Methods and compositions for risk stratification

• US 7,393,656 B2
• Filed: 07/21/2004
• Issued: 07/01/2008
• 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: Nolan, Garry P., Perez, Omar D., Irish, Jonathan M.
• Primary Examiner(s): GABEL, GAILENE
• Application Number:
  US10/898,734
• Publication Number:
  US 20050112700A1
• Time in Patent Office: 1,441 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
• US Class Current: 435/7.21
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/5047 Cells of the immune system
• 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 33/537 with separation of immune complex from unbound antigen or antibody
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/56966 Animal cells
• 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 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]
• CPC Class Codes: Y10T 436/13 Tracers or tags
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The present invention provides an approach for the simultaneous determination of the activation states of a plurality of proteins in single cells. This approach permits the rapid detection of heterogeneity in a complex cell population based on activation states, and the identification of cellular subsets that exhibit correlated changes in activation within the cell population. Moreover, this approach allows the correlation of cellular activities or properties. In addition, the use of potentiators of cellular activation allows for characterization of such pathways and cell populations.

Claims

1. A method of determining a signaling phenotype of a cell population, said method comprising:

contacting said cell population with at least two distinct potentiators in separate cultures;

determining signaling node states of a plurality of signaling nodes in one or more cells from each of said separate cultures, wherein at least two of said plurality of signaling nodes belong to the same signaling pathway comprising;

permeabilizing said one or more cells from each of said separate cultures;

contacting said permeabilized cells from each of said separate cultures with at least one detectible state-specific binding element for each of said plurality of signaling nodes, wherein said detectible state-specific binding elements are distinguishably labeled; and

detecting the presence or absence of binding of each of said distinguishably labeled state-specific binding elements to said plurality of signaling nodes in each of said permeabilized cells from each of said separate cultures;

creating a response panel for said cell population comprising said determined signaling node states; and

determining a signaling phenotype of said cell population based on said response panel.

2. The method according to claim 1, wherein said at least two distinct potentiators are selected from the group consisting of cytokines, interleukins, hormones, bioactive agents, receptor ligands, cells, and therapeutic agents.

3. The method according to claim 1, wherein each of said plurality of signaling nodes is a protein.

4. The method according to claim 3, wherein said protein is an intracellular protein.

5. The method according to claim 3, wherein said protein is an integral membrane protein.

6. The method according to claim 3, wherein said signaling node state being determined is a post-translational state.

7. The method according to claim 6, wherein said post-translational state is a covalent modification.

8. The method according to claim 7, wherein said covalent modification is selected from the group consisting of phosphorylation, prenylation, glycosylation, acetylation, cysteinylation, nitrosylation, methylation, ubiquitination, and sulfation.

9. The method according to claim 1, wherein each of said distinguishably labeled state-specific binding elements is a protein.

10. The method according to claim 9, wherein said protein is an antibody.

11. The method according to claim 10, wherein said antibody is selected from the group consisting of:

anti-PI3K, anti-PIP2, anti-PIP3, anti-AKT-phospho-Ser473 (monoclonal antibody 4E2), anti-phospho AKT (Thr308), anti-p44/42 MAPK (anti-phospho-Thr202/Tyr204, anti-phospho Thr185/Tyr187), anti-phospho TYK2 (Tyr1054/1055), anti-phospho p38 MAPK (Thr180/Tyr182;

monoclonal antibody 28B10), anti-phospho JNK/SAPK (Thr183/Tyr185;

monoclonal antibody G9), anti-phospho-tyrosine (P-tyr-100), anti-phospho-threonine, anti-phospho-PKC-PAN substrate antibody, anti-phospho-PKA-substrate, anti-phospho PYK2 (Tyr402), anti-phospho PKC (phospho Thr638), anti-phospho-Syk (Tyr525/526), anti-phospho Raf1 (Ser259), anti-phospho-stat1 (Tyr701), anti-phospho-Stat3 (Tyr705), anti-phospho-Stat5 (Tyr694), anti-phospho-Stat6 (Tyr641), anti-phospho-p38 (Thr180/Tyr182), and anti-phospho-Erk1/2 (Thr202/Tyr204).

12. The method according to claim 1, wherein one or more of said distinguishably labeled state-specific binding elements is directly labeled with a detectable label.

13. The method according to claim 12, wherein said detectable label is selected from the group consisting of:

radioisotopes, heavy isotopes, fluorescers, FRET labels, enzymes, particles, and chemiluminescers.

14. The method according to claim 1, wherein said detecting step is performed with a detector selected from the group consisting of:

flow cytometer;

microscope having multiple channels of fluorescence;

a plate reader configured to detect fluorescence, ultraviolet and/or visible light, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and/or intensity redistribution;

charge-coupled device (CCD) camera configured to capture and transform data and images into quantifiable formats;

high performance liquid chromatography (HPLC); and

mass spectrometer.

15. The method according to claim 1, wherein for each of said separate cultures, said signaling node states for said plurality of signaling nodes in said one or more cells is determined simultaneously using multiparameter flow cytometry.

16. The method according to claim 1, wherein for each of said separate cultures, said signaling node states for said plurality of signaling nodes in said one or more cells is determined sequentially.

17. The method according to claim 1, wherein said determining step further comprises contacting said cell population with a detectible cell-specific binding element that binds to a subset of cells of interest in said cell population.

18. The method according to claim 17, wherein a signaling phenotype of said subset of cells of interest is determined.

19. The method according to claim 1, wherein one or more of said distinguishably labeled state-specific binding elements is indirectly labeled with a detectible secondary binding moiety.

20. The method according to claim 19, wherein said secondary binding moiety is selected from the group consisting of:

antibody, biotin, avidin, streptavidin, member of a receptor/ligand pair, member of a carbohydrate/binding partner pair, and member of a nucleic acid/nucleic acid binding protein pair.

21. The method according to claim 1, wherein said detecting is quantitative.

22. The method according to claim 1, wherein said detecting is relative to a control value.

23. The method according to claim 22, wherein said control value is the detected binding of said distinguishably labeled state-specific binding elements to said plurality of signaling nodes in cells from said cell population cultured in the absence of a potentiator.

24. The method according to claim 23, wherein said control value is included in said response panel.