CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/609,301, filed September 13, 2004, the content of which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods and compositions for detecting and/or quantifying modifications to or complexes of biological molecules. In particular, components of a signal generating system and their method of use in a homogeneous assay format in fixed samples are provided that generate a signal when brought into proximity by their association with components comprising the modified or complexes biological molecules.

BACKGROUND OF THE INVENTION The formation and disassociation of molecular complexes is a pervasive biological phenomena that is crucial to regulatory processes in living organisms. In particular, signaling pathways between the extracellular environment and the nucleus of a cell involve the formation of many molecular complexes in which multiple proteins are assembled to directly or indirectly induce molecular events, such as phosphorylation or dephosphorylation, which are steps in the signaling process, Gomperts et al, Signal Transduction (Academic Press, New York, 2002). Such pathways and their components have been the subject of intense investigation because of the role aberrant pathway behavior plays in many disease conditions, especially cancer, e.g. McCormick, Trends in Cell Biology, 9/53-56 (1999); Blume- Jensen and Hunter, Nature, 411:355-365 (2001); Nicholson et al, Cellular Signalling, 14:381-395 (2002); and the like. For example, it has been observed that many cancers are associated with an accumulation of mutations or other genetic alterations that affect components of signaling pathways, e.g. by over expression, particularly those pathways involved with cell proliferation, cell motility, differentiation, and cell death, e.g. Blume- Jensen and Hunter (cited above); Evan and Vousden, Nature, 411:342-348 (2001). Signaling pathways have been difficult to study not only because of their complexity and interconnectedness, but also because of the disruptive procedures required for analysis of intracellular complexes, e.g. Weng et al, Science, 284:92-96 (1999); Machida et al, Molecular & Cellular Pr oteomics, 2.4:215-233 (2003); Price et al, Methods in Molecular Biology, 218:255- 267 (2003). A wide variety of techniques have been used to study cellular protein-protein interactions and complexes, including immunoprecipitation, chemical cross-linking, yeast two- hybrid systems, tagged fusion proteins, bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), mass spectroscopy, and the like, e.g. Golemis, editor, Protein-Protein Interactions (Cold Spring Harbor Laboratory Press, New York, 2002); Price et al (cited above); Sorkin et al, Curr. Biol, 10:1395-1398 (2000); McVey et al, J. Biol. Chem., 17:14092-14099 (2001); Salim et al, J. Biol. Chem., 277:15482-15485 (2002); Angers et al, Proc. Natl. Acad. ScL, 97:3684-3689 (2000); Jories et al, Proteomics, 2:76-84 (2002); and Petricoin III, et al, The Lancet, 359:572-577 (2002). Unfortunately, such techniques are difficult to apply, generally lack sufficient sensitivity to provide an accurate picture of the state of a signaling pathway, and/or cannot measure multiple components or interacting components that are crucial for pathway activation. Consequently, measurements made by such techniques have not led to useful biological indicators based on complex formation. In view of the above, the availability of a convenient method and compositions for identifying and/or quantifying complexes, particularly large complexes, formed between or among proteins, enzymes, peptides, nucleic acids, membranes, cellular organelles and other biological compounds and components in abroad array of sample types would advance many fields where understanding intermolecular associations are important, including life science research, medical research and diagnostics, drug development, and the like. One area of particular importance is the analysis of aberrant signaling pathways, especially those involved in cancer growth, which have diagnostic value in determining survival, responsiveness to treatment, metastasis, likelihood of remission or relapse and the like.

SUMMARY OF THE INVENTION The present invention is directed to methods and compositions for identifying analytes in a sample, including samples comprising fixed sample specimens and tissue specimens. In one aspect, the invention provides an improved method for detecting noncovalent complexes by performing proximity assays. The method generally comprises the steps of (a) providing a sample that may contain the analyte of interest, (b) providing a signal generating system comprising two units each capable of binding to a different component of the analyte wherein a first unit is capable of producing a transmitter having an effective proximity and a second unit is dependent upon the transmitter for generating a signal molecule, (c) combining under binding conditions the sample and the signal generating system such that the binding moieties bind their respective components of the analyte and the second unit is within the effective proximity of the transmitter produced by the first unit causing signal molecules to be produced, and (d) analyzing for the presence of signal molecules. In some embodiments, the first unit is capable of enzymatically producing the transmitter having an effective proximity. In other embodiments, the first unit is capable of non-enzymatically, such as chemically, producing the transmitter having an effective proximity. In one embodiment of the invention, the first unit comprises glucose oxidase, the hydrogen peroxide it generates functions as the transmitter, and the second unit comprises horseradish peroxidase, which is capable of generating a signal by catalytic reaction dependent upon the presence of the transmitter hydrogen peroxide. Other enzyme pairs are also contemplated, such as horseradish peroxidase and luminol, and phosphoenol pyruvate kinase and luciferase. In another aspect, the improved analyte detection method employs a non-enzymatic reaction to generate signal molecules. The signal generating system again comprises two units, each capable of binding to a different component of the analyte. The first unit comprises a reaction center, which may be catalytic, non-catalytic or enzymatic, which generates a transmitter. The second unit comprises a molecule bearing at least two signal molecules attached thereto by a cleavable linkage. The transmitter acts within an effective proximity to induce the chemical, i.e. non-enzymatic, cleavage of signal molecules from the second unit and thereby release the signal molecules for subsequent detection. It is an object of the invention that the detectable property of the signal molecule be modified or enabled by the cleavage of the signal molecule from the second unit. In one embodiment of the invention, the first unit comprises horseradish peroxidase (HRP), and the transmitter is a phenylenediamine derivative, produced by the action of HRP on a phenylenediamine derivative. The second unit comprises a polymeric backbone bearing at least two signal molecules linked thereto by a chemical moiety containing a bond cleavable through reaction with the transmitter. Exemplary moieties are phenols, thiophenols, and anilines bearing a signal molecule precursor substituent located ortho or para to the phenol, thiophenol or amine group, respectively. The signal molecule precursor is a leaving group in the reaction of the transmitter with the second unit, and the signal molecule is thus released into solution. In one particular embodiment, the second unit comprises a dextran moiety bearing a hydroxycoumarin derivative linked thereto, via the hydroxyl oxygen, by an aromatic or heteroaromatic ring wherein the hydroxycoumarin leaving group, is located ortho or para to a hydroxy (-OH) group, thiol (-SH) group or amino (-NH2) group. In each of the above aspects, the invention is directed to the detection of noncovalent complexes, such as protein-protein complexes, which may be a soluble complex or a membrane or cell surface-bound complex. The complexes may comprise heterodimers, homodimers, or larger aggregates of cellular components. In a further aspect of the invention the amount of a particular component in a complexed state versus a non-complexed state may be determined. Thus for example the ratio of monomer:dimer for a cellular component is measured by the subject assay methods and compositions. In another aspect of the invention, phosphorylated and non-phosphorylated complexes may be distinguishably detected, such that the phosphorylation state of a particular component or complex may be determined. Another aspect of the subject invention is the use of a signal generating system for performing assays on formalin fixed paraffin-embedded samples or fresh tissue samples or adherent or suspended cell samples. In another aspect, the invention provides a method for detecting complexes by performing proximity assays. The method generally comprises the steps of (a) providing a sample that may contain the analyte of interest, (b) providing a signal generating system comprising two units each capable of binding to a different component of the analyte wherein a first unit is capable of enzymatically producing a transmitter having an effective proximity and a second unit is dependent upon the transmitter for generating a signal molecule, (c) combining under binding conditions the sample and the signal generating system such that the binding moieties bind their respective components of the analyte and the second unit is within the effective proximity of the transmitter produced by the first unit causing signal molecules to be produced, and (d) analyzing for the presence of signal molecules. In some embodiments, the complexes are covalent complexes. The present invention provides methods and compositions for identifying, detecting or measuring analytes that has several advantages over current techniques including, but not limited to, the detection of noncovalent complexes of two or more components, particularly within cells in tissue samples or paraffin-embedded cell samples, by using a signal generating system that relies upon communication between or among components of the system in order to yield a signal. The range over which the components can communicate is tunable, and relatively large complexes, which require a correspondingly large distance for such communication, can be detected. The invention may also be employed to detect the modification state of proteins, such as the phosphorylation state. The method and compositions are employed in a homogeneous format wherein noncovalent complexes are analyzed under conditions that do not substantially perturb the natural equilibrium of the system.

BRIEF DESCRIPTION OF THE DRAWINGS The drawings are cartoon illustrations and are not to scale, the purpose being to convey the principles of the invention. Figures IA and IB show the chemical structure of a family of derivatives useful as a transmitter as a substrate (IA) and as the active transmitter (IB). Figure 2 is a schematic illustration of the combination of species in one embodiment of the invention for detecting an assay target. Figure 3 is a schematic illustration of the combination of species in another embodiment of the invention for detecting an assay target. Figure 4 is a schematic illustration of one embodiment of the invention as practiced for detecting the modification state of a protein. Figures 5A-5C illustrate a protein-protein homodimerization reaction (5A), a set of reagents useful for detecting the homodimerization equilibrium (5B), and the population of possible binding complexes formed among the proteins and the reagents (5C).

DETAILED DESCRIPTION QF THE INVENTION

Definitions As used herein, the term "analyte" refers to a substance, molecule, or component, or a complex of substances, molecules or components in a sample whose presence or absence is to be detected or whose quantity is to be measured in an assay. The term "target" may be used interchangeably with "analyte". Analytes include but are not limited to peptides, proteins, polynucleotides, polypeptides, oligonucleotides, organic molecules, haptens, epitopes, parts of biological cells, posttranslational modifications of proteins, receptors, complex sugars, vitamins, hormones, and the like, and the complexes formed between and among these substances. There may be more than one analyte associated with a particular combination of substances, e.g. different phosphorylation sites within the same complex of proteins, different members within a multicomponent complex, etc. "Antibody" means an immunoglobulin that specifically binds to, and is thereby defined as complementary with, a particular spatial and polar organization of another molecule. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art such as immunization of a host and collection of sera (polyclonal) or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal), or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies. Antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgGl, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular polypeptide is maintained. "Binding moiety" means any molecule that is capable of specifically binding to a portion of an analyte. Binding compounds include, but are not limited to, antibodies, antibody fragments, peptides, proteins, particularly secreted proteins and orphan secreted proteins, nucleic acids, natural and anatural analogues of oligonucleotides and organic molecules having a molecular weight of up to 1000 daltons and consisting of atoms selected from the group consisting of hydrogen, carbon, oxygen, nitrogen, sulfur, and phosphorus. The term "sample" in the present specification and claims is used in a broad sense. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures), or a biological or environmental sample used as the source of material to assay. A sample may include a specimen of synthetic origin. Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may include materials taken from a patient including, but not limited to cultures, blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, semen, needle aspirates, and the like. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, rodents, etc. Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention. On the other hand, "sample" is also meant to refer to the prepared material to be analyzed in the assay. Thus, a volume of solution, an amount of solid material (e.g. tissue) or a mass of cells that is provided for analysis constitutes the sample in the assay method. The sample may be fixed, preserved, denatured, sectioned, frozen, lysed or otherwise pretreated, purified or fractionated prior to being provided in the assay method. Fixed samples, such as formalin fixed, paraffin- embedded samples are preferred as a sample type in the subject invention. "Specific" or "specificity" in reference to the binding of one molecule to another molecule, such as a binding moiety or probe, for a target analyte, means the recognition, contact, and formation of a stable complex between the probe and target, together with substantially less recognition, contact, or complex formation of the probe with other molecules. In one aspect, "specific" in reference to the binding of a first molecule to a second molecule means that to the extent the first molecule recognizes and forms a complex with other molecules, it forms the largest number of complexes with the second molecule. In one aspect, this largest number is at least fifty percent of all such complexes form by the first molecule. Generally, molecules involved in a specific binding event have areas on their surfaces or in cavities giving rise to specific recognition between the molecules binding to each other. Examples of specific binding include antibody-antigen interactions, enzyme-substrate interactions, formation of duplexes or triplexes among polynucleotides and/or oligonucleotides, receptor-ligand interactions, and the like. As used herein, "contact" in reference to specificity or specific binding means two molecules are close enough that weak noncovalent chemical interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules. As used herein, "noncovalent complex" in reference to two or more substances, molecules or components means that such substances, molecules or components form noncovalently linked aggregates, e.g. by specific binding, that under assay conditions are stable. Preferably, the equilibrium between aggregated and non-aggregated states achieved during the assay method substantially reflects the thermodynamic equilibrium found in the sample prior to the start of the assay.

Assay Samples Samples types for use with the invention include cell suspensions, cell lysates, and tissue sections. The invention is particularly well-suited to use with tissue samples owing to the nature of the signal generation mechanism and the fact that the components may be combined and applied without yielding target-independent signal. Tissue samples themselves are valuable for retrospective studies and the ability to store and transport such samples easily. Thus there is a particular benefit gained by coupling the invention to such sample types. Methods of preparing tissue samples for examination or further assay are well-known in the art, having been described in numerous publications, such as U.S. Patent Nos. 3,961,097 issued to Gravlee, and 4,656,047 issued to Kok et ah, the contents of which are incorporated by reference in their entirety. More recently, improved methods for the faster preparation of fixed tissue samples was described by Essenfeld et al., in U.S. Patent No. 6,207,408 in which the preparation time was reduced from overnight to roughly one hour. The use of formalin in fixing the samples was also eliminated in the procedure of Essenfeld. For the purposes of the present invention, the speed of preparation or the use, or not, of formalin and other organic solvents is not critical, and thus samples prepared by any of these conventional methods for fixing samples may be used.

Transmitters Transmitters act generally in one of two ways, (1) by serving as a substrate for the second unit ('substrate-type'), or (2) by reacting with moieties within the second unit to cause the cleavage of a signal molecule from the unit ('reactant-type'). The choice of which type of transmitter is used follows from the choice of signal generation method provided by the second unit, which in turn depends on the preferred detection format to be used. Where a multiplicity of assay targets are to be detected, the latter method of causing the cleavage of signal molecules by the transmitter is preferred because second units bearing distinct signal molecules are more readily prepared than provided a multiplicity of suitable first unit-second unit combinations that can generate distinct signals. Where one target, or possibly two or three targets are to be detected, either type of transmitter may be used, and other considerations will bear on the choice of the signal generation method, and thus transmitter-type. For substrate-type transmitters, the selection will be from molecules that are both the product of one enzymatic reaction and a substrate for a second. Numerous examples exist and have been documented in the literature. One notable example of a pair so coupled is glucose oxidase and a peroxidase. Glucose oxidase catalyzes the reaction between glucose and molecular oxygen, producing hydrogen peroxide, while peroxidase, such as horseradish peroxidase, catalyzes the oxidation of a variety of chromogenic substrates in the presence of hydrogen peroxide. Thus hydrogen peroxide acts as the transmitter. The activity of glucose oxidase provides hydrogen peroxide as a substrate for the peroxidase-catalyzed oxidation of an oxidizable substrate. A suitably chosen oxidizable substrate will serve as a signal molecule, e.g. a molecule that undergoes a color change or a change in fluorescence. The extent of the peroxidase-catalyzed oxidation can then be measured by observing a color change in a chromogenic substrate or a change in fluorescence of a fluorogenic substrate. The presence of a scavenger for hydrogen peroxide, such as catalase, will modulate the effective proximity of the transmitter and help to decrease any background signal caused by the action of peroxidase conjugated to unbound antigen or antibody on hydrogen peroxide in solution. Reactant-type transmitters are generated by an enzyme and then react to induce a cleavage reaction. A particular class of suitable transmitters is the two-electron oxidation products of phenylamine derivatives that are formed by action of a peroxidase enzyme. In an assay or in a kit of reagents for such assays the phenylamine derivatives itself (i.e. in the reduced state) would be provided. These molecules, and functional groups that are cleavable by these molecules in their oxidized state, are discussed at length in U.S. Patent Nos. 5,332,662 and 5,445,944, which are herein incorporated by reference in their entirety. Examples of transmitters include benzidine (4,4'-diamino-biphenyl), p-aminophenol, phenylenediamine, and their derivatives. At least one amino group shall have two hydrogen atoms attached thereto (i.e. an unsubstituted amino group), and at least one position on the aromatic ring ortho to such amino group bears a hydrogen atom substituent. The remaining positions on the amino groups of the benzidine may contain substantially electroneutral substituents such as aryl, alkyl, H, or alkoxy, but not carbonyl, sulfonate or other electron withdrawing groups, that is, anything that causes too large an increase in the oxidation potential and inactivates the benzidine to oxidation. The remaining positions on the aromatic ring may include in addition weakly electron withdrawing groups such as halogen, acylamido, phosphates, etc, where again the primary requirement is that the oxidation of the benzidine be permitted. Further, the additional substituents may contain additional ring systems, as long as substantial planarity of the molecule is maintained so that oxidation of the benzidine is possible. Extensive guidance can be found in the literature for covalently linking enzyme to binding moieties, such as antibodies, e.g. Hermanson, 1996, Bioconjugate Techniques, (Academic Press, New York), and the like. In one aspect of the invention, one or more molecular tags are attached directly or indirectly to common reactive groups on a binding moiety. Common reactive groups include amine, thiol, carboxylate, hydroxyl, aldehyde, ketone, and the like, and may be coupled to molecular tags by commercially available cross-linking agents, e.g. Hermanson (cited above); 2002, Haugland, Handbook of Fluorescent Probes and Research Products, Ninth Edition (Molecular Probes, Eugene, OR), the contents of which are incorporated by reference in its entirety.

Signal Molecules and Detection Modes The signal molecule is selected to provide a detectable property that is distinct from the manifestation of that property in any precursor substrate. The signal may be detected, measured or assayed during its generation, after its generation, after a stop reagent has been added to cease its generation, after an enhancer has been added to enhance its magnitude, according to the nature of the particular signal molecule. Furthermore, the signal may be detected in situ within the same assay medium, or upon further processing, separation or purification of the assay medium, or upon its introduction into an analytical instrument. The detectable property of the signal molecule may be fluorescence intensity, fluorescence wavelength, fluorescence polarization, fluorescence lifetime, absorbance intensity or wavelength, chemiluminescence, mass, electrochemistry, mobility in chromatography, gas chromatography or liquid chromatography, or electrophoretic mobility in electrophoresis. The detectable property may also be a combination of one or more such properties, such as having a particular fluorescence wavelength while having a particular electrophoretic mobility, wherein analysis by capillary electrophoresis using laser-induced fluorescence detection would provide the necessary data. By relying upon a combination of properties greater selectivity and accuracy may be achieved in the assay. The signal molecule may be generated by either (1 ) direct action of an enzyme in the second unit, or (2) by the cleavage of a signal molecule from the second unit through the action of the transmitter. The number of signal molecule precursors per polymer support, that is, per second unit, is a consideration in the second embodiment. Because the total signal capable of being generated from each signal generating system will be limited to the number of signal molecules appended to the second unit, the number of such appended molecules will depend on considerations such as the assay sensitivity required and the sensitivity of the detection instrumentation, the desired dynamic range, the efficiency of the cleavage reaction and the like. Preferably, at least two signal molecules are provided attached to the polymer support. More preferably, at least ten signal precursors are provided, and perhaps as many as 50, or 100 may be used. The signal molecule itself is preferably a water-soluble organic compound that is stable in the presence of the transmitter and any other species present during the assay. In one aspect of the invention, the signal molecules may be electrophoretic tags as described in, for example, Zhang et al, Bioconjugate Chem., 13: 1002-1012 (2002); Giese, Anal. Chern., 2:165-168 (1983); and U.S. Patent Nos. 4,650,750; 5,360,819; 5,516,931; 5,602,273; and U.S. Patent Application No. 10/623,057, published as U.S. Pub. No. US 2004/0126818, the contents of which are incorporated by reference in their entireties. In another aspect, the signal molecule may be a fluorophore that lacks a mobility tag, or a molecule having a distinct mass. Other aspects and advantages of the present invention will be understood upon consideration of the included figures. Figure 2 is a schematic illustration of the relationship among an analyte, a signal generating system, optional substrates and the generated signal molecule according to one embodiment of the invention. The analyte is a noncovalent complex formed from the components 100 and 101. The signal generating system comprises a first unit 102 and a second unit 106. First unit 102 has as a binding moiety an antibody 103, and conjugated to it is enzyme (ei) 104, that acts to convert substrate (s) 111 to a transmitter (t) 112. The enzyme (el) may optionally require a co-factor or second substrate, (u) 110, to perform the enzymatic conversion. Transmitter 112 is a freely diffusing species having an effective range for reacting or influencing the reaction at the second unit 106 dependant upon its lifetime in the given assay medium, as discussed above. Second unit 106 has as a binding moiety an antibody 107, and conjugated to it is enzyme (e2) 108, that in the presence of transmitter 112 is capable of generating a signal molecule 114. As indicated above, either or both first and second units may have a plurality of either binding moiety or reaction center, exemplified here as an enzyme. Transmitter 112 may either be a substrate that is converted to signal molecule 114 or a co-factor for enzyme (e2) in the generation of signal molecule 114. Where transmitter 112 is a substrate, other co-substrates or co-factors, represented by (u') 113, may be required for functioning of the enzyme. Where transmitter 112 is a co-factor, a substrate (u') 113 will be required that is converted to the signal molecule 114. Figure 3 displays a schematic illustration of another embodiment of the relationship among an analyte, a signal generating system, optional substrates and the signal molecule according to the invention. Like numbered elements of Figures 2 and 3 portray the same functional elements of the system, although the particular compositions that fulfill each embodiment may differ and may not be interchangeable. The analyte again is a noncovalent complex formed from the components 100 and 101. The signal generating system comprises a first unit 102 and a second unit 116. First unit 102 has as a binding moiety an antibody 103, and conjugated to it is enzyme (Q{) 104, that acts to convert substrate (s) 111 to a transmitter (t) 112. The enzyme (el) may optionally require a co-factor or second substrate, (u) 110, to perform the enzymatic conversion. Transmitter 112 is a freely diffusing species having an effective range for reacting at the second unit 116 dependant upon its lifetime in the given assay medium, as discussed above. Second unit 116 has as a binding moiety an antibody 117, and conjugated to it is labeled polymer 118, that in the presence of transmitter 112 is capable of releasing a signal molecule 114. As indicated above, the second unit may have a plurality of either binding moiety or labeled polymer. Labeled polymer 118 is comprised of a polymer support backbone 119 and signal molecule precursors 120 that are bonded to the polymer through a cleavable bond. Transmitter 112 reacts at the site of the cleavable bond to become itself bound in whole or in part to the polymer, while displacing the signal molecule 114 as a leaving group in the reaction from the polymer support. One example application of the subject methods and compositions is for assaying a noncovalent complex in a cell signaling pathway, which is illustrated schematically in Figure 4. Cellular component 100 forms a complex with cellular component 101 when the latter adopts one of a plurality of its natural states, as illustrated, 101b, while another at least one state of the component, 101a, does not form any complex with component 100. Thus, an equilibrium is set up for the amount of noncovalent complex 120 (comprising 100 and 101b) present in a sample, which is dependent upon the equilibrium established among the states of component 101 (i.e. 101a, 101b, etc.). The states of component 101 may be regulated, influenced, or determined by transcription factors, up-regulation or down-regulation of gene expression, exogenous substrates, endogenous substrates, phosphorylation, dephosphorylation, and the like. The complex 120, having been formed, is detected using the signal generating system comprising first unit 102, second unit 106, and the required and optional substrates, co-substrates and co-factors, including for example co-factor 110, substrate 111, and co-factor 113. The signal generating system illustrated here is the same as that described in Figure 2, in which transmitter 112 formed by the first unit 102, acts within an effective proximity to promote generation of signal molecule 114 by the second unit 106. Equally applicable is the signal generating system described in Figure 3 that employs a labeled polymer in the second unit as the source of the signal molecule. In another example application, the subject invention is used to determine the extent of formation of a homodimer complex. Referring to Figure 5 A, the equilibrium expression for a protein capable of forming a homodimer complex is shown. The protein 501 is comprised of two distinct binding sites, 505 and 507 that may be, independently, antigenic in that antibodies can be raised against these sites, or receptor binding sites. Two protein molecules interact to form a dimer 503, also referred to as a homodimer, in a reaction that is characterized by an equilibrium constant. The relative amount of the monomeric and dimeric forms will be determined by the concentration of the protein, the temperature, local pH, salt and buffer conditions, the conformation of the protein and perhaps the modification state (/. e. phosphorylated, non-phosphorylated, glycosylated, non-glycosylated, etc.). Importantly, the relative amounts of monomer, dimer, or even larger aggregate may indicate the cell status with respect to disease or other compromise in function. Figure 5B shows a set of reagents useful for analyzing for the presence of biological homodimers. The set is comprised of an enzyme conjugate 510, a first labeled polymer conjugate 520 and a second labeled polymer conjugate 530. The enzyme conjugate is similar in function and structure to the first unit of a signal generating system as described above. An enzyme 512 that is capable of generating a transmitter is conjugated to a binding moiety 511, which may be antibody for binding to an antigenic site, or a substrate for binding to a receptor on the protein target. The first labeled polymer conjugate is conjugated to the same binding moiety 511 as is present in enzyme conjugate 510. Thus the two conjugate species 510 and 520 compete for the same binding site on the protein target. The second labeled polymer conjugate 530 has a binding moiety 531 that is specific for a distinct binding site. Labeled polymer conjugates 520 and 530, in addition to the binding moieties, are comprised of polymeric backbones 522 and 532, respectively, that serve as a scaffold to which detectable labels 523 and 533, respectively, are cleavably bound, again as described earlier. The labels 523 and 533 are different and must provide detectably distinct signals in order to enable determination of the relative amounts of monomeric and dimeric species. The types of signal molecules and compositions that will provide distinct signals are well known to those skilled in the art of analytical chemistry or biochemistry, separation science, spectroscopy and the like. Generally, the molecular characteristics that will be usefully modified and varied to provide a distinct signal will depend upon the measurement method that is contemplated. Distinct signals may take the form of signal molecules having for example the same fluorophores but different electrophoretic mobilities, and thus provide distinct signals when analyzed by electrophoresis with fluorescence detection. Other examples include distinct mass for mass spectral analysis, or distinct fluorescence properties for fluorescence-based analysis. Combining the reagent set with a sample suspected of containing the biological target, e.g. protein, and providing the conditions necessary for binding reactions to occur among the various species will result in range of possible of products. The range of possible products for a protein capable of forming homodimers is shown in Figure 5C. Products containing at least one enzyme conjugate 510 either of labeled conjugate 520 or 530 will generate a signal by the process generally described above in conjunction with Figure 3. Products 550, 570 and 580 will generate a signal, whereas products 560 and 590 will not because they lack the enzyme conjugate. The type of signal and relative amount can be understood by consideration of the products shown in Figure 3. In particular, the detection of the signal 523 from conjugate 520 unambiguously indicates formation of the homodimer. Also, the ratio of the signals for 523 and 533 will be proportional to the ratio of the monomer to the dimer.

Noncovalent Complexes The methods and compositions of the present invention can be used to detect or measure any noncovalent complexes known by one of skill in the art without limitation. For example, the noncovalent complexes can be cell surface receptor complexes, receptor-ligand complexes or intracellular molecular complexes. In one aspect, the amount of the noncovalent complexes is measured. In other aspect, the location of the noncovalent complex in cells or tissues is determined. > The methods and compositions of the present invention can be used to detect or measure cell surface receptor complexes. Cell surface receptor complexes can be used as biomarkers as a reliable indicator of a disease status or condition. See e.g., Chow et al., 2001, Clin. Cancer Res., 7:1957-62 (EGFR or Herl expression). Exemplary cell surface receptor complexes that can be detected by the methods of the present invention are listed in Table 1. In certain embodiments, non-covalent complexes are selected from the receptor dimers listed in Table 1.

Table 1. Exemlar Recetor Comlexes of Cell Surface Membranes

In one aspect, the methods of the present invention can be used to detect or measure Her receptors dimerization or phosphorylation states. Erb receptors or Her receptors are receptor protein tyrosine kinase which belongs to the Erb receptor family and includes EGFR ("Her 1"), ErbB2 ("Her 2"), ErbB3 ("Her 3") and ErbB4 ("Her 4") receptors. It has been shown that Her receptors dimerization or phosphorylation states correlates with the status of a disease, such as a cancer. See e.g., U.S. App. No. 10/812,619, filed April 30, 2004, published as U.S. Pub. No. US 2004-0229293 on November 18, 2004, U.S. App. No. 10/813,412, filed on April 30, 2004, published as U.S. Pub. No. US 2004-0229380 on November 18, 2004,and U.S. App. No. 10/813,417, filed March 30, 2004, published as U.S. Pub. No. US 2004-0229294 on November 18, 2004, the contents of which are incorporated by reference in their entireties. For example, certain Her-containing dimers indicate that a cancer is likely responsive to treatment with, e.g., cetuximab. In certain embodiments, the methods and compositions of the present invention are used to detect the presence and/or the amount of Her receptor complexes. Such receptor complexes include, but are not limited to, one or more of Herl-Herl dimers, Her2-Her2 dimers, Herl-Her2 dimers, Her2-Her3 dimers, Herl-Her3 dimers, Her2-Her4 dimers, Herl-PI3K complexes, Her2- PI3K complexes, Her3-PI3K complexes, Herl-SHC complexes, Her2-SHC complexes, Her3- SHC complexes, Herl-IGF-1R receptor dimers, Her2-IGF-1R receptor dimers, Her3-IGF-1R receptor dimers, Her 1 -PDGFR receptor dimers, Her2-PDGFR receptor dimers, Her3-PDGFR receptor dimers, p95Her2-Her3 receptor dimers, p95Her2-Her2 receptor dimers, p95Her2-Herl receptor dimers, EGFRvIII-Herl receptor dimers, EGFRvIII-Her2 receptor dimers, and EGFRvIII-Her3 receptor dimers. In other embodiments, such Her receptor complexes are selected from the group consisting of Herl-Her2 receptor dimers and Her2-Her3 receptor dimers; or the group consisting of Herl-Her2 receptor dimers, Her2-Her3 receptor dimers, and Herl- Her3 receptor dimers. In other embodiment, the invention includes measurement of complexes comprising a Her receptor and an intracellular adaptor molecule, particularly, intracellular adaptor molecules that form complexes with a Her receptor in response to phosphorylation of such receptor. Exemplary receptor complexes of Her receptors and intracellular adaptor molecules include complexes selected from the group consisting of Her 1 -PI3 K complexes, Her2-PI3K complexes, Her3-PI3K complexes, Herl-SHC complexes, Her2-SHC complexes, and Her3-SHC complexes. The invention further includes the association of receptor heterodimers comprising a Her receptor and another receptor tyrosine kinase to a disease status. Exemplary receptor complexes of Her receptors and other receptor tyrosine kinases include receptor complexes selected from the group consisting of Herl-IGF-1R receptor dimers, Her2-IGF-1R receptor dimers, Her3-IGF- IR receptor dimers, Herl-PDGFR receptor dimers, Her2-PDGFR receptor dimers, and Her3- PDGFR receptor dimers. The invention further includes the association of receptor dimers comprising a full-length Her receptor and a truncated Her receptor to a disease status. Exemplary receptor complexes of full-length Her receptors and truncated Her receptors include receptor complexes selected from group consisting of p95Her2-Her3 receptor dimers, EGFRvIII- Herl receptor dimers, EGFRvIII-Her2 receptor dimers, and EGFRvIII-Her3 receptor dimers. In another aspect, such method of determining disease status includes determining the effectiveness of, or the responsiveness of a patient to, dimer-acting drugs for treating cancer, the dimer-acting drug acting on Her receptor complexes as described above. In other aspect, the methods of the present invention are used to detect or measure PI3K- associated receptor complexes. Detection of PI3K-associated receptor complexes can be used as a surrogate measure of phosphorylation states of a protein. Exemplary PI3K-associated receptor complexes are listed in Table 2. In certain embodiments, non-covalent complexes of the present invention are selected from the PI3K-associated receptor complexes listed in Table 2. Table 2.

PI3K-Associated Recetor Complexes

The methods and compositions of the present inventions can also be used to detect or measure intracellular molecular complexes, for example, those described in U.S. App. No. 10/814,686, filed on April 30, 2004, published as U.S. Pub. No. US 2004-0229299 on November 18, 2004,the contents of which are incorporated by reference in its entirety. Of particular interest are protein-protein complexes that include, but are not limited to, the proteins of Tables 3-5. Preferably, the human forms of the following proteins and protein families are intended.

Table 3. Exemplary Proteins Forming Intracellular Complexes in Apoptotic Pathways

Table 4. Exemplary Protein-Protein Complexes in Apoptotic Pathways (where "protein l//protein 2" indicates a complex comprising protein 1 and protein 2)

Table 5. Exemplary RTK Dimers and Intracellular Complexes (here "protein 1 //protein 2" indicates a complex comprising protein 1 and protein 2)

RTK Dimer Downstream Comlexes

Certain intracellular complexes are indicative of the apoptotic status of a patient suffering from a disease, such as a cancer, characterized by aberrant apoptosis. In one aspect, the methods of the invention may be used to determine whether one or more apoptotic pathways are activated by simultaneously measuring protein-protein complexes. Such intracellular complexes include, but are not limited to, one or more of 14-3-3//BAD, BID//BAX, BAX//BAX, BcI- XL//BAD, Bcl-2//BAD, 14-3-3//BID, BID//BAK, BAX//Bcl-2, BC1-XL//BIK, Bcl-2//BIK, NF- kB//I-kB, BID//Bcl-2, Bcl-XJ/BID, Bcl-2//BID, FADD//caspase-9, BID//Bcl-XL, Bcl-XL//Hrk, Bcl-2//Hrk, TRADD//caspase-9, BID//Al/Bfl-1, Bcl-XJ/BIM, Bcl-2//BIM, Apaf-l//caspase-9, Bcl-XJ/Noxa, Bcl-2//Noxa, Bcl-XL//Bmf, Bcl-2//Bmf, Bcl-XL//Puma, Bcl-2//Puma, BcI- XL//BC1-G, Bcl-2//Bcl-G, Bcl-XL//NIP3, Bcl-2//NIP3, Bcl-XJ/Nix, and Bcl-2//Nix. In another aspect, such intracellular complexes include, but are not limited to, 14-3-3//BAD, Bcl-2//BAD, 14-3-3//BID, BAX//Bcl-2, Bcl-2//BIK, BID//Bcl-2, Bcl-2//BID, Bcl-2//Hrk, Bcl-2//BIM, BcI- 2//Noxa, Bcl-2//Bmf, Bcl-2//Puma, Bcl-2//Bcl-G, Bcl-2//NIP3, and Bcl-2//Nix. In another aspect, such intracellular complexes include, but are not limited to, NF-kB//I-kB. In still another aspect, such intracellular complexes include the measurement of NF-kB//I-kB complexes to determine a disease status of a patient suffering from a cancer or an inflammatory conditions, such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, or asthma. The methods and compositions of the present invention can also be used to detect intracellular complexes that correlates the status of a disease of a patient, such as a cancer, characterized by aberrant signal transduction pathway activation. Such intracellular complexes include, but are not limited to, one or more of Herl//Shc, Grb2//Sos, Herl//Grb7, Herl//RasGAP, Grb2//Shc, Her2//Shc, Her3//PI3K, Her3//Shc, Her3//Grb7, YAP//Her4, IGF- 1R//PI3K, IGF-1R//Shc, IGFR//IRS1, VEGFR//Shc, VEGFR//PI3K, VEGFR//Src, VEGFR//FRS2, PDGFRa//Crk, PDGFR//Grb2, PDGFR//Grb7, PDGFR//Nck; PDGFR//Shc, DGFR//STAT5, PDGFRa//Crk, PDGFRb//GAP, PDGFR//Grb2, PDGFR//Grb7, PDGFR//Nck; PDGFR//Shc, PDGFR//Shp2, PDGFR//RasGAP, PDGFR//STAT5, PDGFRb//GAP, PDGFR//Grb2, PDGFR//Grb7, PDGFR//Nck, PDGFR//Shc, PDGFR//Shp2, PDGFR//RasGAP, PDGFR//STAT5, Kit//Shp-1, Kit//PI3K, Kit//Grb2, Kit//CRKL, FGFR//PLCgl, FGFR//Crk, FGFR//FRS2, GFR//Shp2, FGFR//Shb, Trk//p75NTR, and Trk//PI3K. In another aspect, such intracellular complexes include, but are not limited to, Herl//Shc, Grb2//Sos, Herl//Grb7, Herl//RasGAP, Grb2//Shc, Her2//Shc, Her3//PI3K, Her3//Shc, and Her3//Grb7. In another aspect, such intracellular complexes include, but are not limited to, IGF-1R//PI3K, IGF-1R//Shc, and IGFR//IRS1. In another aspect, such intracellular complexes include, but are not limited to, VEGFR//Shc, VEGFR//PI3K, VEGFR//Src, and VEGFR//FRS2. In another aspect, such intracellular complexes include, but are not limited to, PDGFRa//Crk, PDGFR//Grb2, PDGFR//Grb7, PDGFR//Nck; PDGFR//Shc, DGFR//STAT5, PDGFRa//Crk, PDGFRb//GAP, PDGFR//Grb2, PDGFR//Grb7, PDGFR//Nck; PDGFR//Shc, PDGFR//Shp2, PDGFR//RasGAP, PDGFR//STAT5, PDGFRb//GAP, PDGFR//Grb2, PDGFR//Grb7, PDGFR//Nck, PDGFR//Shc, PDGFR//Shp2, PDGFR//RasGAP, and PDGFR//STAT5.

EXAMPLES Example 1 Measurement of Receptor Dimers in Formalin Fixed Paraffin Embedded Tissue Samples Using Reactant-type Transmitters In this example, exemplary fixed tissues made from pelleted cell lines are assayed for the presence of Her receptor dimers, for example, Herl-Herl dimers. The assay design for Her homodimers is essentially the same as that described in Fig. 5. That is, a signal generating system is employed to detect the Herl-Herl homodimers. The signal generating system is similar in function and structure to the signal generating systems as described above and in Fig. 3. The signal generating system comprises a first unit, an enzyme conjugate 510, and a second unit, a labeled polymer conjugate 520 in Fig. 5. The enzyme conjugate comprises an antibody that binds to an antigenic determinant of Herl (the binding moiety of 103 of Fig. 3). The binding moiety is conjugated to horseradish peroxidase which can convert the phenylamine derivative to the two-electron oxidation products of phenylamine, the transmitter 112 in Fig. 3. The labeled polymer conjugate comprises an antibody that binds to an antigenic determinant of Herl (the binding moiety 117 in Fig. 3), different from the binding site of the binding moiety 103 of the enzyme conjugate. The binding moiety is conjugated to a labeled polymer 118 having a cleavable bond that is cleavable by the transmitter, the two-electron oxidation products of phenylamine, as described in U.S. Pat. no. 5,332,662, the contents of which is incorporated by reference in its entirety. The cleavable bond links a polymer backbone 119 and a signal molecule precursor 120. The signal molecule can be an electrophoretic tags as described in U.S. Patent Application No. 10/623,057, published as U.S. Pub. No. US 2004/0126818, the contents of which are incorporated by reference in its entirety. The presence of the signal molecules indicates the presence of Herl-Herl dimers. The model fixed tissues can be prepared as follows. Cells grown on tissue culture plates can be stimulated with either EGF and/or HRG. Cells can be stimulated with EGF and/or HRG in culture media for 10 minutes at 370C. Exemplary doses of EGF/HRG are 0, 0.032, 0.16, 0.8, 4, 20, or 100 nM. After stimulation, samples are washed and removed by scrapping. The removed cells are centrifuged to form a pellet, after which formalin is added and the mixture is incubated overnight at 40C. The fixed pellet is embedded in paraffin using a Miles Tissue Tek III Embedding Center, after which 10 μm tissue sections are sliced from the pellet using a microtome (Leica model 2145). Tissue sections are placed on positively charged glass microscope slides (usually multiple tissue sections per slide) and baked for 1 hr at 6O0C. Tissue sections on the slides are assayed as follows. Tissue sections on a slide are de- waxed with EZ-Dewax reagent (Biogenex, San Ramon, CA) using the manufacturer's recommended protocol. Briefly, 500 μL EZ-Dewax is added to each tissue section and the sections are incubated at RT for 5 min, after which the slide is washed with 70% EtOH. This step is repeated and the slide is finally rinsed with deionized water, after which the slide is incubated in water at RT for 20 min. The slide is then immersed into a IX Antigen Retrieval solution (Biogenesis, Brentwood, NH) at pH 10, after which it is heated for 15 min in a microwave oven (5 min at high power setting followed by 10 min at a low power setting). After cooling to RT (about 45 min), the slide is placed in a water bath for 5 min, then dried. Tissue sections on the dried slide are circled with a hydrophobic wax pen to create regions capable of containing reagents placed on the tissue sections (as illustrated in Figs. 3H-3I), after which the slide is washed three times in IX Perm/Wash (BD Biosciences). To each section 50-100 μL blocking buffer is added, and the slide is placed in a covered humidified box containing deionized water for 2 hr at 40C, after which the blocking buffer is removed from each section by suction. Blocking buffer is IX Perm/Wash solution with protease inhibitors (Roche), phosphatase inhibitors (sodium floride, sodium vanadate, β-glycerol phosphate), and 10% mouse serum. The slide is then contacted with the signal generation system mixture containing the enzyme conjugate and labeled conjugate and is placed in a humidified box overnight at 40C. The sections are then washed three times with 100 μL Perm/Wash containing protease and phosphatase inhibitors, after which 50 μL of photosensitizer in IX Perm/Wash solution (containing protease and phosphatase inhibitors) is added. The slide is then incubated for 1 -1.5 hr at 40C in the dark in a humidified box, after which the photosensitizer is removed by suction while keeping the slide in the dark. While remaining in the dark, the slide is then immersed in 0.01X PBS and incubated on ice for 1 hr. The slide is remove from the PBS, dried, and to each section, 40-50 μL 0.0 IX PBS with 2 pM fluorescein is added, after which it is illuminated with a high power laser diode (GaAIAs IR emitter, model OD-880W, OPTO DIODE CORP, Newbury Park, CA) for 1 hr. The fluorescein acts as a standard to assist in correlating peaks in an electropherogram with molecular tags. After illumination, the solution covering each tissue section is mixed by gentle pipeting and transferred to a CE plate for analysis on an Applied Biosystems (Foster City, CA) model 3100 capillary electrophoresis instrument. The detection of signal molecules indicates the presence of Herl-Herl dimers. The strength of the signal indicates the amount of Herl-Herl dimers in the samples. Example 2 Measurement of Bcl-2//BAD Apoptotic Complexes in Breast Cell Lines Using Substrate-type Transmitters In this example, an assay is described for measuring Bcl-2 protein//BAD protein in breast cell line culture MCF-7, using the signal generating system as described above. The signal generating system is similar in function and structure to the signal generating systems as described above and in Fig. 2. The signal generating system comprises a first unit 102 and a second unit 106. The first unit comprises an antibody that binds to an antigenic determinant of Bcl-2 (the binding moiety of 103 of Fig. 2). The binding moiety is conjugated to glucose oxidase which can catalyze the reaction between glucose and molecular oxygen, producing hydrogen peroxide. Hydrogen peroxide can function as a transmitter 112 in Fig. 2. The second unit comprises an antibody that binds to an antigenic determinant of BAD protein (the binding moiety 107 in Fig. 2). The binding moiety is conjugated to enzyme horseradish peroxidase. Horseradish peroxidase can catalyze the oxidation of various chromogenic substrates in the presence of the transmitter of hydrogen peroxide, generating a signal molecule 114 of Fig. 2. A range of chromogenic substrate can be used in connection with horseradish peroxidase, including diaminobenzidine (DAB), chloronaphthol, and aminoethycarbazole. The extent of the peroxidase-catalyzed oxidation can be measured by observing a color change in a chromogenic substrate or a change in fluorescence of a fluorogenic substrate. The assays are carried out as follows.

Sample Preparation: 1. Serum-starve breast cancer cell line culture (MCF-7) overnight before use. 2. Stimulate cell lines with HRG in culture media for 10 minutes at 370C. Exemplary doses of HRG are 0, 0.032, 0.16, 0.8, 4, 20, 100 nM for MCF-7 cells. 3. Aspirate culture media, transfer onto ice, and add lysis buffer (described below) to lyse cells in situ. 4. Scrape and transfer lysate to microfuge tube. Incubate on ice for 30 min. Microfuge at 14,000 rpm, 40C, for 10 min. 5. Collect supernatants as lysates and aliquot for storage at -8O0C until use.

Lysis Buffer (made fresh and stored on ice): Final ul Stock 1% Triton X-100 1000 10% 20 mM Tris-HCl (pH 7.5) 200 I M 10O mM NaCl 200 5 M 50 mM NaF 500 I M 50 mM Na beta-glycerophosphate 1000 0.5 M I mMNa3VO4 100 0.1 M 5 mM EDTA 100 0.5 M 10 ug/ml pepstatin 100 1 mg/ml 1 tablet (per 10 ml) Roche Complete protease inhibitor (#1836170) N/A N/A Water 6500 N/A 10 ml Total

The total assay volume is 40 ul. The lysate volume is adjusted to 10 ul with lysis buffer. The antibodies are diluted in lysis buffer up to 20 ul. Typically -5000 to 500,000 cell- equivalents of lysates is used per reaction. Procedure: Working concentrations of pre-mixed antibodies prior to adding into reaction: anti-BAD at 1O nM anti-Bcl-2 at 1O nM Universal Standard US-I at 700 nM are attached directly to antibodies by reacting an NHS-ester of a molecular tag precursor (see Figs. 4A-4J) with free amines on the antibodies using conventional techniques, e.g. Hermanson (cited above). 6. Dissolve 6 mg of Diaminobenzidine (DAB) in 9 ml of 0.05M Tris Buffer (pH 7.6) and add to the antibody mix. 7. To assay 96-well filter plate (Millipore MAGVN2250), add 20 ul antibody mix to 10 ul lysate and incubate for 1 hour at 40C. 8. Add 200 ul wash buffer and apply vacuum to empty. 9. Detect the brown product measured spectrophotometrically using an ELISA-plate reader at an appropriate optical density.

Data Analysis:

1. Normalize relative signal of each signal molecules against that of internal Universal Standard US-I. 2. Subtract RFU of "no lysate" background control from corresponding normalized measurement of signal molecules. The detection of signal molecules indicates the presence of Bcl-2//BAD protein complexes. The strength of the signal indicates the amount of Bcl-2//BAD protein in the samples. The examples provided are for illustrative purposes only, and should not be construed to limit the scope of the invention, which is defined by the claims and specification as a whole. The invention now having been fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications set forth herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporate by reference.