and

a second of said at least two distinguishably labeled activation state-specific antibodies is specific for said activated isoform of said second activatable protein; and

d) using flow cytometry to detect binding of said first and said second distinguishably labeled activation state-specific antibodies to their corresponding activated isoform of said first and second activatable proteins in single cells of said population of cells, wherein said binding of said first distinguishably labeled activation state-specific antibody is indicative of the activation state of said first activatable protein, and said binding of said second distinguishably labeled activation state-specific antibody is indicative of the 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 activated isoform of said first kinase is a first phosphorylated kinase, and said activated isoform of said second kinase is a second phosphorylated kinase.

6. The method according to claim 5, wherein said first distinguishably labeled activation state-specific antibody is specific for said first phosphorylated kinase, and said second distinguishably labeled activation state-specific antibody is specific for said second phosphorylated 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 activated isoform of said first caspase is a cleaved product of a first pro-caspase, and said activated isoform of said second caspase is a cleaved product of a second pro-caspase.

9. The method according to claim 8, wherein said at least two distinguishably labeled activation state-specific antibodies comprise a first distinguishably labeled activation state-specific antibody that is specific for said activated isoform of said first caspase, and a second distinguishably labeled activation state-specific antibody that is specific for said activated isoform of said second caspase.

10. The method according to any one of claims 1 to 9, wherein step a) further comprises contacting said population of cells with an agent that induces the activation of at least said first activatable protein.

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

12. The method according to any one of claims 1 to 9, 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.

13. The method according to claim 12, wherein said first distinguishably labeled activation state-specific antibody comprises a first fluorescent label, wherein said second distinguishably labeled activation state-specific antibody comprises a second fluorescent label, and wherein said sorting is by fluorescent activated cell sorting (FACS).

14. The method according to claim 12, wherein said first distinguishably labeled activation state-specific antibody comprises a first FRET label, wherein said second distinguishably labeled activation state-specific antibody comprises a second FRET label, and wherein said sorting is by fluorescent activated cell sorting (FACS).

15. The method according to claim 12, wherein said first distinguishably labeled activation state-specific antibody comprises a FRET label, wherein said second distinguishably labeled activation state-specific antibody comprises a fluorescent label, and wherein said sorting is by fluorescent activated cell sorting (FACS).

16. The method according to any one of claims 1 to 9, wherein step a) further comprises fixing said cells.

17. The method according to any one of claims 1 to 9, wherein said cells are mammalian cells.

18. The method according to claim 1, wherein said first activatable protein is a protein within the MAPK, AKT, STAT, NFkB, PKC or WNT signaling pathways and said second activatable protein is a protein within the MAPK, AKT, STAT, NFkB, PKC or WNT signaling pathways.

19. The method according to claim 18, wherein said first activatable protein and second activatable protein are independently selected from the group consisting of AKT, PI3K, MAPK, p44/42 MAP kinase, TYK2, p38 MAP kinase, PKC, PKA, SAPK, ELK, JNK, cJun, RAS, Raf, MEK 1/2, MEK 3/6, MEK 4/7, ZAP-70, LAT, SRC, LCK, ERK 1/2, Rsk 1, PYK2, SYK, PDK1, GSK3, FKHR, AFX, PLCg, PLCy, NF-kB, FAK, CREB, α

IIIβ

3, Fc□

RI, BAD, p70S6K, STAT1, STAT2, STAT3, STAT5 and STAT6.

20. The method according to claim 19, wherein said first activatable protein and second activatable protein are independently selected from the group consisting of RAF, ERK 1/2, MEK 1/2, p38, JNK, AKT, PKA substrates, PKC, PLCγ

, STAT1, STAT2, STAT3, STAT5 and STAT6.

21. The method according to claim 1, 4, 5 or 6, wherein said first distinguishably labeled activation state-state specific antibody is selected from the group consisting of anti-phospho-AKT Ser473, anti-phospho-p44/42 MAP kinase (Thr202/Tyr204), anti-phospho-TYK2 (Tyr1054/1055), anti-phospho-p38 MAP kinase (Thr180/Tyr182), phospho-PKC-PAN substrate antibody, phospho-PKA-substrate antibody, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-phospho-tyrosine (P-tyr-100), anti-p44/42 MAPK, anti-phospho-MEK1/2 (Ser217/221), anti-phospho-p90RSK (Ser381), anti-p38 MAPK, anti-JNK/SAPK, anti-phospho-AKT-Thr308, anti-phospho-Raf1 (Ser259), anti-phosphoElk-1 (Ser383), anti-phospho-CREB (Ser133), anti-phosphoSEK1/MKK4 (Thr261), anti-phospho-Jun (Ser 63), anti-phosphoMKK3/MKK6 (Ser189/207), anti-AKT, anti-phospho FKHR, anti-FKHR, anti-phospho-Gsk3 alp21, anti-pAFX, anti-PARP, anti-BAD, anti-BADser112, anti-BADser136, anti-phospho-BADser155, anti-p27, anti-p21, anti-cFLIP, antiMYC, anti-p53, anti-NFKB, anti-Ikkα

, anti-Ikkβ

, anti-phospho-tyrosine, anti-phospho-threonine, anti-STAT1, anti-STAT2, anti-STAT3, anti-STAT5, anti-STAT6, anti-PIP2, and anti-PIP3.

22. The method according to claim 1, wherein the activated isoform of either the first or second activatable protein is created through protein cleavage.

23. The method according to claim 1, wherein said activated isoform of either the first or second activatable protein is created through a covalent addition, structural change, or conformational change.

24. The method according to claim 23 wherein said conformational change causes said activated isoform of either the first or second activatable protein to present at least one epitope that is not present in a non-activated isoform of either the first or second activatable protein.

25. The method according to claim 1 wherein the activated isoform of either the first or second activatable protein is a phosphorylated or a cleaved product of either the first or second activatable protein.

26. The method according to claim 1 wherein the activated isoform of either the first or second activatable protein is created through binding to a third protein or protein complex.

27. The method according to claim 1 wherein the activated isoform of either the first or second activatable protein is created through binding to a soluble small molecule entity.