WO2006031815A2 - Procedes et compositions de dosages de proximite - Google Patents

Procedes et compositions de dosages de proximite Download PDF

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WO2006031815A2
WO2006031815A2 PCT/US2005/032570 US2005032570W WO2006031815A2 WO 2006031815 A2 WO2006031815 A2 WO 2006031815A2 US 2005032570 W US2005032570 W US 2005032570W WO 2006031815 A2 WO2006031815 A2 WO 2006031815A2
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unit
transmitter
signal
complex
binding moiety
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PCT/US2005/032570
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WO2006031815A3 (fr
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Sharat Singh
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Monogram Biosciences, Inc.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • This invention relates to methods and compositions for detecting and/or quantifying modifications to or complexes of biological molecules.
  • 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.
  • the present invention is directed to methods and compositions for identifying analytes in a sample, including samples comprising fixed sample specimens and tissue specimens.
  • 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.
  • the first unit is capable of enzymatically producing the transmitter having an effective proximity.
  • the first unit is capable of non-enzymatically, such as chemically, producing the transmitter having an effective proximity.
  • the first unit comprises glucose oxidase
  • the hydrogen peroxide it generates functions as the transmitter
  • 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.
  • 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.
  • the detectable property of the signal molecule be modified or enabled by the cleavage of the signal molecule from the second unit.
  • 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.
  • 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.
  • 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.
  • the amount of a particular component in a complexed state versus a non-complexed state may be determined.
  • the ratio of monomer:dimer for a cellular component is measured by the subject assay methods and compositions.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • sample is also meant to refer to the prepared material to be analyzed in the assay.
  • 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • peroxidase such as horseradish peroxidase
  • 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.
  • 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.
  • the phenylamine derivatives itself i.e. in the reduced state
  • 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.
  • transmitters examples 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the signal molecule may be a fluorophore that lacks a mobility tag, or a molecule having a distinct mass.
  • FIG. 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 (e 2 ) 108, that in the presence of transmitter 112 is capable of generating a signal molecule 114.
  • enzyme (e 2 ) 108 that in the presence of transmitter 112 is capable of generating a signal molecule 114.
  • 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.
  • transmitter 112 is a substrate
  • other co-substrates or co-factors represented by (u') 113
  • transmitter 112 is a substrate
  • other co-substrates or co-factors represented by (u') 113
  • 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.
  • 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.
  • 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 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.
  • the subject invention is used to determine the extent of formation of a homodimer complex.
  • FIG 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.).
  • 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.
  • 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.
  • 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.
  • FIG. 5C 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.
  • the detection of the signal 523 from conjugate 520 unambiguously indicates formation of the homodimer.
  • 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.
  • the noncovalent complexes can be cell surface receptor complexes, receptor-ligand complexes or intracellular molecular complexes.
  • the amount of the noncovalent complexes is measured.
  • 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.
  • non-covalent complexes are selected from the receptor dimers listed in Table 1.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • protein-protein complexes that include, but are not limited to, the proteins of Tables 3-5.
  • the human forms of the following proteins and protein families are intended.
  • Protein-Protein Complexes in Apoptotic Pathways where "protein l//protein 2" indicates a complex comprising protein 1 and protein 2)
  • Certain intracellular complexes are indicative of the apoptotic status of a patient suffering from a disease, such as a cancer, characterized by aberrant apoptosis.
  • 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- X L //BAD, Bcl-2//BAD, 14-3-3//BID, BID//BAK, BAX//Bcl-2, BC1-X L //BIK, Bcl-2//BIK, NF- kB//I-kB, BID//Bcl-2, Bcl-XJ/BID, Bcl-2//BID, FADD//caspase-9, BID//Bcl-X L , Bcl-X L //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//
  • 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.
  • intracellular complexes include, but are not limited to, NF-kB//I-kB.
  • 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;
  • 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.
  • intracellular complexes include, but are not limited to, IGF-1R//PI3K, IGF-1R//Shc, and IGFR//IRS1.
  • intracellular complexes include, but are not limited to, VEGFR//Shc, VEGFR//PI3K, VEGFR//Src, and VEGFR//FRS2.
  • 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.
  • 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 37 0 C. 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 4 0 C. The fixed pellet is embedded in paraffin using a Miles Tissue Tek III Embedding Center, after which 10 ⁇ m 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 6O 0 C.
  • 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 ⁇ L 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.
  • EZ-Dewax reagent Biogenex, San Ramon, CA
  • 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).
  • IX Antigen Retrieval solution Biogenesis, Brentwood, NH
  • Blocking buffer is IX Perm/Wash solution with protease inhibitors (Roche), phosphatase inhibitors (sodium floride, sodium vanadate, ⁇ -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 4 0 C.
  • the sections are then washed three times with 100 ⁇ L Perm/Wash containing protease and phosphatase inhibitors, after which 50 ⁇ L 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 4 0 C 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 ⁇ L 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.
  • 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.
  • 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.
  • DAB diaminobenzidine
  • 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.
  • MCF-7 Serum-starve breast cancer cell line culture
  • HRG Serum-starve breast cancer cell line culture
  • Exemplary doses of HRG are 0, 0.032, 0.16, 0.8, 4, 20, 100 nM for MCF-7 cells.
  • Lysis Buffer (made fresh and stored on ice):
  • 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.
  • 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 4 0 C.
  • DAB Diaminobenzidine

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Abstract

L'invention concerne des procédés et des compositions destinés à mettre en oeuvre des dosages de proximité dans des échantillons de spécimens fixés. Ces dosages sont, plus particulièrement, utiles dans la détection de complexes multicomposant de molécules biologiques telles que des protéines, des peptides, des hormones, de l'ADN, de l'ARN et analogue. Dans l'un de ses modes de réalisation, le procédé consiste à fournir un système générant des signaux comportant une première unité capable de produire de façon enzymatique un transmetteur et possédant une première fraction de liaison, et une seconde unité qui dépend du transmetteur afin de produire une molécule signal et possédant une seconde fraction de liaison, à combiner dans des conditions de liaison l'échantillon et le système générant des signaux de sorte que les première et seconde fractions de liaison se lient à leur site de liaison respectif sur différents composants du complexe de sorte que la seconde unité se trouve à proximité effective du transmetteur produit par la première unité afin que les molécules signal soient produites, et à analyser la présence de molécules signal, dans lequel le complexe multicomposant est détecté. L'invention concerne également des compositions possédant une unité enzymatique permettant de produire un transmetteur qui génère ensuite de façon non enzymatique un signal au niveau de la seconde unité.
PCT/US2005/032570 2004-09-13 2005-09-13 Procedes et compositions de dosages de proximite WO2006031815A2 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110275097A9 (en) * 2006-09-21 2011-11-10 Prometheus Laboratories Inc. Drug selection for lung cancer therapy using antibody-based arrays
EP2388324A1 (fr) * 2010-05-20 2011-11-23 Siemens Healthcare Diagnostics Inc. Procédé destiné à la régénération de tissus
US20150051107A1 (en) * 2006-09-21 2015-02-19 Nestec S.A. Antibody-based arrays for detecting multiple signal transducers in rare circulating cells
US9274116B2 (en) 2008-02-25 2016-03-01 Nestec S.A. Drug selection for breast cancer therapy using antibody-based arrays
US9664683B2 (en) 2011-09-02 2017-05-30 Pierian Holdings, Inc. Profiling of signal pathway proteins to determine therapeutic efficacy
US9719995B2 (en) 2011-02-03 2017-08-01 Pierian Holdings, Inc. Drug selection for colorectal cancer therapy using receptor tyrosine kinase profiling
CN108226467A (zh) * 2016-12-13 2018-06-29 美天施生物科技有限责任公司 利用具有两个可释放结合部位的缀合物的可逆细胞标记
US10473640B2 (en) 2006-09-21 2019-11-12 Société des Produits Nestlé S.A. Drug selection for gastric cancer therapy using antibody-based arrays

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4281061A (en) * 1979-07-27 1981-07-28 Syva Company Double antibody for enhanced sensitivity in immunoassay
US4758080A (en) * 1986-12-18 1988-07-19 Cornell Research Foundation, Inc. Pointspread retinoscope
US5532138A (en) * 1990-04-26 1996-07-02 Behringwerke Ag Method and kits for determining peroxidatively active catalysts
US5843666A (en) * 1994-09-02 1998-12-01 Lumigen, Inc. Chemiluminescent detection methods using dual enzyer-labeled binding partners
US6159699A (en) * 1994-05-10 2000-12-12 Molecular Light Technology Limited Enzyme linked chemiluminescent assay

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4281061A (en) * 1979-07-27 1981-07-28 Syva Company Double antibody for enhanced sensitivity in immunoassay
US4758080A (en) * 1986-12-18 1988-07-19 Cornell Research Foundation, Inc. Pointspread retinoscope
US5532138A (en) * 1990-04-26 1996-07-02 Behringwerke Ag Method and kits for determining peroxidatively active catalysts
US6159699A (en) * 1994-05-10 2000-12-12 Molecular Light Technology Limited Enzyme linked chemiluminescent assay
US5843666A (en) * 1994-09-02 1998-12-01 Lumigen, Inc. Chemiluminescent detection methods using dual enzyer-labeled binding partners

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9285369B2 (en) * 2006-09-21 2016-03-15 Nestec S.A. Antibody-based arrays for detecting multiple signal transducers in rare circulating cells
US20150051107A1 (en) * 2006-09-21 2015-02-19 Nestec S.A. Antibody-based arrays for detecting multiple signal transducers in rare circulating cells
US9250243B2 (en) * 2006-09-21 2016-02-02 Nestec S.A. Drug selection for lung cancer therapy using antibody-based arrays
US10527622B2 (en) 2006-09-21 2020-01-07 Société des Produits Nestlé S.A. Antibody-based arrays for detecting multiple signal transducers in rare circulating cells
US20110275097A9 (en) * 2006-09-21 2011-11-10 Prometheus Laboratories Inc. Drug selection for lung cancer therapy using antibody-based arrays
US10473640B2 (en) 2006-09-21 2019-11-12 Société des Produits Nestlé S.A. Drug selection for gastric cancer therapy using antibody-based arrays
US10436786B2 (en) 2008-02-25 2019-10-08 Société des Produits Nestlé S.A. Methods for detecting truncated receptors using antibody-based arrays
US9274116B2 (en) 2008-02-25 2016-03-01 Nestec S.A. Drug selection for breast cancer therapy using antibody-based arrays
WO2011144416A1 (fr) * 2010-05-20 2011-11-24 Siemens Healthcare Diagnostics Inc. Matière de contrôle employée pour réaliser des tests faisant intervenir des acides nucléiques
EP2388324A1 (fr) * 2010-05-20 2011-11-23 Siemens Healthcare Diagnostics Inc. Procédé destiné à la régénération de tissus
US9719995B2 (en) 2011-02-03 2017-08-01 Pierian Holdings, Inc. Drug selection for colorectal cancer therapy using receptor tyrosine kinase profiling
US10401364B2 (en) 2011-02-03 2019-09-03 Soiété Des Produits Nestlé S.A. Drug selection for colorectal cancer therapy using receptor tyrosine kinase profiling
US9664683B2 (en) 2011-09-02 2017-05-30 Pierian Holdings, Inc. Profiling of signal pathway proteins to determine therapeutic efficacy
CN108226467A (zh) * 2016-12-13 2018-06-29 美天施生物科技有限责任公司 利用具有两个可释放结合部位的缀合物的可逆细胞标记
CN108226467B (zh) * 2016-12-13 2022-11-01 美天施生物科技有限两合公司 利用具有两个可释放结合部位的缀合物的可逆细胞标记

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