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Kits for multiparametric phospho analysis

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20120276558 patent thumbnailZoom

Kits for multiparametric phospho analysis

As disclosed herein, the present invention provides for kits and a composition for diagnosis, prognosis, drug discovery, drug development, and patient stratification. The kits can comprise a plurality of binding elements for cell surface markers, and a plurality of binding elements for state-specific intracellular markers. The kits can further comprise a plurality of modulators directed for the particular cell function or signaling pathways. The kits can further include fixatives, permeabilizing agent, buffers, containers, instructions, and software for data analysis/compilation.

Browse recent Nodality, Inc. patents - South San Francisco, CA, US
Inventors: David Soper, David Rosen, Todd Covey, Ying-Wen Huang, Wenday Fantl, Ralph Lin
USPTO Applicaton #: #20120276558 - Class: 435 724 (USPTO) - 11/01/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay >Involving A Micro-organism Or Cell Membrane Bound Antigen Or Cell Membrane Bound Receptor Or Cell Membrane Bound Antibody Or Microbial Lysate >Animal Cell >Leukocyte (e.g., Lymphocyte, Granulocyte, Monocyte, Etc.)

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The Patent Description & Claims data below is from USPTO Patent Application 20120276558, Kits for multiparametric phospho analysis.

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This application is a continuation application which claims the benefit of U.S. application Ser. No. 12/730,170, filed Mar. 23, 2010; which claims the priority of U.S. Provisional App. Ser. No. 61/162,673, filed on Mar. 23, 2009 and U.S. Provisional App. Ser. 61/245,000, filed on Sep. 23, 2009 the disclosures of which are hereby incorporated by reference their entirety for all purposes.


Multiparametric analyses of cells provide an approach for the simultaneous determination of the activation states of a plurality of cellular components. The activation status of the plurality of cellular components can be measured after exposure of cells to extracellular modulators and in so doing allows the signaling capacity of signaling networks to be determined when compared to the activation status of those networks in the absence of such modulators. The induced activation status of a protein rather than the frequently measured basal phosphorylation state of a protein has been shown in several studies to be more informative, as it takes into account (and reveals) signaling deregulation that is the consequence of numerous cytogenetic, epigenetic and molecular changes characteristic of transformed cells. For example, multiparameter flow cytometry at the single cell level can measure the activation status of multiple intracellular signaling proteins and can assign activation states of these molecules to the varied cell sub-sets within complex primary cell populations.

However, usually multiparametric analyses of cells, e.g., multiparametric flow cytometry, require the use of multiple reagents at precise concentrations to produce robust and reproducible results. Since these data can be used as tools to inform clinical decisions, as well as therapeutic development, it would be beneficial to provide kits comprised of components relevant to a particular application with accompanying relevant usage information.

Protein phosphorylation is a critical post translational process in controlling many cell functions such as migration, apoptosis, proliferation and differentiation. Site specific phosphorylation of proteins can be detected, for example, by incubating cells with fluorochrome-conjugated phospho-specific antibodies using flow cytometry. However, only reagents whose parameters (including but not limited to, concentration, kinetics, fluorochrome to protein ratio) have been optimized can be used to generate robust and reproducible data that can be applied to a specific purpose. Kits comprising two or more reagents recognizing intracellular markers and/or extracellular markers along with an appropriate modulator or modulators to evoke a signaling response appropriate for the signal transduction pathway, specific cell type, disease state, or cellular function can save the end user from the tedious and often costly process of selecting, optimizing and standardizing reagents thereby providing the user with a more streamlined and cost-saving approach for profiling cellular networks in single cells.

It is therefore an objective of the present invention to provide kits that meet such demands.



The present invention involves the preparation of kits to be utilized in multi-parametric analyses (e.g. flow cytometry) on cell populations for the identification of the activation states of cellular signaling molecules (called nodes) in cells. Profiles of node states in cell populations are useful for diagnosis, prognosis, drug discovery, drug development, patient stratification (for example, who will and who will not respond to a drug) and other applications. Methods for determining cell populations and activation states have been disclosed in U.S. Pat. Nos. 7,381,535, 7,393,656, 7,563,584 and U.S. Ser. No. 61/120,320, which are hereby incorporated by reference in their entirety.

One embodiment of the present invention is a kit comprising a combination of binding element cell surface markers and state-specific intracellular markers. The kit can also comprise one or more modulators, therapeutic agents, fixatives, buffers, physical devices and software as described below.

In some embodiments, kits can be directed toward applications such as prediction of a response to a therapeutic agent, diagnosis and prognosis of various diseases or conditions, profiling signaling in specific cell types, analyzing the functional effects of genetic mutations, etc.

In some embodiments, kits can be prepared based on cell types of interest. For example, a kit can have a panel of antibodies that recognize extracellular markers specific to T cells, B cells, myeloid cells, stromal cells, neuronal cells or epithelial cells.

In some embodiments, the cell-type-specific kits can be supplemented with modulators for different signaling pathways. For example, the kit can include one or more cytokines and or growth factors that activate pathways including, but not limited to, JAK/STAT, PI3K/Akt, Ras/Raf/Erk, phosphatase signaling, metabolism, apoptosis, DNA damage response or transcriptional activation pathways. The kits can further comprise control cells, compounds and/or protocols.

In some embodiments, the invention involves kits for analyzing the effect of a compound on a cancer cell, comprising one or more binding elements that recognize particular surface markers expressed by cells in certain disease states. The kits can also comprise a compound used for treating a condition such as cancer. The kits can also comprise binding elements recognizing the activated state of signaling elements that can be activated in response to a compound, including but not limited to phosphorylated, acetylated, methylated or cleaved proteins.

In some other embodiments of the present invention, kits can additionally comprise consumable hardware, such as plates for holding the reagents or performing reactions, pipette tips, and software or files required to carry out the experiment. In some embodiments, the kit can further comprise a software package for data analysis of cell signaling profiles, which can include reference profiles for comparison with a test profile. The kit can also include software to manage or perform the experiment, including the use of the reagents and protocols for conducting appropriate reactions.

In some other embodiments, kits of the present invention enable the detection of activatable elements by sensitive cellular assay methods, such as immunohistochemistry and flow cytometry, which are suitable for clinical applications in detection, prognosis, and screening of cells and tissues from patients who have a disease involving aberrant signaling networks, for example leukemia.


The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 illustrates some embodiments of apoptosis pathway kits, comprising various intracellular markers involved in intrinsic and extrinsic apoptosis pathways.

FIG. 2 illustrates some embodiments of apoptosis pathway kits, comprising various intracellular markers involved in intrinsic and extrinsic apoptosis pathways.


All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.



The present invention incorporates information disclosed in other applications and texts. The following patent and other publications are hereby incorporated by reference in their entireties: Haskell et al., Cancer Treatment, 5th Ed., W.B. Saunders Co. (2001); Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland Science (2002); Vogelstein and Kinzler, The Genetic Basis of Human Cancer, 2d Ed., McGraw Hill (2002); Michael, Biochemical Pathways, John Wiley & Sons (1999); Weinberg, The Biology of Cancer (2007); Janeway et al., Immunobiology, 7th Ed., Garland Science (2008); Leroith & Bondy, Growth Factors and Cytokines in Health and Disease, Vols. 1A and 1B: A Multi Volume Treatise, (JAI Pr, 1996). Patents and applications that are also incorporated by reference in their entirety include U.S. Pat. Nos. 7,381,535; 7,393,656; 7,563,584 and U.S. Pat. Ser. Nos. 10/193,462; 11/655,785; 11/655,789; 11/655,821; 11/338,957, 12/432,720; 12/229,476; 12/432,239; 12/460,029; 12/471,158; 61/216,825; 61/162,673; 61/157,900; 61/151,387; 61/104,666; 61/226,878; 61/218,718; 61/182,518; 61/170,348; 61/144,684; 61/113,823; 61/181,211; 61/162,598; 61/108,803; 61/182,638; 61/177,935; 61/155,373; 12/293,081; 61/186,619; 61/156,754; 61/106,462; 61/176,420; 12/538,643; 12/501,274; 61/079,537; 12/501,295; 61/146,276; and 61/144,955. Some commercial reagents, protocols, software and instruments that are useful in some embodiments of the present invention are available at the Becton Dickinson Website, and the Beckman Coulter website, Method of performing assays on multiparametric flow cytometry are described in e.g., Krutzik et al., High-content single-cell drug screening with phosphospecific flow cytometry, Nature Chemical Biology (2007) 4:132-142; Irish et al., FLt3 ligand Y591 duplication and Bcl-2 over expression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53, Neoplasia (2007) 109(6):2589-96; Irish et al. Mapping normal and cancer cell signaling networks: towards single-cell proteomics, Nature (2006) 6:146-155; Irish et al., Single cell profiling of potentiated phospho-protein networks in cancer cells, Cell (2004) 118(2):217-18; Schulz, K. R. et al., Single-cell phospho-protein analysis by flow cytometry, Curr Protoc Immunol (2007) 78:8 8.17.1-20; Krutzik, P. O. et al., Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry, J Immunol (2005) 175(4):2357-65; Krutzik, P. O. et al., Characterization of the murine immunological signaling network with phosphospecific flow cytometry, J Immunol (2005) 175(4):2366-73; Stelzer et al., Use of Multiparameter Flow Cytometry and Immunophenotyping for the Diagnosis and Classification of Acute Myeloid Leukemia (John Wiley & Sons, 2000); Krutzik, P. O. et al., Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events, Cytometry A. (2003) 55(2):61-70; Hanahan D. et al., The Hallmarks of Cancer, Cell (2000) 100(1):57-70; Krutzik et al, High content single cell drug screening with phospho-specific flow cytometry, Nature Chemical Biology (2008) 4(2):132-42. Experimental and process protocols and other helpful information can be found at http:/ The articles and other references cited below are also incorporated by reference in their entireties for all purposes.

The present invention relates to the processing of cells for analysis. More specifically, the present invention relates to kits comprising binding elements that can be used, e.g., in multi-parametric flow cytometry in order to determine the activation states of a plurality of proteins in single cells.

In one aspect, the present invention provides a kit comprising one or more binding elements for extracellular markers specifically targeted toward certain diseases, cell types and signaling pathways, which can be used, for example, to facilitate research. A kit of the invention can allow for analysis of relevant activatable elements in specific cell types that can provide the information necessary to make a diagnosis, prognosis, drug discovery, predict the response of disease to a therapeutic agent, and provide information relevant to drug development and patient stratification to a specific condition.

In another aspect, the present invention provides a kit comprising one or more binding elements that recognize one or more cell surface markers and one or more intracellular markers to enable rapid screening of the effects of modulators or therapeutic agents on evoked cell signaling.

In yet another aspect, the present invention provides a kit with one or more binding elements and one or more modulators to develop one or more network profiles, such as a network profile that can predict a response to a therapeutic agent or therapeutic regimen.

In some embodiments, the present invention relates to a kit and/or composition to be used in multiparametric flow cytometry on cell populations and activation states for diagnosis, prognosis, drug discovery, drug development, and patient stratification. A kit can comprise one or more binding elements for the cell surface marker of particular cell type or disease state. A kit can also comprise one or more binding elements that recognize intracellular markers of particular cellular pathway or function, modulators, labeling agent, fixatives, permeabilizing agents, etc. The kit generally can be used for determining the status of an activatable element. The kit might also be used for determining the status of a plurality of activatable elements.

A kit of the present invention can include components necessary to determine the activation state of a plurality of activatable elements from single cells, wherein each cell type is selected based on a target disease cellular state or other area of interest. For example, the target diseases can include, but are not limited to hematological diseases such as acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN); the target states and pathways can include, but are not limited to intrinsic apoptosis pathway, extrinsic apoptosis pathway, DNA damage-induced apoptosis pathway, ABL and BCR/ABL function in CML (chronic myelogenous leukemia), phosphatase function, calcium signaling, Protein Kinase C (PKC) function.

In one embodiment, a kit can be used to monitor and predict disease outcome. In another embodiment, a kit can be used in drug screening to determine whether a drug can be useful for treating a particular disease. In some other embodiments, a kit can also be used in the analysis of drug transport and/or drug metabolism, inflammation, autophagy, metabolism, cell proliferation, cell cycle, cell survival, siRNA function, or other functional characteristic.

A kit can also provide a panel of reagents for the analysis of a targeted therapeutic agent. For example, it could be used to study the effect of aJAK2 inhibitor on JAK/STAT pathway activity; PI3K inhibitor on PI3K/Akt or Ras/Raf/Erk pathway activity; Mek inhibitor on Ras/Raf/Erk pathway activity; mTor inhibitor on the TSC/mTor pathway; IK-Kinase inhibitor on NFkB pathway activity; kinase inhibitor acting on a pathway utilizing a tyrosine kinase, including but not limited to, epidermal growth factor receptor, Fibroblast growth factor, and Src family kinase signaling and nucleoside analogues and alkylating agents on the DNA damage response and apoptosis pathways.

A kit can include a composition for the detection of the activation of an element in a cell. A suitable cell includes cell types implicated in a wide variety of disease conditions, even in non-diseased states. Suitable cell types include, but are not limited to, cancer cells of all types including cancer stem cells, cardiomyocytes, dendritic cells, endothelial cells, epithelial cells, lymphocytes (T cell and B cell), mast cells, eosinophils, basophils, neutrophils, natural killer cells, erythrocytes, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as hematopoietic, neural, skin, and monocyte stem cells. Particularly preferred are primary disease state cells, such as primary cancer cells including circulating tumor cells (CTCs).

One embodiment of the present invention is a kit for classifying cells of a myeloid disorder based on the biology of a cell or group of cells derived from a patient with a myeloid malignancy such as AML, MDS, or MPN.

In some embodiments, the kit of the present invention can be directed towards a particular cell type. Specific examples include, but are not limited to, lymphocytes, myeloid cells, such as mature monocytes (CD45+, CD33+, CD11b+), myeloblasts (CD45+, CD34+, CD11b−), lymphoid subsets, such as T cell, B cell, and nucleated red blood cells (nRBCs).

In some embodiments, a kit of the present invention can be geared towards a particular sample, such as peripheral blood and bone marrow. In some preferred embodiments, the cells used in the present invention are populations of leukemic myeloid cells taken from the bone marrow of a leukemic patient or nucleated red blood cells taken from the bone marrow of a leukemic patient.

The terms “patient” or “individual” as used herein includes humans as well as other mammals.

Cell Surface Markers

Cell surface markers are molecules characteristic of the plasma membrane of a cell or in some cases of a specific cell type. The term “extracellular marker” and “cell surface marker”, and “cell surface antigen” and “phenotypic marker” as used herein, include antigens expressed in healthy and/or diseased cells and can be used interchangeably. In some embodiments, a kit of the present invention can comprise a combination of antibodies that recognize cell surface markers including but not limited to CD3, CD4, CD7, CD8, CD11b, CD11c, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD27, CD33, CD34, CD38, CD40, CD45, CD56, CD69, CD71, CD80, CD117, CD138, CD235a, CD235b, Ter119, GP-130, IgM, IgD, IgE, IgG, IgA, CCR5, CCR3, TLR2, TLR4, TLR9. CD3, also known as T3, is a member of the immunoglobulin (Ig) superfamily that plays a role in antigen recognition, signal transduction and T cell activation. It is found on all mature T lymphocytes, NK-T cells, and some thymocytes. CD4 is also a member of the Ig superfamily, which participates in cell-cell interactions, thymic differentiation, and signal transduction. It is primarily expressed on most thymocytes, a subset of T cell and monocytes/macrophages. CD7 is found on T cells, NK cells, thymocytes, hematopoietic progenitors and monocytes. CD7 is also expressed on ALL and some AML cells. CD11b is a member of the integrin family, primarily expressed on granulocytes, monocytes/macrophages, dendritic cells, NK cells, and subsets of T and B cells. CD14 is a GPI-linked membrane glycoprotein, also known as LPS receptor. It is expressed at high levels on macrophages, monocytes and at low level on granulocytes. CD33 is a sialoadhesion Ig superfamily member expressed on myeloid progenitors, monocytes, granulocytes, dendritic cells and mast cells. It is absent on normal platelets, lymphocytes, erythrocytes and hematopoietic stem cells. CD34 is a type I monomeric sialomucin-like glycophosphoprotein. It is selectively expressed on the majority of hematopoietic stem/progenitor cells, bone marrow stromal cells, capillary endothelial cells, embryonic fibroblasts, and some nervous tissues. It is commonly used marker for identifying human hematopoietic stem/progenitor cells. CD45 is commonly known as the leukocyte common antigen. It is a transmembrane tyrosine phosphatase expressed on all hematopoietic cells, except erythrocytes and platelets. It is a signaling molecule that regulates a variety of cellular processes including cell growth, differentiation, cell cycle, and oncogenic transformation. It plays a critical role in T and B cell antigen receptor-mediated activation. CD71 is a type II heterodimeric transmembrane glycoprotein also known as the transferring receptor. It is expressed on proliferating cells, reticulocytes, and erythroid precursors. CD71 plays a role in the control of cellular proliferation by facilitating the uptake of iron via ferrotransferrin binding and the recycling of apotransferrin to the cell surface. CD235a is also known as glycophorin A and CD235b is also known as glycophorin B, major sialoglycoproteins expressed on the red blood cell membrane and erythroid precursors. Mature, non-nucleated red blood cells are characteristically CD235a and/or CD235b positive, but CD45 and CD71 negative.

In some embodiments, a kit to be used for the analysis of myeloid cells in bone marrow can comprise antibodies that recognize 1, 2, 3, 4, 5, 6 or 7 of the following: CD7, CD11b, CD14, CD15, CD33, CD34, and/or CD45.

In some embodiments, a kit to be used for the analysis of nucleated red blood cells can comprise antibodies that recognize 1, 2, 3, 4, 5 or 6 of the following: CD7, CD14, Cd34, CD45, CD71, CD235a and/or CD235b.

State-Specific Binding Elements for Intracellular Markers

In some embodiments, the kits of the invention are employed to monitor the status of an activatable element, such as a signaling protein, in a signaling pathway known in the art including those described herein. Exemplary types of signaling proteins within the scope of the present invention include, but are not limited to, kinases, kinase substrates (e.g. phosphorylated substrates), phosphatases, phosphatase substrates, binding proteins (such as 14-3-3), receptor ligands and receptors (cell surface receptor tyrosine kinases and nuclear receptors)). Kinases and protein binding domains, for example, have been well described. See, for example, Cell Signaling Technology, Inc., 2002 Catalogue “The Human Protein Kinases” and “Protein Interaction Domains” pgs. 254-279).

In some embodiments, a kit can comprise one or more of the state-specific binding elements specific for the activated element(s) of interest. Exemplary binding elements comprise binding elements specific for PI3-Kinase (p85, p110a, p110b, p110d), JAK1, JAK2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHP1, and SHP2, SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3, TSC1,2, Rheb, mTor, 4E-BP1, p70S6Kinase, S6, LKB-1, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tp12, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, Btk, BLNK, LAT, ZAP70, Lck, Cbl, SLP-76, PLCγ1, PLCγ2, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, FAK, p130CAS, PAKs, LIMK1/2, Hsp90, Hsp70, Hsp27, SMADs, Rel-A (p65-NFKB), CREB, Histone H2B, Histone H3, HATs, HDACs, PKR, Rb, Cyclin D, Cyclin E, Cyclin A, Cyclin B, p15, p16, p21, p14Arf, p27KIP, p21CIP, Cdk4, Cdk6, Cdk7, Cdk1, Cdk2, Cdk9, Cdc25, A/B/C, Abl, E2F, FADD, TRADD, TRAF2, RIP, Myd88, BAD, Bcl-2, Mcl-1, Bcl-XL, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, XIAPs, Smac, Fodrin, Actin, Src, Lyn, Fyn, Lck, NIK, IκB, p65(RelA), IKKα, PKA, PKCα, PKCβ, PKCθ, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK, Chk1, Chk2, ATM, ATR, beta-□catenin, CrkL, GSK3α, GSK3β, FOXO, or glycolytic enzymes including but not limited to M2 pyruvate kinase.

In some preferred embodiments, kits of the present invention comprise one or more of the state-specific binding elements specific for the proteins selected from the group consisting of STAT1, STAT3, STAT5, S6, Erk, Akt, ATM, ATR, Chk1, Chk2, 53BP1, PARP, H2AX, Caspase 3, Caspase 8, CRKL, Histone H3, Cyclin B1, Cyclin D1, Cyclin E, Cyclin A, p15, p16, p21, PLCδ2, p53, SLP-76, and CREB. Other binding elements disclosed in U.S. Pat. No. 7,393,656 are also incorporated here by reference.

Binding Element

In some embodiments of the invention, a kit of the invention can comprise one or more binding elements specific for activation states of activatable elements. The term “binding element” includes any molecule, e.g., peptide, nucleic acid, small organic molecule which is capable of detecting an activation state of an activatable element over another activation state of the activatable element.

In some embodiments, the binding element is a peptide, polypeptide, oligopeptide or a protein. The peptide, polypeptide, oligopeptide or protein can be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, or a combination of both. Thus “amino acid”, or “peptide residue”, as used herein include both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention. The side chains may be in either the (R) or the (S) configuration. In some embodiments, the amino acids are in the D- or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents can be used, for example to prevent or retard in vivo degradation. Proteins including non-naturally occurring amino acids can be synthesized or in some cases, made recombinantly; see van Hest et al., FEBS Lett 428:(1-2) 68-70 May 22, 1998 and Tang et al., Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which are expressly incorporated by reference herein.

A kit of the present invention can be used to detect any particular activatable element in a sample that is antigenically detectable and antigenically distinguishable from other activatable elements which are present in the sample. For example, activation state-specific antibodies can be used in the present kits to identify distinct signaling cascades within a subset or subpopulation of cells within a complex population, and the ordering of protein activation (e.g., kinase activation) in potential signaling hierarchies. Hence, in some embodiments, the expression and phosphorylation of one or more polypeptides can be detected and quantified using a kit of the present invention. In some embodiments, the expression and phosphorylation of one or more polypeptides that are cellular components of a cellular pathway can be detected and quantified using methods of the present invention. As used herein, the term “activation state-specific antibody” or “activation state antibody” or grammatical equivalents thereof, refer to an antibody that specifically binds to a corresponding and specific antigen. Preferably, the corresponding and specific antigen is a specific form of an activatable element. Also preferably, the binding of the activation state-specific antibody is indicative of a specific activation state of a specific activatable element.

In some embodiments, the binding element is an antibody. In some embodiment, the binding element is an activation state-specific antibody.

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