CN1829915A - Surface receptor complexes as biomarkers - Google Patents

Abstract

The invention is directed to a new class of biomarker in patient samples comprising dimers of ErbB cell surface membrane receptors. In one aspect, the invention includes a method of determining the status of a disease or healthful condition by correlating such condition to amounts of one or more dimers of ErbB cell surface membrane receptors measured directly in a patient sample, in particular a fixed tissue sample. In another aspect, the invention includes a method of determining a status of a cancer in a specimen from an individual by correlating measurements of amounts of one or more dimers of ErbB cell surface membrane receptors in cells of the specimen to such status, including presence or absence of a pre-cancerous state, presence or absence of a cancerous state, prognosis of a cancer, or responsiveness to treatment. Preferably, methods of the invention are implemented by using sets of binding compounds having releasable molecular tags that are specific for multiple components of one or more types of receptor dimers. After binding, molecular tags are released and separated from the assay mixture for analysis.

Description

The surface receptor compound is as biomarker

Invention field

The present invention relates generally to biomarker, specifically, relates to and uses the cell surface receptor compound, is used as biomarker as dimer or oligomer

Background of invention

Biomarker is a kind of characteristic body that can measure objectively and calculate, as normal biological process, pathogenic course or at the indicant of the pharmacological reaction of treatment intervention means, Atkinson etc., Clin.Pharmacol.Ther., 69:89-95 (2001).Biomarker in character, measure complexity and fasten and be very different, Frank etc. for example, Nature ReviewsDrug Discovery, 2:566-580 (2003) with the pass of object physiological status.There's a widespread conviction that for people, and the effective biomarker of development of new both can significantly reduce the cost of health care and drug development, can improve the treatment to multiple disease and health again greatly.For this reason, people have done a large amount of effort, are intended to utilize new technology to find the biomarker of newtype, for example, and Petricoin etc., Nature Reviews Drug Discovery, 1:683-695 (2002); Sidransky, Nature Reviews Cancer, 2:210-219 (2002).

The interaction of cell surface film component plays an important role for sending extracellular signal to cell in normal physiologic state and morbid state.Particularly, the cell surface receptor of many types has passed through dimerization, oligomerization or bunch collection effect, these effects and outer activity of born of the same parents or signal, transduction as the ligand-receptor combination is relevant, form the reaction of cell, for example breed, increase or reduce expression of gene or the like, as George etc., Nature Reviews Drug Discovery, 1:808-820 (2002); Mellado etc., Ann.Rev.Immunol., 19:397-421 (2001); Schlessinger, Cell, 103:211-225 (2000); Yarden, Eur.J.Cancer, 37:S3-S8 (2001).The effect of sort signal transduction activity in disorders such as cancers become the closely object of research of people, and causes having developed several new drugs and drug candidate, for example Herbst and Shin, Cancer, 94:1593-1611 (2002); Yarden and Sliwkowski, Nature Reviews Molecular Cell Biology, 2:127-137 (2001); McCormick, Trends in Cell Biology, 9:53-56 (1999); Blume-Jensen and Hunter, Nature, 411:355-365 (2001).

The expression of individual cells surface receptor successfully has been used as biomarker, for example the United States Patent (USP) 4968603 (Her2 expression) of Slamon etc.Yet independent single expression of receptor level is to morbid state or health indicant reliably always, Chow etc. for example, Clin.Cancer Res., 7:1957-1962 (2001) (expression of EGFR or Her1).Although turning into to be used in cell and the lysis, receptor dimerization plays an important role, but also being used, the expression of receptor dimer do not make biomarker, part is because the inconvenience of current measuring technique and shortage sensitivity, and can't or can not actual use this technology come concerning formalin fixed and/or measure for the too little patient's sample of amount analyzing, Price etc. for example, Methods in Molecular Biology, 218:255-267 (2003); Stagljar, Science STKE 2003, pe56 (2003); Koll etc., the open WO 2004/008099 of international monopoly; Golemis, editor, Protein-Protein Interactions (Cold Spring HarborLaboratory Press, New York, 2002); Sorkin etc., Curr.Biol., 10:1395-1398 (2000); McVey etc., J.Biol.Chem., 17:14092-14099 (2001); Salim etc., J.Biol.Chem., 277:15482-15485 (2002); Angers etc., Annu.Rev.Pharmacol.Toxicol., 42:409-435 (2002); Szollosi etc., Reviews in MolecularBiotechnology, 82:251-266 (2002); Matko etc., Meth.in Enzymol., 278:444-462 (1997); The United States Patent (USP) 5192660 of Reed-Gitomer.

In view of the foregoing, according to relating to crucial born of the same parents' internal procedure, as the existence of the cell surface receptor dimer of signal transduction process or compound, do not exist and/or feature or ratio, the biomarker of new kind is provided in patient's sample, it provides new-type instrument for diagnosis, prognosis, patient's classification and drug development, thereby will promote the development of medical domain.

Summary of the invention

The present invention relates to the new biomarker that contains receptor complex of a class, it is present at patient's cell or tissue sample, comprise by conventional method such as sample freezing or that fixation is preserved, cell surface membrane in.On the one hand, the present invention includes a kind of method of measuring morbid state or health status, wherein this class situation is associated with one or more receptor complexes in a large amount of cell surface membrane that comes from individual cells or tissue sample.On the other hand, the present invention includes the method for a kind of mensuration from cancerous state in the sample of individuality, wherein the measured value with one or more a large amount of in sample surface receptor compounds is associated with this situation.The present invention also provides a kind of method of predicting dimer drugs with function effect in addition, and for example, in treatment of cancer, this method is associated the dimeric value volume and range of product of drug responsiveness with the possibility that effect or patient reply.

In a kind of mode, the present invention can measure the disease of patient state, the feature of disease that described patient takes a disease is, the unconventionality expression of one or more cell surface receptor compounds, and step is as follows: (i) measure in patient's sample every kind amount in one or more cell surface receptor compounds; (ii) above-mentioned every kind of amount is compared with its respective amount in standard model; And (iii) the difference of corresponding respectively amount in the amount in patient's sample and the standard model is associated with the disease of patient state.Patient's sample can be fixing or freezing; Yet preferably, patient's sample is fixed for adopting conventional method.

In the special mode of another kind, the invention provides a kind of according to patient's sample, especially the measured value of fixed sample, predict the validity of the dimer drugs with function that is used for the treatment of unusual fiberization state, perhaps the patient is to its reactivity, described dimer drugs with function acts on the pdgf receptor compound, include but not limited to one or more PDGFR α homodimers, PDGFR β homodimer, PDGFR α-PDGFR β heterodimer, PDGFR-SHC, PDGFR-PI3K, the Her1-PDGFR receptor dimer, the Her2-PDGFR receptor dimer, Her3-PDGFR receptor dimer and PDGFR-IGF-1R receptor dimer.In another embodiment, this pdgf receptor compound is selected from the group that PDGFR α homodimer, PDGFR β homodimer and PDGFR α-PDGFR β heterodimer constitutes.

In the special mode of another kind, the invention provides a kind of according to patient's sample, especially the measured value of fixed sample, predict that being used for the treatment of abnormal vascular generates, especially the validity of the dimer drugs with function in the solid tumor, perhaps the patient is to its reactivity, described dimer drugs with function acts on the vegf receptor compound, include but not limited to one or more VEGFR1 homodimers, the VEGFR2 homodimer, the VEGFR1-VEGFR2 heterodimer, the VEGFR2-VEGFR3 heterodimer, the VEGFR2-SHC compound, and VEGFR3-SHC compound.

In another kind of mode, the invention provides a kind of by measuring the amount of one or more cell surface membrane receptors, the perhaps correlative of a plurality of cell surface membrane receptor dimers in these patients' cell or tissue sample, thus determine the method for patient disease state.In one embodiment, at least two kinds of reagent have been adopted, here be called reagent to measuring these dimers, these two kinds of reagent have specificity for every kind of dimeric heterogeneity: a kind of reagent, here be called the cracking probe, have cracking and cause part, it can be initiated with the responsive key of cracking in its direct short range; And another kind of reagent, be called binding compounds here, have one or more molecular labels, by causing the connector connection of part cracking by described cracking.According to this embodiment, no matter when these heterogeneities form dimer, have all introduced the cleavable connector in this cracking causes effective cracking short range partly, make molecular label be released.Then the molecular label that discharges is separated from reaction mixture also quantitatively to measure dimeric formation.

In another aspect of the present invention, reference has been measured the receptor dimer in patient's sample; That is, dimeric amount is the ratio of measured value with the total amount measured value of this dimeric other composition of a kind of composition of existing in this dimer, and no matter whether it is present in this dimer still is that form with monomer exists.In one embodiment, typical measured value comprises the peak heights or the peak area of the electrophoresis pattern centre forward value relevant with the specific molecular label.

In a special embodiment of this mode, the invention provides a kind of by measuring the amount of a plurality of Her receptor dimers in the fixing organization sample come from the patient simultaneously, thereby be determined at the method for cancerous state among this patient.These dimers can utilize at least two kinds every kind of dimeric heterogeneity is had specific reagent to measure: a kind of reagent, be called the cracking probe here, and have cracking and cause part, it can be initiated with the responsive key of cracking in its direct short range; And another kind of reagent, be called binding compounds here, have one or more molecular labels, by causing the connector connection of part cracking by described cracking.According to this embodiment, no matter when the Her receptor dimer forms, and has all introduced the cleavable connector of binding compounds in this cracking causes effective cracking short range partly, makes molecular label be released.Then the molecular label that discharges is separated from reaction mixture and quantitative quantity with mensuration Her receptor dimer.In another embodiment of this mode, measured the relevant amount of a plurality of Her receptor dimers, and interrelated with patient's cancerous state.The cancer example includes but not limited to, breast cancer, oophoroma and prostate cancer.

Biomarker provided by the invention comprises the measured value to receptor complex in patient's sample.Especially, can the figure of receptor complex quantity is relevant with the disease of patient state, and in some embodiments, it can be associated to the reaction possibility for the treatment of with the effect and the patient of prognosis, dimer drugs with function.According to the present invention, by directly measuring the receptor complex in patient's sample, can overcome shortcoming of the prior art, need not to cultivate or otherwise handle the cell or tissue sample, for example heterograft can increase cost and workload like that and brings noise and possible illusion can for the final reading of measuring.The present invention also provides a kind of phosphorylation to intracellular receptor, or the substituting measuring method of other the trim that destroys easily in the specimen preparation process.This substituting measuring method is based on the mensuration to compound, for example PI3K or SHC-receptor complex or the like, and it depends on the above-mentioned modification to its formation, and less is subjected to the influence of specimen preparation process.

The accompanying drawing summary

Figure 1A-1F represents to use releasable molecular label to measure the diagram of receptor dimer quantity.

Fig. 1 G-1H represents to use releasable molecular label to measure the diagram of cell surface receptor compound in the fixing tissue samples.

Fig. 2 A-2E represents the embodiment diagram of the inventive method, and it has described the figure of the dimeric correlative of a plurality of acceptor types.

Fig. 3 A-3C represents molecular label is connected method diagram on the antibody.

Fig. 4 A-4E represents to utilize the SKBR-3 of method mensuration of the present invention and the acceptor heterodimer data of BT-20 cell lysate.

Fig. 5 A-5C represents to utilize the data of the acceptor heterodimer of the human normal and tumour breast tissue sample that method of the present invention measures.

Fig. 6 A and 6B represent that the present invention is used for detecting the determination data of BT-20 cell lysate homodimer and Her1 phosphorylation.

Fig. 7 represents that the present invention measures the Her2 homodimer quantity data of MCF-7 and SKBR-3 clone.

Fig. 8 A-8B represents that the present invention detects the determination data of Her1 and Her3 heterodimer on the cell that the heregulin (HRG) to rising concentration replys.

Fig. 9 A and 9B have represented that under the situation that epidermal growth factor (EGF) concentration raises, the quantity of the Her1-Her3 heterodimer on 22Rv1 and A549 cell increases data respectively.

Figure 10 A-10C has represented the expression data of the heterodimer of IGF-1R and various Her acceptors in the freezing sample of human mammary tissue.

Figure 11 A-11D represents to be used to detect the mensuration design and the test findings of PI3 kinases-Her3 receptor activation compound.

Figure 12 A-12D represents to be used to detect the mensuration design and the test findings of Shc/Her acceptor-aptamers compound.

Figure 13 represents the correlation data between Her2-Her3 heterodimer and PI3K/Her3 compound are expressed in the tumour cell.

Figure 14 A-14B represents the Her1-Her2 that obtains and the measured value of Her2-Her3 receptor dimer quantity from normal galactophore tissue's sample and breast tumor tissues sample.

Figure 15 A-15G has represented the Her1-Her1 in the section of the fixedly ball of cancerous cell line and the measured value of Her2-Her2 homodimer and Her1-Her2 and Her2-Her3 heterodimer.

Definition

" antibody " refers to and can be combined specifically with particular space and the polar structure of another molecule, and it is white therefore to be defined as complementary with it immune globulin. Antibody can be single clone or many clones, and can enough technology well known in the art prepare, such as immune host and gather serum (many clone), or cell system and collection secretion albumen (single clone) by preparing continuous hybrid, or by the clone with express the nucleotides sequence or its mutagenesis type, the described nucleotides sequence amino acid sequence required with natural antibody specific binding of encoding at least. Antibody can comprise white or its fragment of complete immune globulin, and immune globulin comprises all kinds and hypotype in vain, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM etc. Antibody fragment can comprise Fab, Fv, F (ab ') 2, Fab ' etc. In addition, can use the white aggregation of immune globulin, polymer and coupling thing or its fragment in the time of suitably, as long as kept the binding affinity to specific polypeptide. Immunity is measured, comprise that employing can discharge this class mensuration of molecular label (following described), the manufacturing of the antibody of middle use and select guiding technology in textbook and handbook, to find with being easy to, for example, Harlow and Lane, Antibodies:A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1988); Howard and Bethell, Basic Methods in Antibody Production and Characterization (CRC Press, 2001); The Wild chief editor, The Immunoassay Handbook (Stockton Press, New York, 1994), etc.

" antibody binding compositions " refers to comprise molecule or the molecular complex of one or more antibody or its fragment, and its binding specificity comes from this antibody or antibody fragment. The antibody binding compositions includes but not limited to, (i) antibody pair, and wherein first antibody is combined with the target molecular specificity, the constant district specific binding of the 2nd antibody and first antibody; Biotinylated antibody, in target molecule and chain affinity prime, wherein the albumen of the affine element of this chain is become by the part derivation such as molecular label or sensitising agent etc. by biotin moiety (moiety) specific binding for it; (ii) to the target molecular specific and and polymer, such as the antibody of glucan coupling, wherein this polymer is by becoming such as the part derivation of molecular label or sensitising agent, it directly connects or indirectly connects by the chain Avidin-Biotin by covalent bond; (iii) to the target molecular specific and with the antibody of pearl, little pearl or other solid support coupling, wherein this support thing is directly or indirectly by such as the part derivation of molecular label or sensitising agent and become, or contains the latter's polymer.

" antigen determine bunch ", or " table position " refer to molecule, protein molecule normally, the site on the surface, can be combined with single antibody molecule in this site; Usually protein have that several or a plurality of different antigens determine bunch and can from multiple different specificity antibody reaction. Preferred antigen determines bunch to be the phosphorylation site.

Any molecule that " in conjunction with part " refers to be combined with analyte specifically and molecular label is can be directly or indirectly attached to it. The joint portion is divided and is included but not limited to, antibody, antibody binding compositions, peptide, protein, nucleic acid, and the molecular weight organic molecule that reaches 1000 dalton and consisted of by the atom that is selected from the group that following material consists of: hydrogen, carbon, oxygen, nitrogen, sulphur and phosphorus. Preferably, the joint portion is divided into antibody or antibody binding compositions.

" cancer " and " cancer " relates to or described in the mammal typically to be grown to the physiology state of feature without regular cell. The cancer example includes but not limited to, cancer section, lymthoma, blastocyte knurl, sarcoma and leukaemia. The more specifically example of these cancers comprises squamous cell cancer, little cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreas cancer, spongioblast knurl, uterine neck cancer, ovary cancer, liver cancer, bladder cancer, liver cell knurl, breast cancer, colon and rectum carcinoma, uterus endometrial carcinomas, saliva gland cancer, kidney, prostate cancer, vulva cancer, thyroid gland cancer, liver cancer and various types of head and neck cancer.

" compound " refers to aggregate or the aggregation of the molecule that directly or indirectly contacts with each other here. In a kind of mode, " contact " or more particularly, " directly contact " relates to the compound of molecule, perhaps relate to specificity or special combination, refer to that two or more molecules enough approach so that non-covalent attraction effect, such as Fan Dehuali, hydrogen bond, ion and hydrophobic interaction etc., be the leading role of molecular force. In this kind mode, because this compound is more more favourable than non-gathering or non-combined state that it forms molecule at thermodynamics under condition determination, so this molecular complex is more stable. Here employed " compound " is usually directed to the stable aggregate of two or more protein, also is called " protein-protein compound " of equal valuely. Be typically the most, " compound " relates to the stable aggregate of two kinds of protein.

" dimerization body " relates to the cell surface membrane acceptor, refers to the compound of the membrane-bound receptor albumen that two or more are identical or different. The dimerization body of same receptor is called " homotype dimerization body ", and the dimerization body of isoacceptor is not called " special-shaped dimerization body ". The dimerization body is comprised of two kinds of acceptors that contact with each other usually. The dimerization body can be by passive process, such as model ylid bloom action etc., as above described in the definition of " compound ", be formed on the cell surface membrane, perhaps the dimerization body also can form by process initiatively, the dimerization that for example causes by part turns usefulness into, covalently bound, with the interaction of composition in the born of the same parents etc., for example Schlessinger, Cell, 103:211-225 (2000). Term as used herein " dimerization body " should be understood to finger " cell surface membrane receptor dimer ", unless based on context other understanding arranged.

" disease state " includes but not limited to following feature: catch the possibility of disease, whether the existence of disease to be, the prognosis of disease seriousness, and the patient that causes of the special treatment reagent by the receptor complex effect is to the reaction possibility for the treatment of. As for cancer, " disease state " further comprises the detection to pre-cancer or cancer cell or tissue, selection to patient, it is by one or more receptor complexes, such as one or more receptor dimers, may react after the treatment of the treatment agent of effect, and this kind treated the improvement result for the treatment of of agent. In a kind of mode, the disease state relates to the Her receptor complex, refers to that the cancer patient passes through the dimerization body drugs with function of Her, to the reaction possibility for the treatment of. Preferably, this kind cancer patient is breast cancer or ovarian cancer patients, and this kind Her dimerization body drugs with function comprises Omnitarg^TM(2C4), Herceptin, ZD-1839 (Iressa), and OSI-774 (Tarceva). In yet another aspect, the disease state that relates to the PDGFR receptor complex refers to suffer from inappropriate fiber and turns to the patient of disease of feature for the reaction possibility of PDGFR dimerization body drugs with function treatment. Preferably, this class disease comprises cancer and kidney fibrosis. In another kind mode, the disease state that relates to the vegf receptor compound refers to suffer from inappropriate Angiogenesis, is that the patient of disease of feature is for the reaction possibility of VEGF dimerization body drugs with function treatment such as solid tumor.

" ErbB acceptor " or " Her acceptor " is the receptor protein tyrosine kinase that belongs to the ErbB receptor family, comprises EGFR (" Her1 "), ErbB2 (" Her2 "), ErbB3 (" Her3 ") and ErbB4 (" Her4 ") acceptor.The ErbB acceptor generally includes the extracellular region territory that can connect the ErbB part; Lipophilicity is striden diaphragm area; Tyrosine kinase domain in the conservative born of the same parents; But and the c-terminus signal domain of staying the tyrosine residue of having hidden several phosphorylations.The ErbB acceptor can be the ErbB acceptor of native sequences or the variant of its amino acid sequence.The native sequences that preferred this ErbB acceptor is human ErbB acceptor.In a kind of mode, the ErbB acceptor comprises the clipped form of Her acceptor, includes but not limited to, EGFRvIII and p95Her2 are at Chu etc., Biochem.J., 324:855-861 (1997); Xia etc., Oncogene, 23:646-653 (2004); Or the like in have disclosed.

Term " ErbB1 ", " EGF-R ELISA " and " EGFR " and " Her1 " can replace use mutually here, refer to native sequences EGFR, for example at Carpenter etc., disclosed among the Ann.Rev.Biochem.56:881-914 (1987), comprise its variant (for example, deletion mutant of EGFR, as Humphrey etc., PNAS (USA) 87:4207-4211 (1990)).ErbB1 refer to encode the EGFR protein product gene.Can comprise that MAb 579 (ATCC CRL RB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCCCRL 8509) are (referring to U.S. Patent No. 4943533 in conjunction with the antibody example of EGFR, Mendelsohn etc.) with and variant, for example chimerized 225 (C225) and reshaped human antibody 225 (H225) (referring to WO 96/40210, Imclone Systems Inc).

" Her2 ", " ErbB2 ", " c-Erb-B2 " can replace use mutually.Be meant the human protein when except as otherwise noted, using term " ErbB2 ", " c-Erb-B2 " and " Her2 " here.This mankind ErbB2 gene and ErbB2 protein for example have, Semba etc., PNAS (USA) 82:6497-650 (1985) and Yamanoto etc., Nature 319:230-234 (1986) (Genebank accession number X03363).The antibody example that combines with the Her2 specificity is at United States Patent (USP) 5677171; 5772997; Fendly etc., CancerRes. has disclosed among the 50:1550-1558 (1990) etc.

" ErbB3 " and " Her3 " refers to receptor polypeptides, for example U.S. Patent No. 5183884 and 5480968 and PNAS (USA) 86:9193-9197 (1989) such as Kraus in disclosed, comprise its variant.Can in U.S. Patent No. 5968511, describe to some extent in conjunction with the antibody example of Her3, for example 8B8 antibody (ATCC HB 12070).

Term " ErbB4 " and " Her4 " refer to receptor polypeptides here, for example at european patent application No.599274; Plowman etc., Proc.Natl.Acad.Sci.USA, 90:1746-1750 (1993); And Plowman etc., Nature described in the 366:473-475 (1993), comprises its variant, as disclosed Her4 heterogeneous among the WO 99/19488.

" IGF-1R " or " IGF-1R " refers to human receptor's tyrosine kinase, its roughly with EMBO J. such as Ulrich, 5:2503-2512 (1986) or Steele-Perkins etc., J.Biol.Chem., those disclosed in the 263:11486-11492 (1988) are identical.

" separation " relates to polypeptide or protein, is meant basically to isolate from the composition of its natural surroundings.Preferably, the polypeptide of separation or protein are a kind of comparing with its natural surroundings composition by the identical sequence polypeptide of eight ten at least percent weight or the composition that protein constitutes; More preferably, polypeptide or protein that said composition is compared with its natural surroundings composition by the identical sequence of nine ten five percent weight at least constitute, and more preferably, polypeptide or the protein compared with its natural surroundings composition by the identical sequence of nine ten nine at least percent weight of said composition constitutes.Most preferably, the polypeptide of this separation or protein are homogeneous compositions, and by the conventional after separating based on the two-dimensional gel electrophoresis of molecular weight and isoelectric point, it can be differentiated into single point.The scheme that two-dimensional gel electrophoresis by routine carries out this analysis is known for those of ordinary skills, for example, Hames and Rickwood chief editor, Gel Electrophoresis of Proteins:APractical Approach (IRL Press, Oxford, 1981); Scope, Protein Purfication (Springer-Verlag, New York, 1982); The Rabilloud chief editor, Proteome Research:Two-Dimensioned Gel Electrophoresis and Identification Methods (Springer-Verlag, Berlin, 2000).

" kit " refers to any induction system of transporting the material or the reagent that are used for implementing the inventive method.With regard to reaction assay, this induction system comprises and can store, transport to another place or (for example pay reaction reagent from the three unities, probe in appropriate containers, enzyme etc.) and/or the system of support material (for example, damping fluid is to written explanation that realize to measure etc.).For example, kit comprises one or more external packings (as box) that correlated response reagent and/or support material are housed.These contents can be together or are given predetermined acceptance person respectively.For example, first container can contain enzyme used in the mensuration, and second container comprises probe.

" number percent is identical " or similar terms relate to the comparison of canonical sequence and another sequence (i.e. " candidate " sequence) at this, in the best comparison of finger between two sequences, candidate sequence is identical on many subunits site with the canonical sequence that equates the mark percentage composition, described subunit is a nucleotide for the comparison of polynucleotide, is amino acid for the comparison of polypeptide." best comparison " of employed here term aligned sequences is meant the maximum match degree between the subunit and makes up minimizing of the number of gaps that adopts in the comparison.Percentage can adopt the algorithmic tool that can buy on the market to measure mutually on year-on-year basis, this algorithm is at Needleman and Wunsch, J.Mol.Biol., 48:443-453 (1970) (Wisconsin Sequence Analysis Package " GAP " program, Genetics Computer Group, Madison describes in WI) to some extent.Be used in this area make up comparison with calculate percentage mutually on year-on-year basis or other software package of other similarity measurement comprise " BestFit " program, algorithm based on Smith and Waterman, Advances in AppliedMathematics, 2:482-489 (1981) (Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, WI).In other words, for example, for obtaining to have and polypeptide with reference to amino acid sequence at least 95% same acid sequence, have in this canonical sequence nearly that 5% amino acid residue can lack or be replaced by another amino acid, the amino acid that perhaps accounts for canonical sequence total amino acid residue number 5% can insert in this canonical sequence.These conversion of canonical sequence may occur in this with reference to the amino of amino acid sequence or any position between c-terminus site or those endpoint locations, perhaps individually be dispersed in the middle of the residue of canonical sequence as a whole, perhaps be dispersed in this canonical sequence with form than adjacent group.Should be understood that when comparing candidate sequence can be the component or the fragment of big polypeptide or polynucleotide with canonical sequence of the present invention, and for calculate percentage mutually on year-on-year basis this comparison carried out of purpose relate to relevant component or fragment.

" phosphatidylinositols 3 kinase proteins " or " the PI3K albumen " that is equal to refer to a kind of human intracellular protein of human protein in organizing, NCBI accession number NP_852664, NP_852556 and NP_852665, and the identical substantially with it protein of amino acid sequence.

" platelet derived growth factor receptor " or " PDGFR " refers to and PDGFR α or PDGFR β or the substantially the same human receptor's tyrosine-kinase zymoprotein of its variant, at Heldin etc., PhysiologicalReviews describes among the 79:1283-1316 (1999) to some extent.In a kind of mode, the present invention includes by one or more dimers of measuring in following group and determine cancerous state, the early stage state of cancer, fiberization or hardening state: PDGFR α homodimer, PDGFR β homodimer and PDGFR α-PDGFR β heterodimer.Particularly, the fiberization condition comprises lung or kidney fibrosis, and hardening state comprises arteriosclerosis.Cancer includes but not limited to, breast cancer, the carcinoma of the rectum, spongioblastoma and oophoroma.Should be understood to finger " PDGFR α " or " PDGFR β " when relating separately to " PDGFR ".

" polypeptide " is meant a compounds of being made up of amino acid residue, amino acid residue by amido bond removed water between an amino acid carboxyl and another amino amino and chemistry links together.Polypeptide is the condensate of amino acid residue, and it can contain a large amount of this residues.Usually, except being made up of the amino acid of lesser amt, peptide and polypeptide are similar.Peptide is known as oligopeptides sometimes.There is not clear and definite differentiation between polypeptide and the peptide.For convenience's sake, in this open explanation and claim, term " polypeptide " generally will be used in reference to peptide and polypeptide.Amino acid residue can be natural or synthetic.

" protein " refers to polypeptide, and is synthetic by biological cell usually, is folded into fixing three-dimensional structure.The molecular weight of protein is usually about 5; 000 to about 5; 000; 000 or more than; more common about 5; 000 to about 1; 000; 000 molecular weight; and can comprise the modification after the translation; as acetylation; acidylate; the ADP-ribosylation; amidated; covalently bound lactochrome; covalently bound heme moiety; covalently bound nucleotide or nucleotide derivative; covalently bound lipid or lipid derivate; covalently bound phosphatidylinositols; crosslinked; cyclisation; form disulfide bond; farnesylation (farnesylation); demethylation; form covalent cross-linking; form cystine; form the pyroglutamic acid compound; formylation; γ-carboxylated; glycosylation; form the GPI anchor; carboxylated; iodate; methylate; the nutmeg acidylate; oxidation; phosphorylation; isoprenylation; racemic; selenoylation; sulphation; and ubiquinoneization; as Wold; F. Post-translationalProtein Modifications:Perspectives and Prospects; the 1-12 page or leaf; the B.C.Johnson chief editor; Academic Press; New York, 1983.Protein comprises, for example and and unrestricted, cell factor or interleukin, enzyme such as kinases, proteinase, galactosidase or the like, nucleoprotamine, histone, albumin, immunoglobulin (Ig), scleroprotein, phosphoprotein, mucin, chromoprotein, lipoprotein, nucleoprotein, glycoprotein, T-cell receptors, proteoglycan or the like.

" standard model " refers to one or more cells, xenograft or the tissue sample of the normal or non-disease conditions of table, with the measured value of patient's sample and its contrast to determine the amount minimizing whether excessive or in patient's sample of existing of receptor complex.The character of standard model is the problem of design alternative for specific mensuration, can obtain from patient's oneself normal structure or record, and perhaps obtains from the crowd's of healthy individual tissue.Preferably, the value that relates to the receptor complex amount in the standard model is to obtain under the experiment condition substantially the same with analog value in tested patient's sample.Standard model can be from the tissue of the same race with patient's sample, perhaps can be, and can select to have the feature that is consistent with the patient in order to the crowd who obtains this standard model tissue from different types of organizations, and for example age, sex, race or the like.Typically, in mensuration of the present invention, the amount of receptor complex in patient's sample is compared with corresponding value in the standard model, this standard model is made form in advance and average range, mean value and standard deviation is provided, or similar expression mode.

" receptor complex " refers to comprise the compound of at least a cell surface membrane receptor.Receptor complex can comprise the dimer of cell surface membrane receptor, perhaps one or more intracellular proteins, and aptamers albumen for example, it forms connection in various signal paths.The intracellular protein example that belongs to the part of receptor complex includes but not limited to, PI3K albumen, Grb2 albumen, Grb7 albumen, Shc albumen and Sos albumen, Src albumen, Cbl albumen, PLC γ albumen, Shp2 albumen, GAP albumen, Nck albumen, Vav albumen and Crk albumen.In a kind of mode, receptor complex comprises PI3K or Shc albumen.

The human kdr protein that " receptor tyrosine kinase " or " RTK " refers to have the intracellular kinase activity and be selected from protein RTK family, as Schlessinger, Cell, 103:211-225 (2000); Described in Blume-Jensen and Hunter (quote see on).The compound that comprises two kinds of receptor tyrosine kinase proteins in " receptor tyrosine kinase dimer " phalangeal cell surface film.In some modes, the receptor tyrosine kinase dimer can comprise two kinds of covalently bound receptor tyrosine kinase proteins.The dimeric example of RTK is listed in the Table I.The RTK dimer that receives publicity especially has Her receptor dimer and VGEFR dimer.

" sample " or " tissue sample " or " patient's sample " or " patient's cell or tissue sample " or " sample " all are meant the similar cell aggregation that obtains from experimenter or patient's tissue.The source of tissue sample can be a solid tissue, as comes from fresh, freezing and/or corrosion-resistant organ or tissue sample or biopsy or suction gas; Blood or any blood constituent; Body fluid is cerebrospinal fluid, amniotic fluid, ascites or tissue fluid for example; Or come from the experimenter conceived or grow up in the cell in any period.Tissue sample can comprise and the non-compound that mixes naturally of this tissue, and its essence is for example antiseptic, anticoagulant, damping fluid, fixing agent, nutrients, microbiotic or the like.In a kind of mode of the present invention, tissue sample or patient's sample are fixing, particularly the paraffin embedding sample of Chang Gui formalin fixed.During these samples typically were used in the receptor complex of the slice form of fixing organization measured, for example 3-10 μ m was thick, and wherein this fixing organization is arranged on microslide, or on the surface of equivalent.These samples also typically pass through conventional rehydrated process, and selectively, have carried out the part of antigen retrieval process as the determination test preprocessing process.

" separate picture " (separation profile) relates to the separation of molecular label, be meant table, figure, curve, column diagram, perhaps other represents the method that the signal strength data and the parameter of relevant molecular label for example concern between the residence time, the quality etc., and it provides the quantity reading or the measured value of all kinds molecular label that produces in the determination test.Separate picture can be electrophoresis pattern, chromatogram, electrochromatogram, mass spectrogram, or similarly represents according to the datagraphic of the isolation technics that is adopted." peak " or " band " or " district " that relate to separate picture are meant the zone that separating compound is concentrated.A determination test has a plurality of separation charts, for example, when different molecular labels has the different fluorescence labelings of unique emission spectrum, and gathers under a plurality of wavelength and during record data.In a kind of mode, thereby the molecular label that is discharged is separated owing to the difference of electrophoretic mobility forms electrophoresis pattern, and wherein different molecular labels is corresponding to peaks different on the electrophoresis pattern.Measured value to different or less overlapping adjacent peak value in the electrophoresis pattern is " electrophoretic resolution ", and it can be regarded as distance between the adjacent peak maximum divided by 4 times of higher value in two standard deviations of this peak value.Preferably, the resolution of adjacent peak value is at least 1.0, more preferably is at least 1.5, most preferably is at least 2.0.In given separation and detection system, required resolution can obtain by selecting a plurality of molecular labels, the electrophoretic mobility of these molecular label compositions has the difference of differentiating the peak number amount at least, this quantity depends on several factors known to a person of ordinary skill in the art, comprises signal detection system, the character of fluorescence part, the coefficient of diffusion of label, whether the existence of screening matrix, and the characteristic of electrophoresis equipment for example has or not length of passage, split tunnel or the like.Utilize the routine analyzer can the analytical electrophoresis collection of illustrative plates, thereby with the existence of molecular label, do not exist or the quantity data feature is associated, this program be for example disclosed in people's such as William the U.S. Patent Publication 2003/0170734A1.

" SHC " (representative " Src homology 2/ α-collagen is relevant ") refer to Pelicci etc. at Cell, in the aptamers protein family in essentially identical described in the 70:93-104 (1992), the RTK signalling channel (66,52 and 46kDalton) any one.In a kind of mode, SHC refers to the human form of this aptamers albumen.

" signalling channel " or " signal conduction pathway " refers to a series of molecule activity, it begins normally, and cell surface receptor combines with the phosphorylation site of cell surface receptor with the interaction or the interior molecule of born of the same parents of the outer part of born of the same parents, it has triggered a series of interaction of molecules, and wherein this a series of interaction of molecules has caused the regulation and control of gene expression in the pair cell nuclear." Ras-MAPK passage " refers to a kind of signalling channel, and it is included in the phosphorylation that forms after the Ras-GTP compound MAPK albumen." PI3K-Akt passage " refers to a kind of signalling channel, and it comprises by the phosphorylation of PI3K albumen to Akt albumen.

" special " or " specificity " relates to combining of a molecule and another molecule, for example to the binding compounds or the probe of target analyte or compound, refer to identification between probe and the target, contact and form stabilized complex, compound is less discerned, contacts or formed to this probe basically with other molecule simultaneously.In a kind of mode, " special " relates to first molecule and combines with second molecule, is meant that another molecule in first molecule and reaction or the sample discerns and form the degree of compound, and it can form the compound of maximum quantity with this second molecule.In a kind of mode, this maximum quantity accounts for 50% of first whole this compounds that molecule forms at least.In general, the molecule that relates to the specificity cohesive process has some zones in its surface or hole, can produce special identification between the molecule that is bonded to each other.The example of specificity combination comprises that antibody-AI, enzyme-substrate interact, form dual body or triple body, receptor-ligand binding or the like in polynucleotide and/or oligonucleotides.

" spectrally resolved " relates to a plurality of fluorescence labelings, the fluorescent emission wave band that refers to this mark is enough obvious, promptly be enough to not overlap, the molecular label that makes each label connect can be distinguished by the standard photodetector system according to the fluorescence signal that the respective markers thing is produced, for example, adopt bandpass filter and photomultiplier or the like, as exemplarily being described in U.S. Patent No. 4230558; 4811218 or the like, perhaps Wheeless etc., 21-76 page or leaf, in Flow Cytometry:Instrumentation and Data Analysis (AcademicPress, New York, 1985).

" basic identical " relates to the protein compared or the protein amino acid sequence in the related protein family, refer to or a kind of amino acid sequence of protein has five ten at least percent identically with another kind of protein, perhaps a kind of protein is the heterogeneous or the splicing variants of another kind of protein homologous genes.In a kind of mode, a kind of protein of substantially the same finger, or its amino acid sequence have eight ten at least percent identical with another kind of protein or its amino acid sequence.

Here employed " vegf receptor " or " VEGFR " relate to the cell receptor of vascular endothelial growth factor (VEGF), be a kind of being present on the vascular endothelial cell, and maintain in conjunction with the common cell surface receptor on its variant of human VEGF ability.Vegf receptor comprises VEGFR1 (also being called Flt1), VEGFR2 (also being called Flk1 or KDR) and VEGFR3 (also being called Flt4).These acceptors are described in: DeVries etc., Science 255:989 (1992); Shibuya etc., Oncogene 5:519 (1990); Matthews etc., Proc.Nat.Acad.Sci.88:9026 (1991); Terman etc., Oncogene6:1677 (1991); Terman etc., Biochem.Biophys.Res.Commun.187:1579 (1992).The vegf receptor dimer is disclosed in: Shibuya, Cell Structure and Function, 26:25-35 (2001); With Ferrara etc., Nature Medicine, 9:669-676 (2003).In a kind of mode, the present invention includes and estimate unusual angiogenesis, perhaps be generated as the disease of feature with abnormal vascular, wherein measured the dimer in one or more following groups: the VEGFR1 homodimer, VEGFR2 homodimer, VEGFR1-VEGFR2 heterodimer and VEGFR2-VEGFR3 heterodimer.

Detailed Description Of The Invention

The invention provides a kind of with the cell surface receptor compound as the biologic artifact method of the biomarker of morbid state or other physiological status among the mankind particularly.In a kind of mode, directly measure the receptor complex that comes from patient's sample; That is to say, measure and do not cultivate, form xenograft, or similar techniques, this needs additional effort and expense and may introduce illusion and/or noise in measurement.Of the present invention one special aspect, directly in the section of organizing lysate or immobilized patients sample of freezing patient's sample, measure one or more receptor complexes.In a preferred embodiment, in paraffin embedding (FFPE) the sample section of formalin fixed, measure one or more receptor complexes.

In another kind of mode, the invention provides a kind of method of indirect measurement receptor phosphorylation, realize by measuring the compound that its formation depends on this posttranslational modification.

In a kind of mode, a plurality of ErbB receptor complexes, for example receptor dimer in same assaying reaction potpourri, have been measured simultaneously.Preferably, employing is connecting one or more binding compounds that discharge molecular label and is measuring this compound, after the protein in combining compound, this molecular label can discharge from reaction or mensuration potpourri and separate in order to detection and/or quantitative like this.

In a kind of mode, the invention provides a kind of method of measuring the patient disease state, may further comprise the steps: measure one or more receptor dimers amount of every kind in patient's sample; This amount of every kind is compared with the respective amount in its standard model; And the difference of each respective amount in the amount in patient's sample and the standard model is associated with the existence or the seriousness of patient disease state.In a preferred embodiment, determination step comprises: one or more binding compounds (i) are provided, it has specificity to the protein in every kind of one or more receptor dimers, make every kind of binding compounds have one or more molecular labels, connect thereon by the cleavable connector respectively, and make these one or more molecular labels connect different binding compounds with different stalling characteristics, like this by molecular label is separated from different binding compounds, and formed the unique peak on the separate picture; (ii) this binding compounds is mixed with one or more compounds, form detectable compound thereby make binding compounds be incorporated into its corresponding receptor dimer specifically; (iii) cracking forms the cleavable connector of every kind of binding compounds of detectable compound, and (iv) separates and identify that the molecular label that is discharged is to measure whether existing or content of these one or more receptor dimers.

In another kind of mode, the method of amount of measuring the receptor dimer of one or more types may further comprise the steps: i) every kind to the receptor dimer of one or more types provides the cracking probe, its first acceptor of every kind to described one or more receptor dimers has specificity, and the cracking that each cracking probe has effective short range causes part; One or more binding compounds (ii) are provided, its second acceptor of every kind to described one or more receptor dimers has specificity, make every kind of binding compounds have one or more molecular labels, respectively by the cleavable connector connect with its on, and make described one or more molecular labels connect different binding compounds with different stalling characteristics, like this by molecular label is separated from different binding compounds, and formed the unique peak on the separate picture; (iii) cracking probe, binding compounds are mixed with the receptor dimer of described one or more types, be incorporated into first acceptor of receptor dimer with making the cracking probe specificity, and binding compounds is incorporated into second acceptor of receptor dimer specifically, the cleavable connector of binding compounds is in effective short range of cracking probe cracking initiation part like this, and molecular label is released; And (iv) separate and identify the molecular label that discharged whether existing or content with the receptor dimer of measuring these one or more types.Preferably, receptor dimer and first and second acceptor are selected from the receptor dimer that is listed as in the Table I.

In another kind of mode of the present invention, obtain biological sample from the patient, contain the mixed cell population of rare target cell comprising suspection.Then, this biological specimen prepares sample by being mixed with the magnetic-particle of coupling biospecific ligands; described biospecific ligands can with described rare cells on be different from or be not present in antigentic specificity reaction (being called " capture antigen " here) on the haemocyte, can remove other sample composition so substantially.Sample is in the magnetic field, and this magnetic field can be separated the cell of magnetic-particle mark effectively, comprises the target cell of described rareness, if it is present in the sample.Adopt then the molecular label of the special joint portion branch coupling of biomarker is analyzed above-mentioned isolated cells colony, to detect whether existing and/or quantity of rare cells.In a preferred embodiment, the magnetic-particle that uses in this method is the colloid magnetic nanoparticle.Preferably, this rare cells colony is the circulation epithelial cell, and it can utilize epithelium specificity capture antigen to separate from blood samples of patients, and is disclosed in for example following document of this antigen: Hayes etc., Internation J.of Oncology, 21:1111-1117 (2002); Soria etc., Clinical Cancer Research, 5:971-975 (1999); Ady etc., British J.Cancer, 90:443-448 (2004); It all is incorporated herein by reference.Especially, (Dynal A.S., Oslo Norway) catch the human epidermal cell by magnetic-particle can to adopt monoclonal antibody BerEP4.

In another kind of mode, the invention provides a kind of method of the patient's of mensuration cancerous state, may further comprise the steps: (i) contain the epithelial patient's sample of circulation by coming immune magnetic to separate with one or more antibody compositions contacts in the blood samples of patients sample, every kind of antibody compositions has specificity and is connecting magnetic-particle capture antigen; (ii) measure the amount of every kind of one or more receptor complex in patient's sample; Amount in the standard model that every kind of this tittle is corresponding with it is compared; And the difference of each respective amount in the amount in patient's sample and the standard model is associated with the existence or the seriousness of patient's cancer condition.In a preferred embodiment, measuring method comprises step: one or more binding compounds (i) are provided, it has specificity to the protein in every kind of one or more receptor complexes, make every kind of binding compounds have one or more molecular labels, connect thereon by the cleavable connector respectively, and make these one or more molecular labels connect different binding compounds with different stalling characteristics, like this by molecular label is separated from different binding compounds, and formed the unique peak on the separate picture; (ii) this binding compounds is mixed with one or more receptor complexes, thereby the corresponding protein that makes this binding compounds be incorporated into specifically in these one or more receptor complexes forms detectable compound; (iii) cracking forms the cleavable connector of every kind of binding compounds of detectable compound, and (iv) separates and identify that the molecular label that is discharged is to measure whether existing or content of these one or more receptor complexes.

In another kind of mode, the method of measuring one or more receptor complex amounts may further comprise the steps: i) every kind to one or more receptor complexes provides the cracking probe, its first albumen of every kind to described one or more receptor complexes has specificity, and the cracking that each cracking probe has effective short range causes part; One or more binding compounds (ii) are provided, its second acceptor of every kind to described one or more receptor complexes has specificity, make every kind of binding compounds have one or more molecular labels, respectively by the cleavable connector connect with its on, and make described one or more molecular labels connect different binding compounds with different stalling characteristics, like this by molecular label is separated from different binding compounds, and formed the unique peak on the separate picture; (iii) cracking probe, binding compounds are mixed with a kind of or many compound, make first albumen of cracking probe specificity ground bind receptor compound, and binding compounds second albumen of bind receptor compound specifically, the cleavable connector of this binding compounds is in effective short range of cracking probe cracking initiation part like this, and molecular label is released; And (iv) separate and identify that the molecular label that is discharged is to measure whether existing or content of these one or more receptor complexes.

Receptor dimer biomarker and dimer drugs with function example

Biomarker of the present invention comprises the dimer and the oligomer of following acceptor.

Table I.

The receptor complex example of cell surface membrane

The dimer dimer

Her1-Her1	β 2-adrenocepter homodimer
		Her1-Her2	ATII acceptor homodimer
Her1-Her3	BradykinininB2 acceptor homodimer
		Her1-Her4	CCR2 acceptor homodimer
Her2-Her2	D1 dopamine receptor homodimer
		Her2-Her3	D2 dopamine receptor homodimer
Her2-Her4	α 2-adrenaline-β 2-adrenaline heterodimer
		Her3-Her4	GABA BR1-GABA BR2
Her4-Her4	The cholescystokinin-dopamine
		The IGF-R1 homodimer	M2-M3muscarinic
VEGFR1(Flt1)-VEGFR2(KDR)	μ-δ opium
		VEGFR2(KDR)-VEGFR2(KDR)	5-HT 1B-5-HT 1D
PDGFRα-PDGFRα	α 2c-adrenaline-M3 muscarinic
		α 2-adrenocepter homodimer	Kit/SCFR (homodimer)
PDGFRα-PDGFRβ	FGFT (particularly FGFR1 homodimer)
		PDGFRβ-PDGFRβ	NGFR(TrkA)-NGFR(TrkA)
D3 dopamine receptor homodimer	β 2-adrenaline-δ opium
		Glutamic acid R1 α acceptor homodimer	β 2-adrenaline-κ opium
Human chorionic is urged sexual gland acceptor, homodimer	A1 adenosine-D1 dopamine
		H2 histidine acceptor homodimer	The SSTR1-SSTR5 growth hormone-release inhibiting factor
Muscarinic acceptor homodimer	The opiate receptors homodimer
		The platelet activating factor receptor homodimer	D2 dopamine-SSTR5 growth hormone-release inhibiting factor

V2 vasopressin receptor homodimer	The IGF-1R-Her1 heterodimer
		The IGF-1R-Her2 heterodimer	The IGF-1R-Her3 heterodimer
The IGF-1R-PDGFR heterodimer	The Her1-PDGFR heterodimer
		The Her2-PDGFR heterodimer	The Her3-PDGFR heterodimer
α v β 3 heterodimers	α v β 5 heterodimers
		Her1-PI3K	Her2-PI3K
Her3-PI3K	Her1-SHC
		Her2-SHC	Her3-SHC
IGF-1R-PI3K	IGF-1R-SHC
		The IGF-1R-insulin receptor	PDGFR-PI3K
PDGFR-SHC

The mechanism of action of the many medicines that come into operation or developing need suppress one or more functions of receptor dimer, for example receptor component is combined into dimeric structure, perhaps based on the function of dimerization effect, as enzymatic activity, for example kinase activity, or autophosphorylation.This medicine is referred to herein as " dimer effect " medicine, perhaps " dimerization " medicine.Treating or finishing quantity, type, the formation of the receptor dimer in patient's cell of treatment and/or decomposing relevant with the effect or the applicability of the specific dimer drugs with function of use.Below receptor dimer be with shown in the relevant biomarker of medicine.In a kind of mode, the present invention provides biomarker for monitoring dimer drugs with function to the effect of morbid state.

Table II.

The medicine relevant with the dimer of cell surface membrane

The dimer medicine

Her1-Her1、Her1-Her2、Her1- Her3、Her1-Her4、Her1-IGF-1R	Cetuximab(Erbitux)， Trastuzumab(Herceptin)，h-R3 (TheraCIM)，ABX-EGF，MDX-447， ZD-1839(Iressa)，OSI-774 (Tarceva)，PKI 166，GW572016， CI-1033，EKB-569，EMD 72000
		Her2-Her1、Her2-Her3、Her2- Her2、Her 2-Her4	4D4Mab，Trastuzumab (Herceptin)，2C4，GW572016
The VEGFR dimer	PTK787/K222584，ZD6474，

	SU6668，SU11248，CHR200131， CP547632，AG13736， CEP7055/5214，KRN633
		The PDGFR dimer	SU6668，SU11248，AG13736， CHR200131
The FGFR dimer	CP547632，CHR200131

Dimer drugs with function listed in the Table II has been described: Traxler, Expert Opin.Ther.Targets, 7:215-234 (2002) in the following document; The Baselga chief editor, OncologyBiotherapeutics, 2:1-36 (2002); Nam etc., Current Drug Targets, 4:159-179 (2003); Seymour, Current Drug Targets, 2:117-133 (2001); Or the like.

Table III

The receptor complex that PI3K is relevant

The dimer dimer

Her1-Her1	β 2-adrenocepter homodimer
		Her1-Her2	ATII acceptor homodimer
Her1-Her3	BradykinininB2 acceptor homodimer
		Her1-Her4	CCR2 acceptor homodimer
Her2-Her2	D1 dopamine receptor homodimer
		Her2-Her3	D2 dopamine receptor homodimer
Her2-Her4	α 2-β 2-adrenaline heterodimer
		Her3-Her4	GABA BR1-GABA BR2
Her4-Her4	The cholescystokinin-dopamine
		The IGF-R1 homodimer	M2-M3muscarinic
VEGFR1(Flt1)-VEGFR2(KDR)	μ-δ opium
		VEGFR2(KDR)-VEGFR2(KDR)	5-HT 1B-5-HT 1D
PDGFRα-PDGFRα	α 2c-adrenaline-M3muscarinic
		α 2-adrenocepter homodimer	Kit/SCFR (homodimer)
PDGFRα-PDGFRβ	FGFT (particularly FGFR1 homodimer)
		PDGFRβ-PDGFRβ	NGFR(TrkA)-NGFR(TrkA)
D3 dopamine receptor homodimer	β 2-adrenaline-δ opium

Glutamic acid R1 α acceptor homodimer	β 2-adrenaline-κ opium
		Human chorionic is urged sexual gland acceptor, homodimer	A1 adenosine-D1 dopamine
H2 histidine acceptor homodimer	The SSTR1-SSTR5 growth hormone-release inhibiting factor
		Muscarinic acceptor homodimer	The opiate receptors homodimer
The platelet activating factor receptor homodimer	D2 dopamine-SSTR5 growth hormone-release inhibiting factor
		V2 vasopressin receptor homodimer	The IGF-1R-Her1 heterodimer
The IGF-1R-Her2 heterodimer	The IGF-1R-Her3 heterodimer
		The IGF-1R-PDGFR heterodimer	The Her1-PDGFR heterodimer
The Her2-PDGFR heterodimer	The Her3-PDGFR heterodimer

Specimen preparation

The sample that contains molecular complex can come from widely and is applicable to various source of the present invention, thereby makes the quantity of receptor complex relevant with morbid state or health status, comprises cell culture, animal or plant tissue, patient's slicer or the like.Preferably, sample is the human patients sample.Can utilize conventional technology to prepare sample and be used for mensuration of the present invention, used technology can be determined according to the source of obtaining sample.

A. solid tissue's sample.For biopsy and medical science sample, below with reference to the technological guidance is provided in the document: Bancroft JD ﹠amp; Stevens A chief editor, Theory and Practice of HistologicalTechniques (Churchill Livingstone, Edinburgh, 1977); Pearse, Histochemistry.Theory and applied. the 4th edition (Churchill Livingstone, Edinburgh, 1980).

In the cancerous state zone, the patient tissue samples example that can adopt includes but not limited to, mammary gland, prostate, ovary, colon, lung, endometrium, stomach, salivary gland or pancreas.Described tissue sample can obtain by several different methods, includes but not limited to surgical excision, suction or slicer.Tissue can be fresh or freezing.In one embodiment, mensuration of the present invention is to implement on fixing and tissue sample with paraffin or analog embedding; Therefore, in this embodiment, slough the step of paraffin.Tissue sample can be fixed (promptly anticorrosion) by the method for routine [referring to for example, " Manual of HistologicalStaining Method of the Armed Forces Institute of Pathology ", the 3rd edition (1960), Lee G.Luna, HT (ASCP) chief editor, The Blakston Division McGraw-HillBook Company, New York; The Armed Forces Institute of Pathology AdvancedLabotatory Methods in Histology and Pathology (1994) Ulreka V.Miked chief editor, Armed Forces Institute of Pathology, American Registry ofPathology, Washington, D.C..It will be appreciated by those skilled in the art that the selection of fixing agent is according to being organized dyeing or making other purposes of analyzing used tissue and decide.Those skilled in the art be to be further appreciated that fixing length depends on the size and the employed fixing agent of tissue sample.In the example, available neutral buffered liquid formalin, bouin's solution or paraformaldehyde fix tissue sample.

In general, at first the fixing organization sample by the ethanol dehydration of ascending series, with paraffin or other section medium osmosis and embedding, makes this tissue sample form section then.Perhaps, tissue can be cut into slices and the fixing section that is obtained.In the example, the method for available routine in paraffin embedding and handle this tissue sample (referring to for example, " Manual of Histological Staining Method of the Armed ForcesInstitute of Pathology ", supra).Spendable paraffin example includes but not limited to, Paraplast, Broloid and Tissuemay.In case tissue sample by embedding after, this sample just can be cut into slices by microtome or analogous instrument (referring to for example, " Manual of HistologicalStaining Method of the Armed Forces Institute of Pathology ", supra).In this method example, slice thickness can about three microns to about 12 microns scope, preferably, thickness about 5 microns to about 10 microns scope.In a kind of mode, the section area can be from about 10mm ²To about 1cm ²In case after the section, can this section be added on the microslide by the method for some standards.The example of microslide bonding agent includes but not limited to silane, gel, poly-L-Lysine or the like.In the example, this paraffin-embedded tissue can be pasted on the microslide of positively charged and/or be coated with on the microslide with poly-L-Lysine.

If used paraffin as embedded material, in general this histotomy will be sloughed paraffin and carry out rehydrated with water.This histotomy can be sloughed paraffin by the standard method of several routines.For example, can adopt dimethylbenzene and gradually the ethanol of descending series (Manual of Histological Staining Method of theArmed Forces Institute of Pathology ", supra).Perhaps, can adopt can buy on the market take off the paraffin inorganic reagent, as Hemo-De  (CMS, Houston Tex.).

For mammiferous tissue culture cells, flesh tissue or similar source, can prepare sample (0.14M NaCl for example, 1.5mM MgCl by the lysis technology of routine ₂, 10mM Tris-Cl (pH 8.6), 0.5%Nonidet P-40 and required proteinase and/or inhibitors of phosphatases).For fresh mammalian tissues, the preparation of sample can also comprise organizes the depolymerization step, for example broken, mince, grinding, sonicated or the like.

B. the magnetic resolution of cell.In some applications, for example when mensuration comes from dimer on the rare metastatic cell of blood samples of patients, can before carrying out surface receptor dimer quantitative measurement, implement the step of an enrichment.Can utilize various technology known in the art and material to carry out immunomagnetic isolation or enrichment process,, introduce it as a reference: the United States Patent (USP) 6365362 of Terstappen etc. as disclosed in the following representative references; The United States Patent (USP) 5646001 of Terstappen etc.; The United States Patent (USP) 5998224 of Rohr etc.; The United States Patent (USP) 5665582 of Kausch etc.; The United States Patent (USP) 6048515 of Kresse etc.; The United States Patent (USP) 5508164 of Kausch etc.; The United States Patent (USP) 5691208 of Miltenyi etc.; The United States Patent (USP) 4452773 of Molday; The United States Patent (USP) 4375407 of Kronick; Radbruch etc., Methods in CellBiology, the 23rd chapter among the Vol 42, (Academic Press, New York, 1994); Uhlen etc., Advances in Biomagnetic Separation (Eaton Publishing, Natick, 1994); Safarik etc., J.Chromatography B, 722:33-53 (1999); Miltenyi etc., Cytometry, 11:231-238 (1990); Nakamura etc., Biotechnol.Prog., 17:1145-1155 (2001); Moreno etc., Urology, 58:386-392 (2001); Racila etc., Proc.Natl.Acad.Sci., 95:4589-4594 (1998); Zigeuner etc., J.Urology, 169:701-705 (2003); Ghossein etc., Seminars in Surgical Oncology, 20:304-311 (2001).

Be preferred for implementing the particle that magnetic-particle of the present invention is character such as colloid.This particle is characterised in that the particle size of its sub-micro level of confidentiality, and usually less than about 200 nanometers (nm) (0.20 micron), and its stability can continue the long period for the gravity separation in the solution.Except many other advantages, this range of size also makes its analytical technology commonly used in cell analysis invisible basically.Expectation in the 90-150nm scope and the particle of magnetic mass between 70-90% be applicable to the present invention.The magnetic-particle that is fit to by around constitute round the superparamagnetic material nucleus of molecule, this molecule is for example by physisorption or covalently boundly be attached to magnetic core in the heart and give its stable colloid property.The preferred applied amount of coating material should prevent biomacromolecule in the biological sample and the non-specific interaction between the magnetic core effectively.This biomacromolecule can comprise the lip-deep sialic acid residues of non-target cell, lectin, glycoprotein and other film component.In addition, this material should comprise magnetic mass/nano particle as much as possible.The magnetic crystal size that comprises core should make it not contain complete magnetic domain enough for a short time.Enough little its Blang's energy that makes of the size of nano particle has surpassed its magnetic moment.As a result, the alignment in the arctic, the South Pole and attracting each other/repulsive interaction afterwards appear in these colloid magnetic-particles hardly, also are that this helps the stability of its solution under the powerful magnetic field of appropriateness even.At last, this magnetic-particle should be separated in the external field separation vessel of high magnetic field gradients.This feature helps sample preparation, and has than the advantage more economical than the complex internal gradient column that loads ferromagnetism pearl or steel wool.Magnetic-particle with above-mentioned characteristic can prepare by the stock described in the U.S. Patent No. 4795698,5597531,5698271, introduces these patents as a reference.

Employing can discharge the determination test of molecular label

Employing can discharge molecular label to be measured dimeric quantity and have many advantages, comprising: (1) is separated the molecular label that is discharged and can be reduced background greatly and significantly improve sensitivity from measure potpourri; And (2) use is easy to record simultaneously a plurality of receptor complex compositions like this for simplifying the diversification performance that separation and the specially designed molecular label of testing process are provided convenience in same mensuration.Adopted the mensuration of this label can have the United States Patent (USP) 6627400 of various ways and disclosed in below with reference to document: Singh etc.; The U.S. Patent Publication 2002/0013126 of Singh etc.; And 2003/0170915 and Williams etc. 2002/0146726; With the open WO2004/011900 of the international monopoly of Chan-Hui etc., all documents are hereby incorporated by.For example, can adopt the multiple isolation technics of distinguishing molecule according to the physics between the separated molecule, chemistry or optical characteristics difference, described characteristic includes but not limited to electrophoretic mobility, molecular weight, shape, solubleness, pKa, hydrophobicity, electric charge, lotus/matter ratio, polarity or the like.In a kind of mode, a plurality of or a component subtab is different on electrophoretic mobility and optical detection characteristic, thereby separate by electrophoresis.In another kind of mode, an a plurality of or component subtab can be different on molecular weight, shape, solubleness, pKa, hydrophobicity, electric charge, polarity, thereby separate by positive or reversed-phase HPLC, ion-exchange HPLC, Capillary Electrophoresis, mass spectroscopy, vapor-phase chromatography or the like technology.

Molecular label in groups is provided, after binding compounds discharges, is separated into different bands or peak by isolation technics at it.Identify that also quantitative these peaks can provide measured value or the image to receptor dimer kind and amount.One component subtab can have different chemical property; Yet for simplicity, the common chemical property of molecular label in groups is relevant.For example, it can all be a peptide, and perhaps it can be made of the various combination of identical base structure district or monomer, and perhaps it can adopt the identical base skeleton with different substituents to synthesize to give different stalling characteristics, as following described in detail.The quantity of a plurality of molecular labels can be different according to some factors, comprises the clastotype of employing, the certification mark thing that uses on the molecular label, the sensitivity of bound fraction, efficient of cracking cleavable connector or the like.In a kind of mode, the quantity of a plurality of molecular labels that is used to measure receptor dimer quantity is in 2 to 10 scope.In alternate manner, the quantity of a plurality of molecular labels can be 2 to 8,2 to 6,2 to 4, or in 2 to 3 the scope.

Can detect receptor dimer in the promptly heterogeneous determination test at homogeneous phase or non-homogeneous.In homogeneous phase, without any need for step specificity is incorporated in the binding compounds of binding compounds combination never of target compound and separates.In a preferred embodiment, the reagent that homogeneous phase adopts is to comprising (i) and have one or more binding compounds that can discharge molecular label and (ii) can produce at least one cracking probe of active substance, and this active substance can react with molecular label and discharge molecular label in effective short range of this cracking probe.

Receptor dimer can also be measured by the determination test that adopts heterogeneous form.Heterogeneous technology is usually directed to separating step, wherein will have in the binding compounds of intracellular complexes combination never of binding compounds of specificity combination to separate, and selectively from other sample composition, as separating in protein, the film fragment etc.Can realize detachment process by variety of way, for example adopt that it can distinguish the compound and the unconjugated binding compounds of combination in conjunction with the reagent of solid support.Solid support can be a chamber wall, and for example the hole of microtitre orifice plate, kapillary, flat board, microslide, microballon comprise magnetic bead, liposome or the like.The principal character of this solid support is that (1) can separating and combining and unconjugated binding compounds, and (2) do not disturb the formation of binding compounds, perhaps other operation when measuring receptor dimer.Usually, in fixed sample, unconjugated binding compounds can easily be got rid of by washing.

When in heterogeneous form, utilizing molecular label to detect, after washing, sample and solvent can be discharged molecular label therein.According to the character and the cleavage method of cleavable key, can contain any additive reagent that is useful on cracking in this solvent.Under the situation that does not need lytic reagent, this solvent can be easily as dissociating buffer, for example, and the electrophoretic separation medium.For example, by the active substance that photosensitizer produces, this cleavable connector have photosensitivity or easily during decomposability, can be with this medium of rayed of suitable wavelength so that this molecular label be discharged in the damping fluid.

No matter under which kind of form,, then may before cracking and isolated molecule label, remove or exchange this assaying reaction damping fluid if the condition of assaying reaction has been disturbed the isolation technics that is adopted.For example, in certain embodiments, condition determination comprises salinity (for example specificity is in conjunction with required), and when molecular label separated on the electrophoretic mobility basis, this salinity it can make the performance of separation reduce.In this embodiment, before release and the isolated molecule label, substitute with dissociating buffer or medium and measure damping fluid.

Adopted the determination test that can discharge molecular label and cracking probe can make multiple different form and structure according to compound detected or that measure.According to present disclosure specification, selecting the quantity of specific binding compounds and cracking probe and specificity to those skilled in the art is a kind of design alternative.

In a kind of mode of the present invention, Figure 1A and 1B have represented that use can discharge molecular label and measure the dimeric diagram of cell surface membrane.To have molecular label " mT ₁" and " mT ₂" binding compounds (100) and the cracking probe (102) with photosensitizer " PS " mix with biological cell (104).Has molecular label " mT ₁" binding compounds pair cell surface receptor R ₁(106) have specificity, have molecular label " mT ₂" binding compounds pair cell surface receptor R ₂(108) has specificity.Cell surface receptor R ₁And R ₂As monomer, as (106) and (108), and dimer (110) exists in cell surface membrane (112).These are measured composition after the incubation, makes to have formed (114) stabilized complex between its corresponding acceptor target of binding compounds and between cracking probe and its acceptor target in suitable binding buffer liquid.As shown in the figure, preferred combination compound and cracking probe comprise the antibodies component respectively, and it makes molecular label and cracking cause part target film component specifically.In a kind of mode, this antibodies component is a monoclonal antibody.In these embodiments, connect damping fluid and can comprise the damping fluid that uses in the conventional elisa technique, or analog.Binding compounds and cracking probe become stabilized complex (116) afterwards, and this measures potpourri to cause photosensitizer (120) generation singlet oxygen irradiation (118).Singlet oxygen promptly reacts with the mensuration mix ingredients, and its effective short range (122) that is used for the cleavable connector of cracking molecular label is subjected to the restriction on the space like this, makes to have only lucky molecular label in this effective short range to be released (124).As shown in the figure, have only those and R ₁-R ₂Molecular label on the binding compounds of dimer and cracking probe formation stable compound is released.The molecular label (126) that discharges is removed from measure potpourri and separated (128), in separate picture (132), form the peak (130) of uniqueness like this according to stalling characteristic.According to the present invention, this removal can be same step with separating.Selectively, can remove this binding buffer liquid and use the damping fluid that is more suitable for separating before irradiation, promptly dissociating buffer replaces.For example, the salinity that binding buffer liquid has usually can reduce some isolation technics, Capillary Electrophoresis for example, thereby performance influence the isolated molecule label to form tangible peak value.In certain embodiments, the exchange of sort buffer liquid can realize by membrane filtration.

Fig. 1 C has represented the embodiment of a reference mensuration heterodimer, has wherein adopted extra binding compounds to come the total amount of albumen in the working sample (1104).Reaction reagent of the present invention (1122) comprises (i) cracking probe (1108), first binding compounds (1106) and second binding compounds (1107), wherein first binding compounds (1106) has specificity to albumen (1102), and second binding compounds (1107) has specificity to albumen (1104) at the antigenic determinant place different with cracking probe (1108) specificity.After association reaction reagent, cracking probe (1108) is activated the generation active substance, its can be in effective short range of photosensitizer the cleavable connector of this molecular label of cracking.In this example, molecular label and is connecting reagent (1108) and is connecting individually or simultaneously on the heterodimer of reagent (1106) and (1107) and discharging from the monomer that is connecting reaction reagent (1107) and (1108) albumen (1104) simultaneously.The molecular label (1123) that discharges is separated, and the peak in the separate picture (1126) (1118 and 1124) are relevant with the amount of release molecular label.In this embodiment, the height of relevant peaks or area can reflect the affinity difference of (i) first and second binding compounds to its corresponding antigens determinant, and/or the existence that (ii) binding compounds is had a specific antigenic determinant whether.No matter when binding compounds is used to monitor state after the translation of protein, as phosphorylation state, latter event is all very important.

Mensuration to homodimer can be as shown in Fig. 1 D.As mentioned above, mensuration can comprise three kinds of reaction reagents (1128): cracking probe (1134), first binding compounds (1130) and second binding compounds (1132).First binding compounds (1130) is fabricated in the paired samples with cracking probe (1134) and exists the last identical antigenic determinant (1135) of albumen (1138) of (1140) to have specificity with homodimer (1136) or monomer (1138) form.Promoting to form between reaction reagent and its respective target under the condition of stable compound, after reaction reagent (1128) and the sample mix, measure in the potpourri and formed multiple compound (1142 to 1150).Because cracking probe (1134) is specific to identical antigenic determinant (1135) with binding compounds (1130), four kinds of various combinations (1144 to 1150) of reaction reagent can form the compound with homodimer.In the compound in measuring potpourri, the molecular label (1151) that the compound that has only those (1143) to have cracking probe (1134) and at least one binding compounds simultaneously can be contributed release is in order to separate and to detect (1154).In this embodiment, the size at peak (1153) is directly proportional with the amount of homodimer in the mensuration potpourri, and the size of peak (1152) is directly proportional with the total amount of albumen (1138) in the mensuration potpourri, and its existing monomeric form (1142) has homodimer form (1146 and 1148) again.Measure the cell surface receptor that is used for forming heterodimer like Fig. 1 E representation class at cell surface membrane (1161).It will be appreciated by those skilled in the art that dimer can be measured in the lysate of cell or tissue, perhaps can be by fixation procedure infiltration or removed in the fixed sample of cell membrane and carried out.In this case, the outer or intracellular region territory of the born of the same parents that binding compounds can pair cell surface film acceptor has specificity.

As shown in Fig. 1 E and 1F, releasable molecular label can also be used for detecting simultaneously or measuring the multiple dimer and the intracellular complexes of cell sample.With cell (160), it can come from vitro culture or come from the patient tissue sample, and cracking (172) to be providing come-at-able molecular complex, and it is connecting the posttranslational modification site in the tenuigenin zone of cell membrane and/or membrane molecule, as phosphorylation site.After the cracking, the lysate that obtains (174) mixes with the mensuration reagent (176) that comprises a plurality of cracking probes (175) and a plurality of binding compounds (177).Select (178) condition determination to make its corresponding target-specific combination of reagent (176), like this behind overactivation, the cleavable connector that cracking causes in effective short range (180) of part is cleaved, and discharges (182) molecular label.As mentioned above, after the cracking, the molecular label that discharges is separated (184) and identify in as electrophoresis pattern, and obtain the image of selected molecular complex in the sample cell according to measured molecular label number and amount at separate picture (186).

Fig. 1 G and 1H have represented that the present invention measures the embodiment of receptor complex in the fixing or freezing tissue sample.With fixing tissue sample (1000), the paraffin embedding sample of formalin fixed for example, utilize the cutting of microtome or analogous instrument to obtain section (1004), be placed on surface (1006), dewax and rehydration is measured reagent so that add as after on the micro-microslide.Enlarged drawing (1007) has been represented part (1008) section (1004) on part (1014) micro-microslide (1006).Shown receptor dimer molecule (1018) is embedded in the residual film structure (1016) of fixed sample.According to this mode of the present invention, cracking probe and binding compounds make them combine with its target molecule with the fixed sample incubation.For example, cracking probe (1012) (being expressed as the antibody that is connecting photosensitizer (" PS ") among the figure) (is expressed as and is being connected molecular label " mT with first binding compounds (1010) ₁" antibody) specifically in conjunction be common to dimeric acceptor shown in all (1011), second binding compounds (1017) (has " mT ₂") specifically in conjunction with acceptor (1015), and the 3rd binding compounds (1019) (has " mT ₃") specifically in conjunction with acceptor (1013).Washing adds damping fluid (1024) (being called " irradiation damping fluid " among the figure) to remove not after the binding compounds and cracking probe of specificity in conjunction with its respective target molecule.For simplicity, can form hydrophobic carrier, for example use wax crayon, damping fluid (1024) is included on section (1004) or its part by going up at microslide (1006).The irradiation photosensitizer also discharges molecular label (1026) afterwards, the current damping fluid that contains the molecular label (1025) of release is transferred in the tripping device, capillary electrophoresis apparatus for example is in order to separate the molecular label that (1028) and discriminating are discharged in for example electrophoresis pattern (1030).

To the mensuration of directly on tissue sample, carrying out, especially shown in Fig. 1 G and 1H, can carry out standardization, it comprises cell representative target cell or the tissue of mensuration to characteristic hypotype in total cell number and/or the sample in the sample.Preferred even must carry out extra mensuration, because cell in patient's sample, the especially tumor sample and tissue have unevenness, it may comprise Normocellular basic fragment.For example, among Fig. 1 H, the ratio that the total amount value of acceptor (1011) can be used as following two measured values provides: molecular label (" mT ₁") peak (1032) area and with sample in the total cell or tissue composition of all cells, as tubulin, the respective peaks area of relevant molecular label.In some cases, when all cells all is epithelial cell in the sample, can adopt cytokeratin.Therefore, can comprise extra step, the binding compounds that is specific to standardization albumen such as tubulin (having unique molecular label) promptly is provided based on the method that can discharge molecular label.

Fig. 2 A-2E has represented an alternative embodiment of the invention, is used to describe the dimerization process between a plurality of acceptor types.Fig. 2 A has summarized the basic step of this determination test.Being used for cell membrane (200) that the cell surface receptor dimer detects and several groups of binding compounds (202) and (204) and cracking probe (206) mixes.Film fragment (200) contains three kinds of dissimilar monomeric acceptor molecules (" 1 ", " 2 " and " 3 ") in its cell membrane, it is in conjunction with forming three kinds of different heterodimers: 1-2,1-3 and 2-3.Three kinds of antibody reagents (202) and (204) combine with film fragment (200), every kind of antibody reagent has binding specificity to a kind of in three kinds of acceptor molecules, wherein antibody (206) is specific to acceptor molecule 1, and antibody (204) is specific to acceptor molecule 2, and antibody (202) is specific to acceptor molecule 3.The antibody covalent coupling of first acceptor molecule is labeled as PS in photosensitizer molecule.Second is being connected two kinds of different labels with the antibody of the 3rd acceptor molecule, is labeled as T respectively ₂And T ₃, connect by the connector of the active substance cracking that can partly be produced by photosensitizer.

After the mixing, make the lip-deep molecule of antibodies (208) film.Photosensitizer is activated (210), and cracking sensitizer molecule works apart from interior label and the connector between the antibody, thereby label is discharged in the mensuration medium.Separating substances (212) in will reacting then is for example by Capillary Electrophoresis, as shown in the figure.Shown in Fig. 2 A bottom, label T ₂And T ₃Be released, will occur two by electrophoretic separation and be with accordingly with these labels.Because these labels of design have the known electrophoretic mobility, thereby every band can be represented employed a kind of label in the mensuration uniquely.

Shown in Fig. 2 A, have only two kinds can therefore have only these two kinds of materials can produce the label of release in three kinds of heterodimers that exist in the cell membrane simultaneously in conjunction with antibody that contains photosensitizer and the antibody that contains label.Yet, need test of many times to measure different dimeric correlatives.Tabulation provides five kinds of different mensuration combinations among Fig. 2 B.Fig. 2 C represents that every kind is measured composition results.Measure I and represent all results of mensuration, shown in Fig. 2 A.In measuring II, do not connecting acceptor molecule 1 specific antibody of photosensitizer.This mensuration does not produce signal, shows to measure the T that obtains among the I ₂And T ₃The signal demand photosensitive reagents.Similarly, measure V and show that need there be film in label signal.Measure III and IV and show that every kind is not needed to exist another kind of cleaved by the reagent of label.Lump together when considering that these as a result, the existence of the acceptor heterodimer that exists in the membrane of can deriving and the conclusion of combination as given among Fig. 2 C, promptly exist 1-2 and 1-3 heterodimer simultaneously.And, can estimate the relative populations of every kind of heterodimer by the relative signal intensity of every kind of label.

Yet, can not draw the conclusion that the 2-3 heterodimer exists according to the agent combination of using in this mensuration.Can not obtain to represent the signal of this compound, no matter whether this compound exists, because do not have the photosensitive reagents combination thereon.In order to derive every kind of possible dimer combination of three kinds of monomers, must use can navigate to by monomer 1, the 4th kind of reagent on 2 and/or 3 every kind of possible oligomer that constitute, otherwise three kinds of binding reagents that use in this test must be coupled to different combinations on label and the sensitizer molecule.A kind of scheme in back is as shown in Fig. 2 D and 2E.Three kinds of possible combinations that photosensitizer and label distribute in the middle of three kinds of antibody reagents have been listed in the form on Fig. 2 D left side.First kind of combination comprises the photosensitizer that is coupled on monomer 1 specific antibody, and it is identical with the combination of using shown in Fig. 2 A-2C, and with Fig. 2 C in dimer quantity phasor.Second kind of combination comprises the photosensitizer that is coupled on monomer 2 specific antibodies, and its quantity image obtains adding the identical number of heterodimer 2-3 with heterodimer 1-2.The third combination comprises the photosensitizer that is coupled on monomer 3 specific antibodies, and its quantity image obtains the number identical with 2-3 with heterodimer 1-3, such as preceding two the combination acquisition.Can be in conjunction with these results to obtain all heterodimer quantity images, as given among Fig. 2 E.

Fig. 2 F represents to be used to measure the correlative preferred embodiment of the receptor dimer that contains total component acceptor.Measure in the design at this, can be according to two kinds of different receptor dimers of this total composition reference measurement (" 1-2 " (240) and " 2-3 " (250)), wherein every kind all contains total composition " 2 ".Shown mensuration design is to be used for measuring comprising the acceptor heterodimer (240) of acceptor " 1 " (222) and acceptor " 2 " (220), and the acceptor heterodimer (250) that comprises acceptor " 2 " (220) and acceptor " 3 " (224).The key feature of this embodiment is that cracking probe (227) has specificity to the right total acceptor of heterodimer.The binding compounds (228) that is specific to acceptor " 2 " provide with measure in the relevant signal (234) of acceptor " 2 " total amount, and the signal (being respectively 232 and 236) that the binding compounds (226) that is specific to acceptor " 1 " and the binding compounds that is specific to acceptor " 3 " (230) provide the amount with acceptor " 1 " and acceptor " 3 " is relevant respectively, and described acceptor " 1 " and acceptor " 3 " only exist only in the heterodimer that forms with acceptor " 2 ".The design of Fig. 2 F can be generalized in the two or more receptor complex that contains total composition, only needs to add the binding compounds that is specific to extra compound composition.

A. binding compounds

As mentioned above, provide the potpourri that contains multiple different binding compounds, wherein every kind of different binding compounds has the molecular label that one or more connect by the cleavable connector.The character of binding compounds, cleavable connector and molecular label can in very large range change.Binding compounds can contain antibody connect component, antibody, peptide, at peptide or non-peptide part, albumen, oligonucleotides, oligonucleotide analogs such as the peptide nucleic acid of cell surface receptor, lectin, perhaps other can specificity in conjunction with or form stable compound with target analytes, as protein complex, molecular entity.In a kind of mode, binding compounds can be represented with following general formula, comprises that one or more are connecting the molecular label of bound fraction.

B-(L-E) _k

Wherein B is the coupling part; L is the cleavable connector; E is a molecular label.In homogeneous determination, cleavable connector L can be the connector of easy oxidation, and being more preferably can be by the connector of singlet oxygen cracking.Partly " (L-E) _k" represent that single binding compounds can have a plurality of molecular labels, connect by the cleavable connector.In a kind of mode, k is the integer more than or equal to, and in further embodiments, and k can be greater than hundreds of, for example 100 to 500, and perhaps k is greater than hundreds of and reach several thousand, and for example 500 to 5000.Usually each of a plurality of dissimilar binding compounds all has different molecular label E.The cleavable connector, for example easy oxidation connector is connected on the B by the chemical method of routine with molecular label E.

Preferably, B is that specificity is incorporated into target, as the predetermined antigens determinant of target protein, as cell surface receptor, the antibodies composition.This composition is easy to be made by the antibody that can buy on the market, and it can be a monoclonal and polyclonal, and target protein is had specificity.Especially, disclose in the following patent EGF-R ELISA has been had specific antibody, introduced it as a reference: 5677171; 5772997; 5968511; 5480968; 5811098.United States Patent (USP) 6488390 is incorporated herein by reference here, and it discloses has specific antibody to g protein coupled receptor CCR4.United States Patent (USP) 5599681 is incorporated herein by reference here, and its phosphorylation site that discloses protein has specific antibody.Supplier such as CellSignaling Technology (Beverly, MA); Biosource International (Camarillo, CA), and Upstate (Charlottesville VA), also provides multiple acceptor is had specific monoclonal and polyclonal antibody.

Cleavable connector L can be in fact any cytotoxic compounds, its can do not degrade discharge the structure of molecular label E or influence under its condition that detects characteristic cracking takes place.No matter when the cracking probe is used for all to mensuration, cleavable connector L can be by the decomposition agent cracking of this cracking probe generation, and wherein the cracking probe works in short distance and makes that only cracking can take place the cleavable connector in the direct short range of this cracking probe.Typically, this reagent must just can be activated by reaction mixture generation physics or chemical change, makes the of short duration active substance of this reagent generation be diffused into cleavable connector place and carries out splitting action.In homogeneous phase, decomposition agent is preferably connecting bound fraction, and as antibody, it made the specific site of lytic reagent target before activation, and this site is positioned at the short range with the binding compounds that can discharge molecular label.In this embodiment, decomposition agent is referred to herein as " cracking initiation part ", and it will more detailed below argumentation.

Non-homogeneous mutually in owing to separate in the binding compounds of the binding compounds combination never of specificity combination, so can select to use the cleavable connector more widely and be connected reagent.The cleavable connector not only comprises easily and the local action reactive material, the connector that reacts as hydrogen peroxide, singlet oxygen etc., also comprise the unsettled connector of the reagent in the entire reaction potpourri, but for example to the connector of alkali labile connector photodestruciton, can by the connector of reduction reaction cracking, by the connector of oxidation reaction cracking, to the unsettled connector of acid, can be by peptide bond of specific proteins enzymatic lysis or the like.Described many these class connectors in the list of references, comprised Greene and Wuts, Protective Groups in Organic Synthesis, second edition (John Wiley ﹠amp; Wons, New York, 1991); Hermanson, BioconjugateTechniques (Academic Press, New York, 1996); And the United States Patent (USP) 5565324 of Still etc.

In a kind of mode, the present invention can adopt the cleavable reactant system that can buy on the market.For example, can between antibodies composition and molecular label, utilize assorted sense reagent to introduce the disulfide bond connector, these reagent such as N-succinimide 3-(2-pyridine two sulfo-s) propionic ester (SPDP), succinimidyloxycarbonyl-Alpha-Methyl-α (2-pyridine two sulfo-s) toluene (SMPT) or the like, available from supplier such as Pierce Chemical Company (Rockford, IL).Handle the disulfide bond that can destroy by this connector introducing with reductive agent, described reductive agent is dithiothreitol (DTT) (DTT), dithioerythritol (DTE), 2 mercapto ethanol, sodium borohydride or the like for example.Effectively the typical concentration of the reductive agent of cracked disulfide bond arrives in the scope of 100mM 10.Utilization is with bifunctional NHS ester crosslinking chemical, two succinimide tartrates (DST) (available from Pierce), wherein contain easily by the suitable-glycol of sodium metaperiodate cracking (for example 15mM periodic acid under physiological pH 4 hours), can between antibodies composition and molecular label, introduce the unsettled connector of oxygenant.The connector that contains esterification sept composition can be with strong nucleopilic reagent cracking, azanol for example, 0.1N azanol for example, 8.5,37 ℃ of following 3-6 of pH hours.By with bifunctional crosslinking chemical as (Rockford, ethylene glycol bisthioglycolate IL) (succinic acid succinimide ester) (EGS) can be introduced this sept available from Pierce.Example can be introduced alkali labile connector with sulfuryl.The same bifunctional crosslinking chemical that can be used for introducing sulfuryl in the cleavable connector comprises two [2-(succinimidyloxycarbonyloxy) ethyl] sulfone (BSOCOES) and 4,4-two fluoro-3,3-dinitro benzene sulfone (DFDNPS).The alkali condition example of cracking comprises the 0.1M sodium phosphate, add Tris alkali is adjusted to pH 11.6, contains 6M urea, 0.1%SDS, and 2mM DTT, 37 ℃ of following incubations 2 hours.But the photodestruciton connector comprises disclosed those materials in the United States Patent (USP) 5986076 of Rothschild etc.

Easy when oxidized as L, L can be thioether or its selenium analog; Or containing the alkene of carbon-to-carbon double bond, wherein two keys are cracked into oxy radical, discharge molecular label E.The United States Patent (USP) 6627400 of Singh etc., the U.S. Patent Publication 2002/0013126 and 2003/0170915 of Singh etc., and the example that discloses easy oxidation connector in people's such as Willner the United States Patent (USP) 5622929, all documents are hereby incorporated by.

When by vapor-phase chromatography or mass spectroscopy a plurality of molecular labels being separated, molecular label E can comprise the electrophoresis mark in the present invention, described in following document: Zhang etc., Biocon jugate Chem., 13:1002-1012 (2002); Giese, Anal.Chem., 2:165-168 (1983); And United States Patent (USP) 4650750; 5360819,5516931,5602273 or the like.

Molecular label E is preferably active substance especially singlet oxygen stable, and comprises the water-soluble organic compounds of detection or reporter group.In addition, the size of E and structure can in very large range change.In a kind of mode, the molecular weight of E is about 50 to about 2500 dalton's scopes, more preferably, about 50 to about 1500 dalton.The preferred structure of E has more detailed description below.E can comprise that detection moiety is in order to produce galvanochemistry, fluorescence or color signal.In utilizing the embodiment of quality testing, E can not contain the separating part that is used for testing goal.Preferably, detection moiety produces fluorescence signal.

In many amounts, select molecular label to make and all have unique stalling characteristic and/or unique optical characteristics each other member in same amount.In a kind of mode, chromatogram or electrophoretic separation characteristic are the residence time under a cover standard separation condition of this area routine, and described condition is pressure, post pressure, post type, mobile phase, electrophoretic separation medium or the like for example.In another kind of mode, optical characteristics is a fluorescent characteristic, for example fluorescence intensity of emission spectrum, fluorescence lifetime, setted wavelength or wave band or the like.Preferably, fluorescent characteristic is a fluorescence intensity.For example, every kind of molecular label in many amounts can have identical fluorescence radiation characteristic, but every kind since unique retention time and with other differentiate mutually.On the other hand, the molecular label of two or more in perhaps many amounts can have the identical mobility or the residence time, but they have unique fluorescent characteristic, for example spectrally resolved emission spectrum, all members in many like this amounts are by molecular separation and fluorimetric combination and can be distinguished.

Preferably, the molecular label of release can record by the fluorescence of electrophoretic separation and detection moiety.In these embodiments, the molecular label with basic identical fluorescent characteristic has different electrophoretic mobilities, can form different peaks on the electrophoresis pattern like this under separation condition.Preferably, a plurality of molecular labels of the present invention separate by the capillary electrophoresis of routine, carry out under the situation that has or do not exist conventional screening matrix.The example of capillary electrophoresis comprises Applied Biosysterms (Foster City, CA) 310,3100 and 3700 types; Beckman (Fullerton, CA) P/ACE MDQ type; Amersham Biosciences (Sunnyvale, CA) MegaBACE 1000 or 4000; SpectruMedix genetic analysissystem, or the like.Electrophoretic mobility and q/M ^2/3Be directly proportional, wherein q is the charge number on the molecule, and M is a molecular mass.Ideally, the difference of the mobility under condition determination between the immediate electrophoresis label is approximately 0.001 at least, is generally 0.002, be more typically about at least 0.01, and can be for more than 0.02.Preferably, in this conventional equipment, the electrophoretic mobility of the molecular label in many amounts has centesimal difference at least, and more preferably scope is 1 at least percent to 10.Can differentiate and the quantitative molecular label that by analyzing separate picture perhaps more specifically, electrophoresis pattern and these values can be associated with the amount and the kind of the receptor dimer that exists in the sample.For example, in the electrophoretic separation process or afterwards, by fluorescence signal and the transit time (or migration distance) that writes down separated compound, perhaps relative fluorescence and the migration precedence diagram (for example as electrophoresis pattern) by making up molecular label can detect or identify molecular label.Preferably, the existence of molecular label whether and/or amount can adopt one or more standards to measure, disclosed among the U.S. Patent Publication 2003/0170734A1 as people such as Williams, be hereby incorporated by.

A plurality of molecular labels can also be designed to separate by the chromatography based on one or more physical characteristicss, comprising singly being not limited to molecular weight, shape, solubleness, pKa, hydrophobicity, electric charge, polarity or the like, as disclosed in the U.S. Patent Publication 2003/0235832, be hereby incorporated by.Chromatographic separation technology is selected according to parameter, and for example post type, stationary phase, moving phase or the like are selected to separate a plurality of molecular labels that form different peaks or band afterwards again in single operation.Which kind of HPLC technology is some factors determined to select be used for the present invention, the number (being the amount of described many amounts) that comprises detected molecular label, the estimator of every kind of molecular label that may generate in the mensuration, synthetic possibility and the easy degree that is used for one group of candidate molecules label of polynary mensuration, the test format that adopts, and HPLC instrument, post and solvent availability, durable degree, cost and processing ease degree.In general, preferably those are suitable for analyzing post and the technology that the finite quantity sample also can provide the separation of highest resolution.Can in following document, find the technological guidance who carries out this selection: Snyder etc. for example, Practical HPLC Method Development, (Johe Wiley ﹠amp; Sons, New York, 1988); Millner, " High Resolution Chromatography:A Practical Approach ", Oxford University Press, New York (1999), Chi-San Wu, " Column Handbook for Size Exclusion Chromatography ", Academic Press, San Diego (1999) and Oliver, " HPLC of Macromolecules:A Practical Approach; Oxford University Press ", Oxford, England (1989).Especially, can take measures,, fixedly equate to carry out system development and optimization as the post type to the specified criteria of chromatographic resolution, for example, Haber etc., J.Chromatogr.Sci., 38:386-392 (2000); Outinen etc., Eur.J.Pharm.Sci., 6:197-205 (1998); Lewis etc., J.Chromatogr., 592:183-195 and 197-208 (1992) or the like.The example that is applicable to HPLC instrument system of the present invention have the serial HPLC of Agilent 1100 system (Agilent Technologies, Palo Alto, CA).

In a kind of mode, molecular label E be (M, D), wherein M move to change part, D is the test section.Symbol " (M, D) " be used to show that the order of M and D part is such, wherein every part can be adjacent to cleavable connector L.That is to say that " B-L-(M, D) " represented any in two kinds of forms of binding compounds: " B-L-M-D " or " B-L-D-M ".

Test section D can be fluorescence labeling or dyestuff, colour developing mark or dyestuff, electrochemical label or the like.Preferably, D is a fluorescent dye.The example that is used for fluorescent dye of the present invention comprises water-soluble rhodamine, fluorescein, 4,7-dichlorofluorescein, benzo xanthene dyestuff and energy transfer dye, as below with reference to disclosed in the document: Handbook of Molecular Probes and Research, the 8th edition, (Molecular Probes, Eugene, 2002); The United States Patent (USP) 6191278 of Lee etc.; The United States Patent (USP) 6372907 of Lee etc.; The United States Patent (USP) 6096723 of Menchen etc.; The United States Patent (USP) 5945526 of Lee etc.; Lee etc., Nucleic Acids Research, 25:2816-2822 (1997); Hobb, the United States Patent (USP) 4997928 of Jr.; The United States Patent (USP) 4318846 of Khanna etc.; Or the like.Preferably, D is fluorescein or fluorescein derivative.

In the embodiment shown in Fig. 3 A, binding compounds comprises the biotinylated antibody (300) as bound fraction.Molecular label is connecting bound fraction (300) by Avidin or streptavidin bridge (306).Preferably, in operation, bound fraction (300) at first reacts with the target compound, adds (304) Avidin or streptavidin afterwards to form antibody-biotin-avidin compound (305).Add (308) biotinylation molecular label (310) to form binding compounds (312) to this compound (305).

In another embodiment shown in Fig. 3 B, binding compounds comprises the antibody (314) of being derived by multifunctional part (316), this multifunctional part contains polyfunctional group (318), and it can obtain being connected the final binding compounds of a plurality of molecular labels (322) with molecular label precursors reaction (320).The example of multifunctional part comprises glycosaminoglycan and similar substance.

In case after every kind of binding compounds was derived respectively by different molecular labels, it promptly converged with other binding compounds and forms a plurality of binding compounds.Usually, each different types of binding compounds is present in the composition with identical ratio; Yet, can change as the design alternative ratio, make that one class or the group of particular combination compound exist with higher or lower ratio according to the expectation or the needs of specific embodiment or mensuration.The factor that may influence this design alternative includes but not limited to, to the antibody compatibility and the affinity of particular target, the relative dominance of target, fluorescent characteristic of molecular label test section or the like.

B. the cracking that produces active substance causes part

Cracking causes part or decomposition agent is a kind of group that can produce active substance, and described active substance can cracking cleavable connector, preferably the mode by oxidation.Preferably, this active substance is to show active in short-term chemical substance, and its cracking initiation effect only works in the short range in its generation site like this.This active substance life-span itself is short, will can not produce tangible background owing to exceeded its generation short range like this, or adopt the scavenger that can effectively remove this active substance, makes it can not react with the cleavable connector outside its generation site short distance.Exemplary active substance comprises singlet oxygen, hydrogen peroxide, NADH, and hydroxyl, phenoxy group, superoxides or the like.Amino acid-pyrroles N-the conjugate that comprises polyalkenes, carotenoid, vitamin E, vitamin C, tyrosine for the quencher example of the active substance that produces oxidation reaction, histidine and glutathione or the like, Beuther etc. for example, Meth.Enzymol., 319:226-241 (2000).

Adopt cracking to cause in the determination test of part and cleavable connector in design, an important consideration is that the distance that they are removed each other when being incorporated into receptor complex can not be too far away, to such an extent as to should cause active substance that part produces this cleavable connector of cracking effectively by cracking.In a kind of mode, the cleavable connector preferably causes within the 1000nm of part in the cracking of institute's combination, and preferably within 20-200nm.More preferably, cause part for the photosensitizer cracking that generates singlet oxygen, the cleavable connector is arranged within about 20-100nm of photosensitizer of receptor complex.Cracking causes partly effectively, and the scope of cracking cleavable connector (that is, the molecular label that cracking is enough is to generate detectable signal) is called its " effectively short range " at this.Those of ordinary skills should understand, effective short range of specific sensitizer depends on the details of particular assay design and can measure and change by the experiment of routine.

Sensitizer is a kind of can be directed producing reaction intermediates or material, usually the compound of position singlet oxygen.Preferably, sensitizer used according to the invention is a photosensitizer.Other sensitizer that is included in the scope of the present invention has by heat, light, ionising radiation, or chemical activation and after activating, and will discharge the compound of singlet oxygen molecular.The member who knows most in this compounds comprises endoperoxide, for example 1, and 4-biscarboxyethyl-1,4-naphthalene endoperoxide, 9,10-biphenyl anthracene-9,10-endoperoxide and 5,6,11,12-tetraphenyl naphthalene 5,12-endoperoxide.Thereby these compound heating or direct absorbing light discharge singlet oxygen.Other sensitizers are disclosed in the following document: Di Mascio etc., FEBS Lett., 355:287 (1994) (peroxidase and oxygenase); Kanofsky, J.Biol.Chem.258:5991-5993 (1983) (milk peroxidase); Pierlot etc., Meth.Enzymol., 319:3-20 (2000) (thermal cracking of endoperoxide) or the like.The mode that bond is connected to cracking initiation part can be direct or indirect, covalently or non-covalently, and can realize that it can obtain in many documents by technique known.Referring to for example, " Immobilized Enzymes ", Ichiro Chibata, Halsted Press, NewYork (1978); Cuatrecasas, J.Biol.Chem., 245:3059 (1970).

As mentioned above, preferred cracking initiation part is the photosensitizer that can produce singlet oxygen according to the present invention.Here employed " photosensitizer " relates to the light absorption molecule, and it converts molecular oxygen to singlet oxygen when photoactivation.Photosensitizer can be connected on the bond of class specific reaction thing directly or indirectly by connector covalently or non-covalently.For making up this composition, especially the technological guidance as the antibody of bond can obtain in the literature, for example in light dynamic property therapy, immunodiagnosis or the like field.Below be exemplary list of references: Ullman etc., Proc.Natl.Acad.Sci.USA 91,5426-5430 (1994); Strong etc., Ann.New York Acad.Sci., 745:297-320 (1994); Yarmush etc., Crit.Rev.Therapeutic Drug Carrier Syst., 10:197-252 (1993); The United States Patent (USP) 5709994 of Pease etc.; The United States Patent (USP) 5340716 of Ullman etc.; The United States Patent (USP) 6251581 of Ullman etc.; United States Patent (USP) 5516636 of McCapra or the like.

Existed a large amount of light sources in order to the photoactivation photosensitizer to produce singlet oxygen.Polychrome and monochromatic source can adopt, as long as this light source has enough intensity to produce enough singlet oxygens in the duration of reality.Illumination wavelength depends on the character of photosensitizer, the character of cleavable connector, the power of irradiation source, with and to distance of sample or the like.In general, irradiation time can be less than an about microsecond to reaching 10 minutes, usually about one millisecond to about 60 seconds scope.The intensity and the wavelength of irradiation should enough excite about at least 0.1% photosensitizer molecule, usually about at least 30% photosensitizer molecule, preferably nearly all photosensitizer molecule.Light source example comprises, for example and also says laser instrument without limitation, as he-Ne laser, argon laser, YAG laser instrument, He/Cd laser instrument and ruby laser, photodiode, mercury, sodium and xenon vapour lamp, incandescent lamp is tungsten and tungsten/halogen for example, flashlamp or the like.Among the embodiment, adopted the photoactivation device that discloses among the open WO 03/051669 of international monopoly of Bjornson etc.In brief, the photoactivation device is a kind of array that is installed in the light emitting diode (LED) in the shell, and it can illuminate all plate holes in 96 orifice plates simultaneously.Be applicable to that LED of the present invention has high power GaAIAs IR transmitter, for example by OPTO DIODECORP. (Newbury Park, CA) the OD-880W type of Sheng Chaning.

Can be used for photosensitizer example of the present invention and should have above-mentioned characteristic, be set forth in the following document: the United States Patent (USP) 5536834 of Singh and Ullman; The United States Patent (USP) 5763602 of Li etc.; Martin etc., MethodsEnzymol., 186:635-645 (1990); Yarmush etc., Crit.Rev.Therapeutic DrugCarrier Syst., 10:197-252 (1993); The United States Patent (USP) 5709994 of Pease etc.; The United States Patent (USP) 5340716 of Ullman etc.; The United States Patent (USP) 6251581 of Ullman etc.; The United States Patent (USP) 5516636 of McCapra; The European patent of Thetford discloses 0484027; Sessler etc., SPIE, 1426:318-329 (1991); The United States Patent (USP) 5565552 of Magda etc., United States Patent (USP) 6001673 of Roelant or the like.

In certain embodiments, as sensitizer, photosensitizer can combine with solid support, covalently or non-covalently is attached on the surface of holder or adds in the holder main body.In general, the amount of the photosensitizer that combines with holder must be able to obtain the singlet oxygen of aequum.In general, the amount of photosensitizer is rule of thumb determined.

In one embodiment, photosensitizer is added in the latex particle to form photosensitizer glue pearl, for example the United States Patent (USP) 5709994 of Pease etc. has disclosed among the open WO 01/84157 of the PCT of the United States Patent (USP) 6346384 of Pollner and Pease etc.Perhaps, preparation photosensitizer glue pearl can be by the chloromethyl on the latex bead with photosensitizer, is connected on 0.5 micron the latex bead as rose-red, thereby the ester linking group is provided, as J.Amer.Chem.Soc., described in the 97:3741 (1975).The use of this photosensitizer glue pearl as shown in Figure 3.As the detection that is used for heteroduplex described in Fig. 1 C, formed compound (330) after reagent (1122) and the sample mix.Implementing this reaction can carry out in 96 for example conventional holes or 384 hole titer plate or the like, wherein has a wall that forms plate hole, as the bottom, filtering membrane, make reaction reagent to remove by applying negative pressure.Be different from the singlet oxygen generation or separate required damping fluid in conjunction with required damping fluid if be used for the binding compounds specificity, above-mentioned technology is the conversion damping fluid easily.For example, under situation, need the damping fluid of high salt based on the binding compounds of antibody.If release mark is adopted electrophoretic separation, this damping fluid is changed into be applicable to that damping fluid electrophoresis, that have than low salt concn then can obtain more performance.In this embodiment, for replacing directly photosensitizer being connected on binding compounds such as the antibody, the cracking probe can comprise two kinds of compositions: with catching the antibody (332) that part derives, biological example element (being designated as " bio " among Fig. 3 C), with photosensitizer glue pearl (338), the surface of this glue pearl is used and is caught reagent (334) that part combines as Avidin or streptavidin specificity and derive and obtain.Then, catch (335) as biotin (336) with the part of catching of compound (330) by photosensitizer glue pearl.Be that if select the aperture of filtering membrane to make the intransitable words of photosensitizer glue pearl (338), then buffer-exchanged also plays the effect of removing unconjugated binding compounds, the signal that is improved so easily.If necessary, add the suitable damping fluid that is used to separate, shine photosensitizer glue pearl (338) afterwards and make that generating (342) singlet oxygen also discharges (344) molecular label.The molecular label (346) that separates this release then forms separate picture (352), and comes the quantitative dimer of reference according to peak (348) and (350).Photosensitizer glue pearl can be used for mensuration even or heterogeneous mutually in.

Preferably, when the check and analysis thing, during as the antigen in cell surface receptor or the fixed sample, the cracking probe can comprise elementary haptenization antibody and derive the secondary antihapten that obtains in conjunction with albumen with a plurality of photosensitizer molecules.Preferred elementary haptenization antibody is biotinylated antibody, and preferred secondary antihapten can be anti-biotin antibodies or streptavidin in conjunction with albumen.Other combination of this primary and secondary reactant is well known in the art, Hauland for example, Handbook of Fluorescent Probes and Research Reagents, the 9th edition (Molecular Probes, Eugene, OR, 2002).The example combinations of this reactant is shown in Fig. 3 E.But having release mark (is " mT among the figure ₁" and " mT ₂") binding compounds (366 and 368), and be incorporated into specifically on the different epi-positions of receptor dimer (362) in the film (360) by one anti-(368) that biotin (369) is derived.Biotin binding proteins specific (370), for example streptavidin is connected on the biotin (369), a plurality of photosensitizers (372) is introduced in effective short range of binding compounds (366 and 368).Biotin binding proteins specific (370) can also be anti-biotin antibodies, and by conventional coupling chemical method, as Hermanson (quoting as above), photosensitizer can be connected by the free amine group group on the protein.The photosensitizer example of this purposes has, and the European patent of pressing Shimadzu etc. discloses the NHS ester of the methylene blue of the method preparation disclosed in 0510688.

Condition determination

Below for illustrative purposes and unrestricted to the roughly argumentation of method, specified conditions and material.Those of ordinary skills should understand how to make method described here be applicable to other application, particularly to adopt the application of different samples, cell type and target compound.

In implementing method of the present invention, the mixed determining composition comprises sample, binding compounds and selectable cracking probe.In general, measuring composition can mix with any order.Yet in some applications, the order of adding has relation.For example, the someone wishes to monitor competitive combination, for example in quantitative measurement.Perhaps wish to monitor the stability of the compound of being formed by the people.In these are used, can react by proceed step by step, and before all potpourris combine, perhaps before starting cracking reaction, may need the incubation process.

The amount of every kind of reaction reagent rule of thumb comes to determine usually.In the mensuration used sample size by the pre-quantitation of the target compound that exists and the separation and the detection method that are used to monitor measured signal determined.In general, the molal quantity of binding compounds that is provided and cracking probe amount should relative pre-metering greater than target molecule in the sample, and molal quantity should excessive at least 1.5 times usually, more preferably excessive 10 times or more.In application-specific, the concentration of use can be higher or lower, decides according to the target molecule desired number that exists on the compatibility of bond and the individual cells.Measuring under the situation of chemical compound to the effect of formation oligomerization cell surface compound, this compound can add before adding probe simultaneously or afterwards, decided according to monitored effect.

Be used for probe is connected under the condition of cell surface molecule, mix and incubation mensuration potpourri, normally in aqueous medium, generally under physiological pH (pH of similar cultured cell), keep by the damping fluid of concentration in about 10 to 200mM scopes.Can adopt common damping fluid, and other required common additives, for example salt, nutrient culture media, stabilizing agent etc.Usually adopt physiology and stationary temperature.The normal range of heated culture temperature is at about 4 ° to 70 ℃, and is generally at about 15 ° to 45 ℃, more common at 25 ° to 37 ℃.

Measuring potpourri forms and carries out incubation and make after probe is attached on the cell surface molecule, handle this potpourri with the activation decomposition agent, thereby the cracking from the binding compounds of the label in the effective short range of decomposition agent is got off, corresponding mark is discharged into the solution from cell surface.The character of this processing depends on the mechanism of action of decomposition agent.For example, adopting under the situation of photosensitizer as decomposition agent, the cracking activation is included in this potpourri of irradiation under the wavelength light that is applicable to employed specific sensitizer.

After the cracking, then analytic sample is to measure d/d mark identity.Under the situation that adopts a plurality of binding compounds, it will separate the mark that is discharged before detecting usually.The method of separating and detecting is definite in the process of the mark that is designed for this mensuration.Preferred separate mode is to adopt electrophoresis, promptly separates various marks according to the known difference of its electrophoretic mobility.

As mentioned above, in certain embodiments,, just need before cracking and isolated molecule label, remove or change the assaying reaction damping fluid if the condition of assaying reaction may be disturbed the isolation technics that adopts.For example, condition determination can comprise salinity (for example specificity is in conjunction with required), and it can reduce separating property when isolated molecule label on the electrophoretic mobility basis.Therefore, can remove this high-salt buffer, for example before the cracking molecular label, and by filter, suction, dilution or other means replace with the another kind of damping fluid that is fit to electrophoretic separation.

Embodiment

Employed material source among the embodiment

The Her acceptor is had specific antibody, aptamers molecule and standardized standard can be buied from the market supply merchant, comprises Labvision, Cell Signaling Technology and BDBiosciences.Clone is buied from ATCC.All human quick-frozen tissue samples are available from WilliamBaibridhe Genome Foundation (Seattle, WA) or Bio Research Support (Boca Raton, and passed through Institutional Reseach Board (IRB) approval of suppliers FL).

Below employed molecular label-antibody coupling matter form by the step reaction of the free amine group on the NHS ester of molecular label and the indication antibody by routine.Molecular label below is labeled as title " Pro_N ", has disclosed in below with reference to document: the U.S. Patent Publication 2003/017915 and 2002/0013126 of Singh etc., introduce it as a reference.In brief, following binding compounds is molecular label-monoclonal antibody conjugate that the free amine group by the NHS ester of molecular label and antibody forms by popular response.

Embodiment 1

The special-shaped dimerization of the Her-2 of pair cell lysate

Analysis with receptor phosphorylation

In this embodiment, measured Her1-Her2 and Her2-Her3 heterodimer and phosphorylation state in the cell lysate that comes from several clones, this clone is handled with the epidermal growth factor (EGF) and the heregulin (HRG) of multiple concentration.Adopted three kinds of binding compounds and cracking probe to measure, as described below.

Sample separation:

1. the breast cancer cell line of serum starvation overnight incubation before use.

2. in nutrient culture media, it is 10 minutes under 37 ℃ with EGF and/or HRG irritation cell.(SKBR-3) dosage of EGF/HRG for example 0,0.032,0.16,0.8,4,20 for MCF-7 for example, T47D, and 100nM should increase to 500nM to the maximum dose of BT20, because can not reach capacity with the EGF of 100nM to all cells system except that BT20.

3. the suction nutrient culture media is transferred on ice, and adds lysis buffer with cracking original position cell.

4. scrape and get and shift lysate to micro-centrifuge tube.Ice is educated 30min.14000rpm, 4 ℃ of following little centrifugal 10min.(can select to carry out centrifugal)

5. collect supernatant as pyrolysis product, packing be stored in-80 ℃ to be used.

Measure;

Measure design: shown in diagram among Fig. 4 A, the combination according to cracking probe (902) and binding compounds (904), (906) and (908) comes the quantitative Her2-Her3 heterodimer of reference (900).The photosensitizer that will be labeled as " PS " is connected on the cracking probe (902) by Avidin-biotin connector, and distinguishes mark binding compounds (904), (906) and (908) with molecular label Pro14, Pro10 and Pro11.Binding compounds (904) has specificity to the phosphorylation site on the Her3.

Total mensuration volume is 40ul.With lysis buffer the lysate volume is adjusted to 30ul.Antibody is diluted to 10ul with lysis buffer.Typically, use the pyrolysis product that is equivalent to-5000 to 15000 cells in each reaction.Detection limit is the pyrolysis product that is equivalent to-1000 cells.

Step: the ultimate density of the binding compounds that is pre-mixed in the reaction (being antibody coupling matter molecular label or biotin):

The anti-Her-2:0.1ug/ml of Pro4_

The anti-Her-1:0.05-0.1ug/ml of Pro10_

The anti-Her-3:0.1ug/ml of Pro11_

Anti-phosphorus-the Tyr:0.1ug/ml of Pro2_

Biotin _ anti-Her-2:1-2ug/ml

1. in 96 holes of measuring, add 10ul mixtures of antibodies incubation 1 hour in the lysate of 30ul and under the room temperature.

2. add cracking probe (final concentration 2ug/ hole) that the streptavidin of 2ul derives in the hole of measuring and incubation 45min.

3. in the PBS that adds 150ul and 1% BSA to the 96 hole titer plate (MilliporeMAGVN2250) and at room temperature incubation 1hr is in order to block.

4. by vacuum suction sucking filtering plate.Transfer to assaying reaction on the filter and apply negative pressure and find time.

5. add the 200ul lavation buffer solution and apply negative pressure and find time.Repeat once.

6. add 200ul irradiation damping fluid and apply negative pressure and find time.Repeat once.

7. adding 30ul shines damping fluid and shone 20 minutes.

8. shift in each reaction and analyze on 10ul to the CE assay plate, adopt 22cm ABI3100CE instrument capillaceous (injecting condition: 5kV, 75sec, 30 ℃; Service condition: 600sec, 30 ℃).

The mensuration damping fluid is as follows:

Lysis buffer (newly make and preserving on ice)

Final concentration The ul storage capacity

1％Triton X-100 1000 10％

20mM Tris-HCl(pH 7.5) 200 1M

100mM NaCl 200 5M

50mM NaF 500 1M

50mM β-phosphoglycerol Na 1000 0.5M

1mM Na ₃VO ₄ 100 0.1M

5mM EDTA 100 0.5M

10ug/ml pepsin inhibitor 100 1mg/ml

1 (every 10ml) Roche Complete protease inhibitors (#1836170) N/A N/A

Water 6500 N/A

Amount to 10ml

Lavation buffer solution (4 ℃ of storages)

Final concentration The ul storage capacity

1％NP-40 50 10％

1x PBS 50 10x

150mM NaCl 15 5M

5mM EDTA 5 0.5M

Water 380 N/A

Amount to 500ml

The irradiation damping fluid:

Final concentration The ul storage capacity

0.005x PBS 50 1x

CE standard 3 100x

10mM Tris-HCl(pH8.0) 0.1M

10pM A160 1nM

10pM A315 1nM

10pM HABA 1nM

Water 10000 N/A

Amount to 10ml

Data analysis:

1. make relative fluorescence unit (RFU) signal standardization of each molecular label according to CE normative reference A315 (the desoxyadenossine list phosphoric acid that a kind of fluorescein is derived, its known peak position is with relevant according to the molecular label in the mensuration of electrophoretic separation).

2. from corresponding molecular label signal, deduct the RFU of " no pyrolysis product " background contrast.

3. the special-shaped dimerization degree of report Her-1 or Her-3, as corresponding RFU with come from use biotin anti--Pro4_ in the mensuration hole of Her-2 is anti--reference of Her-2RFU.

4. report Her-1,2,3 receptor phosphorylation degree, as Pro2_PT100 anti--RFU of phosphorus-Tyr with come from use biotin anti--Pro4_ in the mensuration hole of Her-2 is anti--reference of Her-2RFU.

Measurement result is shown in Fig. 4 B-4H, and Fig. 4 B represents that the amount of Her1-Her2 heterodimer increases on the MCF-7 cell along with the increase of EGF concentration, and same dimeric amount is along with the increase not variation basically of HRG concentration.Fig. 4 C has represented the adverse consequences on the Her2-Her3 heterodimer.That is to say that the amount of Her2-Her3 heterodimer increases on the MCF-7 cell along with the increase of HRG concentration, and same dimeric amount is along with the increase not variation basically of EGF concentration.Fig. 4 D and 4E represent that the amount of Her1-Her2 heterodimer increases respectively along with the increase of EGF concentration on SKBR-3 cell and BT-20 cell.

Embodiment 2

To the special-shaped dimerization of the Her-2 that organizes lysate

Analysis with receptor phosphorylation

In this embodiment, measured Her1-Her2 and Her2-Her3 heterodimer and the phosphorylation state in the lysate organized that comes from the human breast cancer sample.

Sample separation:

1. by cutting Mechanical Crushing quick-frozen tissue under freezing state.

2. be transferred to tissue in the micro-centrifuge tube and add the lysis buffer (appendix I) of 3x tissue volume, stir then so that tissue is dispersed in the damping fluid.

3. ice is educated 30min and stirring and evenly mixing frequently.

4. 14000rpm, 4 ℃ of centrifugal 20min down.

5. collect supernatant as pyrolysis product and use an aliquot to measure total protein concentration by BCA determination experiment (Pierce)

6. the packing surplus materials is to be used to be stored in-80 ℃.

Measure design:

1. always measuring volume is 40ul.

2. mensuration lysate, it becomes continuous titration series 40,20,10,5,2.5,1.25,0.63, total equivalent of 0.31ug, and with lysis buffer volume is adjusted to 30ul.From the data acknowledgement of this titration series the specificity of dimerization or phosphorylation signal.

3. comprise that all universal antibody potpourris that are diluted in the eTAg-antibody in the lysis buffer use under following concentration.

4. will add in the reaction respectively at the independent biotin-antibody of each acceptor.

5. organize lysate to carry out three kinds of eTag with each and measure, every kind is used the different biotin-antibody corresponding to tested special receptor dimerization.

6. measure every kind of receptor expression level in the difference mensuration that contains this receptor specific biological element-antibody.

Only in the mensuration that contains biotin-anti-Her-2 reference measure dimerization and phosphorylation signal.

Measure contrast: with MCF-10A and the negative and positive qualitative contrast of conduct respectively of MCF-7 clone.Clone or irriate not, or stimulate with 100nM EGF or 100mM HRG.When replacing tissue sample, comprised that lysis buffer contrasts as background.

The ultimate density of the antibody that is pre-mixed in the reaction:

The universal antibody potpourri:

The anti-Her-2:0.1ug/ml of Pro4_

The anti-Her-1:0.05-0.1ug/ml of Pro10_

The anti-Her-3:0.1ug/ml of Pro11_

Anti-phosphorus-the Tyr:0.01ug/ml of Pro2_

Independent biotin antibody:

Biotin _ anti-Her-1:2ug/ml

Biotin _ anti-Her-2:2ug/ml

Biotin _ anti-Her-3:2ug/ml

Step:

1. in the universal antibody potpourri, add biotin antibody with preparation antibody response potpourri.

2. in 96 holes of measuring, add the general reaction mixture of 10ul incubation 1 hour in the lysate of 30ul and under the room temperature.

3. add cracking probe (final concentration 2ug/ hole) that the streptavidin of 2ul derives in the hole of measuring and incubation 45min.

4. in the PBS that adds 150ul and 1% BSA to the 96 hole titer plate (MilliporeMAGVN2250) and at room temperature incubation 1hr is in order to block.

5. by vacuum suction sucking filtering plate.Transfer to assaying reaction on the filter and apply negative pressure and find time.

6. add the 200ul lavation buffer solution and apply negative pressure and find time.Repeat once.

7. add 200ul irradiation damping fluid and apply negative pressure and find time.Repeat once.

8. adding 30ul shines damping fluid and shone 20 minutes.

9. shift in each reaction and analyze on 10ul to the CE assay plate, adopt 22cm ABI3100 capillary electrophoresis apparatus capillaceous (injecting condition: 5kV, 75sec, 30 ℃; Service condition: 600sec, 30 ℃).

Data analysis:

1. make RFU signal standardization of each molecular label according to CE normative reference A315.

2. measure the cutoff (respectively at dimerization or phosphorylation degree) of RFU, do not calculate the ratio under it, because signal value is too low and unreliable.Under this cutoff, the RFU signal in the tested cracking dilution series is not titratable.Available expectation dimerization and phosphorylation signal do not exist or low-down one big group normal structure is measured this value.These values are also represented the basic horizontal of dimerization on the normal structure that will compare with tumor tissues or acidifying.

3. if any, be higher than the normal structure of cutoff, measure the independent RFU level and the reference reading at Her-1 that surveys of institute or Her-3 abnormal shape dimerization or phosphorylation peak in order to monitor the RFU value.These samples are represented exceptional value, and it should be used as the donor contrast that corresponding neoplasmic tissue sample is complementary during record.

4. to all tumor samples that shows titratable RFU signal, the lowest signal of phosphorylation degree that adopts each Her-1, Her-2, Her-3 or organize the lysate titration series is as background.From the molecular label of high dose lysate (as 40ug), deduct this background to obtain special RFU signal.If there is not the signal dose response in the titration series, then regards all signals (very low usually) as background and do not have specific signals can be used as the reference analysis.

5. the special-shaped dimerization degree of report Her-1 or Her-3, as corresponding special RFU with come from Pro4_ anti--reference of the special RFU of Her-2.If do not obtain special RFU, then the dimerization degree is negative.

6. report Her-1,2,3 receptor phosphorylation degree is as specificity RFU and the reference that comes from the specificity RFU of the anti-Her-2 of Pro4_ of the anti-phosphorus-Tyr of Pro2_.If do not obtain special RFU, then phosphorylation degree is negative.

A plurality of patients' that data represented usefulness mensuration of the present invention shown in Fig. 5 A-5C records breast tissue sample.The clinical Her-2 situation that comes from immunohistochemistry (DAKO Herceptest) in 10 tumor samples, have 9 negative, show or not detect Her-2 painted, tumour cell less than 10% is painted, perhaps on the parts of fine after birth greater than 10% tumour cell painted faint hardly as seen.Her-1, Her-2 and Her-3 that determination experiment of the present invention has been measured on normal and the tumor tissues simultaneously express.The special-shaped dimerization degree of Her-1 and Her-2 and Her-2 and Her-3 only detects in tumor tissues, does not all record in any normal structure.

Embodiment 3

The Her1 of pair cell lysate or Her2 homotype dimerization

Analysis with receptor phosphorylation

The operation of specimen preparation is basically as described in the embodiment 2.Handle clone to cause the homotype dimerization of Her1 with EGF and TGF α.For the homotype dimerization of the Her2 that does not have part, compare with the not irriate MCF-7 cell of low expression level Her2 crossing the not irriate SKBR-3 or the MDA-MD453 cell of expressing Her2.

Measure design: will to acceptor have specific monoclonal antibody respectively with molecular label or biotin coupling (being connected with photosensitizer by the Avidin bridge again after the biotin coupling), make cracking probe and binding compounds competitiveness in conjunction with epi-position same among this embodiment.The another kind of binding antibody that uses comprises the second antibody of overlapping epi-position on the energy identification receptor, can generate the measured value of reference signal as the homotype dimerization like this.The signal that obtains from second antibody also provides the measured value of acceptor total amount in the sample.The acceptor total amount is measured in independent mensuration hole.Receptor phosphorylation can or always be subjected to scale of construction quantitative measurement together with the homotype dimerization.

Step: the mensuration volume is 40ul, and key step is similar to embodiment 2.Each sample is set up two measure hole A and B so that quantitative dimerization degree and acceptor total amount respectively.

For the Her1-Her1 homodimer quantitatively:

Measure the mixtures of antibodies final concentration among the A of hole:

The anti-Her-1:0.05-0.1ug/ml of Pro12_

Biotin _ anti-Her-1:1-2ug/ml

Measure the mixtures of antibodies final concentration among the B of hole:

The anti-Her-1:0.05-0.1ug/ml of Pro10_

Anti-phosphorus-the Tyr:0.1ug/ml of Pro2_

Biotin _ anti-Her-1:1-2ug/ml

For the Her2-Her2 homodimer quantitatively:

Measure the mixtures of antibodies final concentration among the A of hole:

The anti-Her-1:0.05-0.1ug/ml of Pro4_

Biotin _ anti-Her-1:1-2ug/ml

Measure the mixtures of antibodies final concentration among the B of hole:

The anti-Her-1:0.05-0.1ug/ml of Pro4_

Anti-phosphorus-the Tyr:0.1ug/ml of Pro2_

Biotin _ anti-Her-1:1-2ug/ml

Data analysis:

1. make RFU signal standardization of each molecular label according to CE normative reference A315.

2. from corresponding molecular label signal, deduct the RFU of " no pyrolysis product " background contrast.

3. report the homotype dimerization degree of Her-1 or Her-2, as the reference that always is subjected to accordingly scale of construction standardization RFU among corresponding standard RFU among the mensuration hole A and the mensuration hole B.

4. report the receptor phosphorylation degree of Her-1 or Her-2 homodimer, as standardization RFU that measures the anti-phosphorus-Tyr of Pro2_PT100 among the B of hole and the reference that always is subjected to scale of construction standardization RFU that comes among the same mensuration hole B.

Measurement result such as Fig. 6 A-6B and shown in Figure 7, Fig. 6 A represent that the amount of Her1-Her1 homodimer increases on the BT-20 cell along with the increase of EGF concentration.Fig. 6 B represents that the amount of Her1 phosphorylation in the BT-20 cell increases along with the increase of EGF concentration.Signal by in the MCF-7 cell of Her2 expression reduction on signal in the SKBR-3 cell of relatively expressing Her2 and the cell surface has proved the detection to the Her2-Her2 homodimer.In the chart as shown in Figure 7, do not detect special titratable Her2-Her2 homodimer signal, and clearly be positioned on the signal from the MCF-7 cell from the Her2-Her2 homodimer signal of SKBR-3 cell with the MCF-7 cell.

Embodiment 4

The special-shaped dimerization of the Her-1-Her3 of pair cell lysate

Analysis with receptor phosphorylation

Sample separation is as follows:

1. the breast cancer cell line of serum starvation overnight incubation before use.

2. in nutrient culture media, it is 10 minutes under 37 ℃ with the HRG irritation cell.For the dosage of T47D cell HRG for example 0,0.032,0.16,0.8,4,20,100nM.

3. the suction nutrient culture media is transferred on ice, and adds lysis buffer with cracking original position cell.

4. scrape and get and shift lysate to micro-centrifuge tube.Ice is educated 30min.14000rpm, 4 ℃ of following little centrifugal 10min.(can select to carry out centrifugal)

5. collect supernatant as pyrolysis product, packing be stored in-80 ℃ to be used.

Measure design: always measuring volume is 40ul.With lysis buffer the lysate volume is adjusted to 30ul.Antibody is diluted to 5ul with lysis buffer.Typically, use the pyrolysis product that is equivalent to-5000 to 15000 cells in each reaction.The ultimate density of the antibody that is pre-mixed in the reaction:

The anti-Her-1:0.05-0.1ug/ml of Pro10_

The anti-Her-3:0.1ug/ml of Pro11_

Anti-phosphorus-the Tyr:0.1ug/ml of Pro2_

Biotin _ anti-Her-3:1-2ug/ml

1. in 96 holes of measuring, add 5ul mixtures of antibodies incubation 1 hour in the lysate of 30ul and under the room temperature.

2. add molecule that the streptavidin of 5ul derives and cut (molecular scissor) (final concentration 4ug/ hole) in the hole of measuring and incubation 45min.

3. in the PBS that adds 150ul and 1% BSA to the 96 hole titer plate (MilliporeMAGVN2250) and at room temperature incubation 1hr is in order to block.

4. by vacuum suction sucking filtering plate.Transfer to assaying reaction on the filter and apply negative pressure and find time.

5. add the 200ul lavation buffer solution and apply negative pressure and find time.Repeat once.

6. add 200ul irradiation damping fluid and apply negative pressure and find time.Repeat once.

7. adding 30ul shines damping fluid and shone 20 minutes.

8. shift in each reaction and analyze on 10ul to the CE assay plate, adopt 22cm ABI3100 capillary electrophoresis apparatus capillaceous (injecting condition: 5kV, 425sec, 30 ℃; Service condition: 600sec, 30 ℃).

Data analysis:

1. make RFU signal standardization of each eTag acceptor according to CE normative reference A315.

2. from corresponding eTag receptor signal, deduct the RFU of " no pyrolysis product " background contrast.

3. report special-shaped dimerization degree, Pro10 RFU that derives as Her-1 and reference from the Pro11 RFU of anti--Her-3.

4. report receptor phosphorylation degree, as from Pro2_PT100 anti--RFU of phosphorus-Tyr with come from use biotin anti--Pro11_ in the mensuration hole of Her-3 is anti--reference of Her-3RFU.

Measurement result is shown in Fig. 8 A and 8B.Data show that special-shaped dimerization degree of Her1-Her3 and dimer phosphorylation degree increase along with the increase of HRG concentration.

Embodiment 5

The increase that the Her1-Her3 receptor dimer is expressed in the cancerous cell line

Increase with the answer list skin growth factor

In this embodiment, measured the Her1-Her3 heterodimer in the cell lysate that comes from cancerous cell line 22Rv1 and A549, this clone is handled with the epidermal growth factor (EGF) of multiple concentration.Adopted three kinds of binding compounds and cracking probe to measure, as described below.

Sample separation:

1. the breast cancer cell line of serum starvation overnight incubation before use.

2. in nutrient culture media, it is 10 minutes under 37 ℃ with the EGF irritation cell.The EGF dosage that applies to two clones for example changes between 0-100nM.

3. the suction nutrient culture media is transferred on ice, and adds lysis buffer with cracking original position cell.

4. scrape and get and shift lysate to micro-centrifuge tube.Ice is educated 30min.14000rpm, 4 ℃ of following little centrifugal 10min.(can select to carry out centrifugal).Measure protein concentration.

5. collect supernatant as pyrolysis product, packing be stored in-80 ℃ to be used.

Measure design basically with identical shown in Fig. 4 A, except following difference: binding compounds (904), (906) and (908) are used molecular label Pro10, Pro11 and Pro2 mark respectively.Total mensuration volume is 40ul.With lysis buffer the lysate volume is adjusted to 30ul.Antibody is diluted to 5ul with lysis buffer.Typically, use the pyrolysis product that is equivalent to-5000 to 15000 cells in each reaction.Detection limit is the pyrolysis product that is equivalent to-1000 cells.Step: the ultimate density of the binding compounds that is pre-mixed in the reaction (being antibody coupling matter molecular label or biotin):

The anti-Her-1:0.05-0.1ug/ml of Pro10_

The anti-Her-3:0.1ug/ml of Pro11_

The anti-phosphorus of Pro2_-Tyr:0.1 to 0.2ug/ml

Biotin _ anti-Her-3:1-2ug/ml

1. in 96 holes of measuring, add 5ul mixtures of antibodies incubation 1 hour in the lysate of 30ul and under the room temperature.

2. add cracking probe (final concentration 4ug/ hole) that the streptavidin of 5ul derives in the hole of measuring and incubation 45min.

3. in the PBS that adds 150ul and 1% BSA to the 96 hole titer plate (MilliporeMAGVN2250) and at room temperature incubation 1hr is in order to block.

4. by vacuum suction sucking filtering plate.Transfer to assaying reaction on the filter and apply negative pressure and find time.

5. add the 200ul lavation buffer solution and apply negative pressure and find time.Repeat once.

6. add 200ul irradiation damping fluid and apply negative pressure and find time.Repeat once.

7. adding 30ul shines damping fluid and shone 20 minutes.

8. shift in each reaction and analyze on 10ul to the CE assay plate, adopt 22cm ABI3100 CE capillaceous instrument (injecting condition: 5kV, 70sec, 30 ℃; Service condition: 425sec, 30 ℃).

The mensuration damping fluid is as follows:

Lysis buffer (newly make and preserving on ice)

Final concentration The ul storage capacity

1％Triton X-100 1000 10％

20mM Tris-HCl(pH 7.5) 500 1M

100mM NaCl 200 5M

50mM NaF 500 1M

50mM β-phosphoglycerol Na 500 1.0M

1mM Na ₃VO ₄ 100 0.1M

5mM EDTA 100 0.5M

10ug/ml pepsin inhibitor 100 1mg/ml

1 (every 10ml) Roche Complete protease inhibitors (#1836170) N/A N/A

Water 7ml N/A

Amount to 10ml

Lavation buffer solution (4 ℃ of storages): 0.5%Triton X-100 among the 1x PBS

The irradiation damping fluid:

Final concentration The ul storage capacity

0.005x PBS 50 1x

CE standard 1 (A27, ACLARA Biosciences, Inc., Mountain View, CA) 4 5000x

CE standard 2 (fluorescein) 4 5000x

Water 9942 N/A

Amount to 10ml

Data analysis:

1. make relative fluorescence unit (RFU) signal standardization of each molecular label according to CE normative reference 2.

2. from corresponding molecular label signal, deduct the RFU of " no pyrolysis product " background contrast.

3. the special-shaped dimerization degree of report Her-1, as corresponding RFU with come from use biotin anti--Pro11_ in the mensuration hole of Her-3 is anti--reference of Her-3RFU.

4. report Her-1,2,3 receptor phosphorylation degree, as Pro2_PT100 anti--RFU of phosphorus-Tyr with come from use biotin anti--Pro11_ in the mensuration hole of Her-3 is anti--reference (data not shown) of Her-3RFU.

The amount that Fig. 9 A and 9B represent the Her1-Her3 heterodimer is along with the increase of EGF concentration increases on 22Rv1 and A549 cell respectively.

Embodiment 6

The tool Her1, the Her2 that occur in the breast tumor tissues lysate

IGF-1R heterodimer with Her3

In this embodiment, adopted and substantially the same determination test shown in Fig. 4 A, measured and come from Her1-IGF-1R, Her2-IGF-1R and the dimeric existence of Her3-IGF-1R in 12 human breast tumor tissues cells of difference.The specimen preparation operation is as follows:

1. by cutting Mechanical Crushing quick-frozen tissue under freezing state.

2. be transferred to tissue in the micro-centrifuge tube and add the lysis buffer of 3x tissue volume, stir then so that tissue is dispersed in the damping fluid.

3. ice is educated 30min and stirring and evenly mixing frequently.

4. 14000rpm, 4 ℃ of centrifugal 20min down.

5. collect supernatant as pyrolysis product and use an aliquot to measure total protein concentration by BCA determination experiment (Pierce)

6. the packing surplus materials is to be used to be stored in-80 ℃.

Determination test is set up as follows:

1. always measuring volume is 40ul.

2. mensuration lysate, it becomes continuous titration series 40,20,10,5,2.5,1.25,0.63, total equivalent of 0.31ug, and with lysis buffer volume is adjusted to 30ul.From the data acknowledgement of this titration series the specificity of dimerization.

3. comprise that all binding compounds that are diluted in the lysis buffer and the universal antibody potpourri of biotin antibody use under following concentration.

The ultimate density of the antibody that is pre-mixed in the reaction:

The anti-Her-2:0.1ug/ml of Pro10_

The anti-Her-1:0.1ug/ml of Pro14_

The anti-Her-3:0.1ug/ml of Pro11_

The anti-IGF-1R:0.1ug/ml of Pro7_

Anti-phosphorus-the Tyr:0.2ug/ml of Pro2_

Biotin _ anti-Her-2:2ug/ml

Step:

1. in 96 holes of measuring, add the general reaction mixture of 5ul incubation 1 hour in the lysate of 30ul and under the room temperature.

2. add molecule that the streptavidin of 5ul derives and cut (final concentration 4ug/ hole) in the hole of measuring and incubation 45min.

3. in the PBS that adds 150ul and 1% BSA to the 96 hole titer plate (MilliporeMAGVN2250) and at room temperature incubation 1hr is in order to block.

4. by vacuum suction sucking filtering plate.Transfer to assaying reaction on the filter and apply negative pressure and find time.

5. add the 200ul lavation buffer solution and apply negative pressure and find time.Repeat once.

6. add 200ul irradiation damping fluid and apply negative pressure and find time.Repeat once.

7. adding 30ul shines damping fluid and shone 20 minutes.

8. shift in each reaction and analyze on 10ul to the CE assay plate, adopt: (i) CE instrument: ABI3100,22cm kapillary, (ii) CE injecting condition: 5kV, 70sec, 30 ℃, (iii) CE service condition: 425sec, 30 ℃.

Data analysis:

1. make RFU signal standardization of each molecular label according to CE normative reference 1.

2. seek the titratable signal of each molecular label.Signal that can not titration is assumed to non-specific signal and can not be used for data parsing.The dimerization signal does not exist or low-down one big group normal structure is measured cutoff on the estimation.These values have also been represented the dimerization basic horizontal on the normal structure that will compare with tumor tissues.

3. the special-shaped dimerization degree of report IGF-1R and Her-1 or Her-2 or Her-3 is as corresponding RFU.

There are two to express Her1-IGF-1R, Her2-IGF-1R and Her3-IGF-1R heterodimer in 12 tumors of breast measuring, as shown in Figure 10 A-C.Lines in each drawing show tested acceptor heterodimer amount and to the trend between the amount of the lysate of surveying in two tumor of breast samples being positive of indication heterodimer.

Embodiment 7

PI3K/Her-3 receptor activation compound

In this embodiment, measure design as shown in Figure 11 A and 11C, in order to measure the receptor complex that comprises Her2, Her3 and PI 3K among the breast cancer cell line MCF-7.Have first molecular label and (be " mT among the figure ₁"; following for " eTag1 ") binding compounds (1106) has specificity to the extracellular region territory of Her3 acceptor (1102); have second molecular label (being " mT2 " among the figure; followingly be " eTag2 ") binding compounds (1110) the p185 composition of PI3K albumen (1100) is had specificity; and the cracking probe (1108) that is connected with photosensitizer has specificity to the intracellular region territory of Her3 acceptor (1102); " H2 " expression Her2 acceptor (1104) wherein, " H3 " represents Her3 acceptor (1102), " p85 " and " p110 " is the composition of PI3 kinases (1100), and it partly is incorporated into the phosphorylation site (being expressed as " P ") of H3 by its p85.Measure design class seemingly for these two, except cracking probe in the design of Figure 11 A is specific to the Her3 acceptor, and the cracking probe is specific to outside the kinase whose p85 composition of PI3 in the design of Figure 11 C.This measurement operation is as follows.

Specimen preparation:

1. the breast cancer cell line of serum starvation overnight incubation before use.

2. in nutrient culture media, it is 10 minutes under 37 ℃ with the HRG irritation cell.The HRG dosage that is used for the MCF-7 cell for example 0,0.032,0.16,0.8,4,20,100nM.

3. the suction nutrient culture media is transferred on ice, and adds aforesaid lysis buffer with cracking original position cell.

4. scrape and get and shift lysate to micro-centrifuge tube.Ice is educated 30min.14000rpm, 4 ℃ of following little centrifugal 10min.(can select to carry out centrifugal)

5. collect supernatant as pyrolysis product, packing be stored in-80 ℃ to be used.

Lysis buffer (newly make and preserving on ice)

Final concentration The ul storage capacity

1％Triton X-100 1000 10％

20mM Tris-HCl(pH 7.5) 200 1M

100mM NaCl 200 5M

50mM NaF 500 1M

50mM β-phosphoglycerol Na 1000 0.5M

1mM Na ₃VO ₄ 100 0.1M

5mM EDTA 100 0.5M

10ug/ml pepsin inhibitor 100 1mg/ml

1 (every 10ml) Roche Complete protease inhibitors (#1836170) N/A N/A

Water 6500 N/A

Amount to 10ml

Measure design: the formation that comes the quantitative receptor complex of reference according to the diagram shown in each figure.That is to say that measuring reading is the peakedness ratio of molecular label eTag2/eTag1.

Total mensuration volume is 40ul.With lysis buffer the lysate volume is adjusted to 10ul.Antibody is diluted to 20ul with lysis buffer.Typically, use the pyrolysis product that is equivalent to-5000 to 15000 cells in each reaction.

Step: the antibody working concentration that was pre-mixed before the adding reaction is:

For the Her-3/PI3K compound (design of Figure 11 A) that has the cracking probe at the Her-3 place

The anti-Her-3 of eTag1_ is 10nM (eTag1 is Pro14 in this mensuration)

The anti-PI3K of eTag2_ is 10nM (eTag2 is Pro1 in this mensuration)

Biotin _ anti-Her-3 is 20nM

Universal standard US-1 is 700nM

[described universal standard US-1 is the BSA that biotin and molecular label Pro8 in coupling, and it is used for making the amount standardization of mensuration streptavidin-photosensitizer glue pearl].Utilize routine techniques for example Hermanson (quoting as above) make the NHS-ester of molecular label precursor and the reaction of the free amine group on the antibody, thereby molecular label is directly connected on the antibody.

For the Her-3/PI3K compound (design of Figure 11 C) that has the cracking probe at the PI3K place

The anti-PI3K of eTag1_ is 10nM (eTag1 is Pro1 in this mensuration)

The anti-Her-3 of eTag2_ is 10nM (eTag2 is Pro14 in this mensuration)

Biotin _ anti-PI3K is 20nM

Universal standard US-1 is 700nM

9. in the 96 hole titer plates of measuring (Millipore MAGVN2250), add 20ul mixtures of antibodies 4 ℃ of following incubations 1 hour also in the lysate of 10ul.

10. add cracking probe (final concentration 4ug/ hole) that the streptavidin of 10ul derives in the hole of measuring and incubation 45min.

Find time 11. add the 200ul lavation buffer solution and apply negative pressure.

12. add 30ul irradiation damping fluid and irradiation.

Analyze on 10ul to the CE assay plate 13. shift in each reaction.

Data analysis:

1. make relative fluorescence unit (RFU) signal standardization of each molecular label according to its general international standard US-1.

2. from corresponding standard eTag receptor signal, deduct the RFU of " no pyrolysis product " background contrast.

3. the formation of report receptor complex is as the reference (shown in Figure 11 B and 11D) of standardization eTag2/eTag1 signal.

Embodiment 8

Shc/Her-3 acceptor-aptamers interacts

In this embodiment, measure design as shown in Figure 12 A and 12C.Among Figure 12 A, Her2 acceptor (1200) and Her3 acceptor (1202) form dimer in cell surface membrane (1204), and each represented acceptor all has phosphorylation site (1209 and 1210).Shc albumen (1206 and 1208) is incorporated into respectively on phosphorylation site (1210) and (1209).First binding compounds (1214) and cracking probe (1216) have specificity to the different antigenic determinants in Her2 acceptor (1200) extracellular region territory.Second binding compounds (1212) has specificity to Shc albumen (1206 and 1208).The mensuration design class of Figure 12 A and 12C seemingly, except cracking probe in the design of Figure 12 A is specific to the Her2 acceptor, and the cracking probe is specific to outside the Her3 acceptor in the design of Figure 12 C.Therefore, in the previous case, measure total Her2 acceptor, measured total Her 3 acceptors in then a kind of situation.Measurement operation is as follows.The preparation manipulation of sample is (embodiment 7) as above.

Measure design: the formation that comes the quantitative receptor complex of reference according to the diagram shown in each figure.That is to say that among Figure 12 B and the 12D, the mensuration reading is the mT as the function of HRG concentration ₂/ mT ₁Peakedness ratio.

Total mensuration volume is 40ul.With lysis buffer the lysate volume is adjusted to 10ul.Antibody is diluted to 20ul with lysis buffer.Typically, use the pyrolysis product that is equivalent to-5000 to 15000 cells in each reaction.

Step: being pre-mixed the antibody working concentration before adding reaction is:

For the Her-3/Shc compound (design of Figure 12 A) that has the cracking probe at the Her-3 place:

The anti-Her-3 of eTag1_ is 10nM (eTag1 is Pro14 in this mensuration)

The anti-Shc of eTag2_ is 10nM (eTag2 is Pro12 in this mensuration)

Anti-phosphorus-the Tyr of eTag3_ is 10nM (eTag3 is Pro2 in this mensuration)

Biotin _ anti-Her-3 is 20nM

Universal standard US-1 is 700nM

For the Her-2/Shc compound (design of Figure 12 A) that has the cracking probe at the Her-2 place:

The anti-Her-2 of eTag1_ is 10nM (eTag1 is Pro14 in this mensuration)

The anti-Shc of eTag2_ is 10nM (eTag2 is Pro12 in this mensuration)

Anti-phosphorus-the Tyr of eTag3_ is 10nM (eTag3 is Pro2 in this mensuration)

Biotin _ anti-Her-2 is 20nM

Universal standard US-1 is 700nM

1. in the 96 hole titer plates of measuring (Millipore MAGVN2250), add 20ul mixtures of antibodies 4 ℃ of following incubations 1 hour also in the lysate of 10ul.

2. add cracking probe (final concentration 4ug/ hole) that the streptavidin of 10ul derives in the hole of measuring and incubation 40min.

3. add the 200ul lavation buffer solution and apply negative pressure and find time.

4. add 30ul irradiation damping fluid and irradiation.

5. shift in each reaction and analyze on 10ul to the CE assay plate.

Data analysis:

1. make relative fluorescence unit (RFU) signal standardization of each molecular label according to its general international standard US-1.

2. from molecular labeling corresponding standard signal, deduct the RFU of " no pyrolysis product " background contrast.

3. the formation of report receptor complex is as standardization mT ₂/ mT ₁The reference of signal (shown in Figure 12 B and 12D), and receptor phosphorylation degree (data not shown goes out) is as the mT3/mT1 signal.

Embodiment 9

In the tumor of breast sample Her2-Her3 heterodimer measured value with

Relation between the Her3-PI3K compound measured value

In this embodiment, utilize said method to measure the human mammary tumor sample respectively with the amount of definite Her2-Her3 heterodimer and the amount of Her3-PI3K compound.Figure 13 has represented the data of acquisition from this mensuration, shows that these two measured values are relevant.

Embodiment 10

Her1-Her2 in tumor of breast lysate and the normal structure lysate

Expression with the Her2-Her3 heterodimer

Freezing human mammary tumor sample and normal structure sample from William BainbridgeGenomic Foundation (Bainbridge Island, WA).The mensuration form is carried out in 32 neoplasmic tissue sample and 30 normal structure samples as shown in Fig. 3 E.Tumor tissues is made of tumour and Normocellular potpourri, provides the pathology data of tissue according to supplier, and normal structure accounts for about 25% to more than 90%.Specimen preparation and implement measuring basically as described in embodiment 2 and 6.The data of report are that described binding compounds is incorporated into the acceptor relative with the cracking probe specifically, promptly is equivalent to " mT among Fig. 3 E according to the intensity of peak area or the isolated molecule label that discharges from binding compounds ₁" molecular label.The signal value that does not need tumour cell percentage in the standardization per sample and produce.

The data that obtain from these are measured are as shown in Figure 14 A (Her1-Her2 opposite sex dimer measured value) and Figure 14 B (Her2-Her3 opposite sex dimer measured value), wherein hollow square () expression is to the measured value of tumor tissues, and solid diamond (◆) expression is to the measured value of normal structure.Data show, with respect in the normal structure sample cell expression, the tumor cells expression in the basic segment of neoplasmic tissue sample a large amount of Her1-Her2 heterodimer and Her2-Her3 heterodimers.

Embodiment 11

Receptor dimerization body measurement in the paraffin-embedded tissue sample of formalin fixed

In this embodiment, measure in the typical fixing organization of making by globuli cell system whether have the Her receptor dimer.To the mensuration of heterodimer design substantially the same with described in Fig. 4 A, except the difference of the following stated.That is to say, four kinds of compositions have been adopted: comprise that (i) it (is the streptavidin that photosensitizer is derived among this embodiment that cracking initiation part in coupling, shown in Fig. 3 E) and the cracking probe that a kind of acceptor in the dimer had specific biotinylation monoclonal antibody, the (ii) monoclonal antibody that derives by first molecular label, its specificity to acceptor is same as the cracking probe, the (iii) monoclonal antibody that derives by second molecular label, its to the specificity of acceptor in contrast to the cracking probe, and the monoclonal antibody that (iv) derives with the 3rd molecular label, it has specificity to phosphorylated tyrosine in the born of the same parents.Mensuration to homodimer designs basically with identical described in Fig. 1 D, except the difference of the following stated.

In each case, typical fixing organization is prepared as follows: stimulate the cell of growing on the tissue culture medium (TCM) with EGF or HRG, as described in the previous embodiment, afterwards with its washing and scrape and get.The centrifugal cell that takes off is spherical to form, and adds formalin then and under 4 ℃ potpourri is incubated overnight.Be somebody's turn to do fixedly ball with MilesTissue Tek III Embedding Center embedding in paraffin, use microtome (Lecia 2145 types) to downcut the histotomy of 10 μ m from this ball afterwards.Histotomy is placed on the micro-microslide of glass of positively charged (usually every on a plurality of histotomies are arranged) and at 60 ℃ of baking 1hr down.

Measure as follows to the histotomy on the microslide: take the method for manufacturer recommendation to slough the paraffin of histotomy on the microslide with EZ-Dewax reagent (Biogenex, San RamonCA).In brief, in each histotomy, add 500 μ L EZ-Dewax and at room temperature incubation should section 5min, wash this microslide with 70% EtOH afterwards.Repeat this step and, afterwards with this microslide incubation 20min in the water at room temperature at last with this microslide of deionized water rinsing.(Biogenesis, Brentwood NH), heat 15min (high power set under 5min 10min under low-power settings then) afterwards in micro-wave oven then microslide to be immersed the 1X AntigenRetrieval solution of pH 10.(approximately 45min) is placed on water-bath 5min in the water with microslide behind the cool to room temperature, and be dry then.Thereby the histotomy on the dry microslide can be held the zone (shown in Fig. 1 H-1I) that places the reagent on the histotomy with having formed on the hydrophobic wax crayon circle, afterwards washed three times in 1X Perm/Wash (BD Biosciences).Add 50-100 μ L blocking-up damping fluid to each section, this microslide is placed on 4 ℃ of following 2hr in the moist box of the sealing that contains deionized water, remove blocking-up damping fluid in each section by suction afterwards.(the blocking-up damping fluid is for containing the 1X Perm/Wash solution of protease inhibitors (Roche), inhibitors of phosphatases (fluoridize receive, sodium vanadate, β-phosphoglycerol) and 10% mouse serum).Add the mixtures of antibodies (every kind 5 μ g/mL, biotin-Ab5 (anti-Her1) is the 10 μ g/mL in Her1-Her2 measures) contain binding compounds and cracking probe to each section, and microslide is placed in the moist box spends the night under 4 ℃.Contain the Perm/Wash washing slice three times of proteinase and inhibitors of phosphatases then with 100 μ L, add the photosensitizer of 50 μ L in 1XPerm/Wash solution (containing proteinase and inhibitors of phosphatases) afterwards.Then, protecting the adusk suction removal photosensitizer that passes through simultaneously of special microslide afterwards with microslide 4 ℃ of following incubation 1-1.5hr in the moist box of dark.When keeping in the dark, then microslide is immersed also to ice among the .01X PBS and educate 1hr.From PBS, take out microslide, drying, and add the 0.01XPBS that 40-50 μ L has the 2pM fluorescein to each section, use high-power laser diode (GaAIAs IR transmitter, model OD-880W, OPTO DIODECORP afterwards, Newbury Park, CA) irradiation 1hr.Fluorescein helps the electrophoretogram upward peak is associated with molecular label as a standard.After the irradiation, with the solution that is wrapped in each histotomy with inhaling gently mixing and transfer to Applied Biosystems (Foster City CA) analyzes on the CE plate on the 3100 type capillary electrophoresis apparatus of ball.

Figure 15 A has represented that wherein clone is prepared from by unprovoked cell or with 100nM EGF stimulated cells to the Her1-Her1 homodimer in the fixedly ball section of breast cancer cell line MDA-MB-468 (ATCC accession number no.HTB-132) and the analysis data of receptor phosphorylation.Resist as one of cracking probe (be used for the streptavidin (as mentioned above) that the cracking methylenum careuleum is derived, connect) with the biotinylated anti-Her1 monoclonal antibodies of 2 μ g/mL (Labvision) by biotin.The anti-Her1 monoclonal antibody of deriving with 2 μ g/mL Pro10 is measured the Her1 of homotype dimerization.Measure total Her1 with the anti-Her1 monoclonal antibody (Labvision) that 0.8 μ g/mL Pro1 derives.Also comprise the unlabelled antibody A b-5 of 3.2 μ g/mL in the reaction.Measure phosphorylation degree in the born of the same parents with the monoclonal antibody (anti-phosphorylation Tyr, CellSignaling) that 0.5 μ g/mL Pro2 derives.The data acknowledgement that measure to obtain from fixing organization has also met the measurement of cell lysate, because the stimulation of EGF has caused the Her1-Her1 homodimer to express and the increase of the interior phosphorylation of born of the same parents.

Figure 15 B represents the Her2-Her2 homodimer in the fixedly ball section of breast cancer cell line MCF-7 and SKBR-3 and the analysis data of receptor phosphorylation.All are used as the working concentration of cracking probe or binding compounds monoclonal antibody at 5 μ g/mL.For producing better lytic effect, two kinds of cracking probes have been adopted in this mensuration, a kind of born of the same parents' exoantigen determinant, a kind of born of the same parents' endoantigen determinant at Her2 at Her2.From the data acknowledgement that fixing organization records, the SKBR3 cell than MCF-7 cellular expression higher H er2-Her2 homodimer level.

Figure 15 C represents that wherein clone is prepared from by unprovoked cell or with 40nM EGF stimulated cells to the Her1-Her2 heterodimer in the fixedly ball section of breast cancer cell line MCF-7 and the analysis data of receptor phosphorylation.Adopted two kinds of cracking probes, a kind of anti-Her1 monoclonal antibody (5 μ g/mL) that comprises, another kind comprises anti-Her1 monoclonal antibody (10 μ g/mL) (all from Labvision), so that improve the speed that molecular label discharges.Data show because the stimulation of EGF has detected the Her1-Her2 heterodimer and expressed in fixing organization.

Figure 15 D represents that wherein clone is prepared from by unprovoked cell or with 100nM EGF stimulated cells to the Her1-Her2 heterodimer in the fixedly ball section of breast cancer cell line 22Rv1 and the analysis data of receptor phosphorylation.Measurement to fixing organization has proved once more with the incremental adjustments that causes Her1-Her2 dimer and Her receptor phosphorylation after the EGF processing.

Figure 15 E represents that wherein clone is prepared from by unprovoked cell or with 40nM HRG stimulated cells to the Her2-Her3 heterodimer in the fixedly ball section of breast cancer cell line MCF-7 and the analysis data of receptor phosphorylation.In this embodiment, association reaction and cracking reaction are carried out in containing the test tube of section, rather than micro-microslide.In addition, this method is substantially the same with the dimeric method of detection Her1-Her2.(for example, by centrifugal enforcement washing step) these data show because the stimulation of HRG has detected the increase that the Her2-Her3 heterodimer is expressed in fixing organization.

Figure 15 F represents Her2-Her3 heterodimer and the dimeric analysis data of PI3K-Her3 in the section of the fixedly ball of MCF-7 cell, wherein cell irriate or be subjected to 40nM HRG and stimulate not.Should design basically described in Figure 11 A at the mensuration of PI3K-Her3.All then carry out the said fixing method in both cases, except all handling the sample without antigen retrieval reagent.Data show by HRG to be handled, and the Her2-Her3 dimer increases, but the dimeric amount of PI3K-Her3 remains unchanged substantially.

Figure 15 G represents total PI3K, total Her2-Her3 dimer, and the analysis data of total Her3, and all the amount with tubulin is relevant for it.Sandwich determination method with routine is measured tubulin, and this method has adopted the cracking probe and had the binding compounds of molecular label.Measure tubulin and be used for the step that testing needle measures the standardization dimer of the target of total cell number in the representative sample, this may need when mensuration has the sample of different cell types.The ratio that data show PI3K-Her3 and Her2-Her3 and tubulin is qualitative identical with the mensuration of directly carrying out on PI3K-Her3 and Her2-Her3.

Claims (26)

1. method of measuring the patient disease state, disease that this patient takes a disease is characterised in that the unconventionality expression of one or more cell surface receptor compounds, this method may further comprise the steps:

Directly measure in patient's sample every kind amount in one or more cell surface receptor compounds;

Above-mentioned every kind of amount is compared with its respective amount in standard model; And

The difference of corresponding respectively amount in the amount in patient's sample and the standard model is associated with the disease of patient state.

2. the described method of claim 1, wherein said patient's sample is described fixing tissue sample or described freezing tissue sample.

3. the described method of claim 2, wherein said one or more cell surface receptor compounds are that one or more receptor tyrosine kinase compounds and wherein said disease are cancers.

4. the described method of claim 1, wherein said one or more cell surface receptor compounds are one or more pdgf receptor compounds.

5. the described method of claim 1, wherein said one or more pdgf receptor compounds are selected from the group that following material constitutes: PDGFR α homodimer, PDGFR β homodimer, PDGFR α-PDGFR β heterodimer, PDGFR-SHC compound, PDGFR-PI3K compound, Her1-PDGFR receptor dimer, Her2-PDGFR receptor dimer, Her3-PDGFR receptor dimer and PDGFR-IGF-1R receptor dimer.

6. according to claim 4 or 5 described methods, wherein said disease is that cancer or wherein said disease are relevant with unusual fiberization state.

7. the described method of claim 6, wherein said cancer are selected from the group that following illness constitutes: breast cancer, oophoroma and spongioblastoma.

8. the described method of claim 7, wherein said one or more pdgf receptor compounds are measured by following steps:

Every kind to described one or more pdgf receptor compounds provides a kind of reagent right, this reagent causes the cracking probe of part to comprising the cracking with effective short range, and one or more binding compounds, every kind has one or more molecular labels, connect thereon by the cleavable connector, the described molecular label of different binding compounds has different separation characteristics;

With described cracking probe with at one or more binding compounds and patient's sample mix of every kind of described one or more pdgf receptor compounds, make cracking probe and one or more binding compounds specificitys be incorporated on its corresponding pdgf receptor compound, and the cleavable connector of described one or more binding compounds is in cracking to be caused in effective short range partly, and molecular label is released; And

Separate and identify the molecular label that is discharged, thereby whether or amount the existence that is determined at one or more pdgf receptor compounds described in described patient's sample.

9. the described method of claim 1, wherein said one or more cell surface receptor compounds are one or more vegf receptor compounds.

10. the described method of claim 9, wherein said one or more vegf receptor compounds are selected from the group that following material constitutes: VEGFR1 homodimer, VEGFR2 homodimer, VEGFR1-VEGFR2 heterodimer, VEGFR2-VEGFR3 heterodimer, VEGFR2-SHC compound and VEGFR3-SHC compound.

11. according to claim 9 or 10 described methods, wherein said disease is that cancer or wherein said disease are relevant with unusual angiogenesis.

12. the described method of claim 11, wherein said one or more vegf receptor compounds are measured by following steps:

Every kind to described one or more vegf receptor compounds provides a kind of reagent right, this reagent causes the cracking probe of part to comprising the cracking with effective short range, and one or more binding compounds, every kind has one or more molecular labels, connect thereon by the cleavable connector, the described molecular label of different binding compounds has different separation characteristics;

With described cracking probe with at one or more binding compounds and patient's sample mix of every kind of described one or more vegf receptor compounds, make cracking probe and one or more binding compounds specificitys be incorporated on its corresponding vegf receptor compound, and the cleavable connector of described one or more binding compounds is in cracking to be caused in effective short range partly, and molecular label is released; And

Separate and identify the molecular label that is discharged, thereby whether or amount the existence that is determined at one or more vegf receptor compounds described in described patient's sample.

13. the described method of claim 1, wherein said one or more cell surface receptor compounds are the receptor dimers that are selected from the group of following material formation: VEGFR1 (Flt1)-VEGFR2 (KDR), VEGFR2 (KDR)-VEGFR2 (KDR), PDGFR α-PDGFR α, PDGFR α-PDGFR β, PDGFR β-PDGFR β, Kit/SCFR homodimer, FGFR homodimer, NGFR (TrkA)-NGFR (TrkA), α ₂-adrenocepter homodimer, α ₂-adrenaline-β ₂-adrenaline heterodimer, β ₂-adrenocepter homodimer, GABA _BR1-GABA _BR2 acceptor heterodimer, ATII acceptor homodimer, cholescystokinin-dopamine receptor homodimer, bradykinininB2 acceptor homodimer, M2-M3muscarinic acceptor heterodimer, CCR2 acceptor homodimer, μ-δ opiate receptors heterodimer, D1 dopamine receptor homodimer, 5-HT 1B-5-HT 1D acceptor heterodimer, D2 dopamine receptor homodimer, α _2c-adrenaline-M3 muscarinic acceptor heterodimer, D3 dopamine receptor homodimer and β ₂-adrenaline-δ opiate receptors heterodimer.

14. the described method of claim 13, wherein said one or more receptor dimers are measured by following steps:

Every kind to described one or more receptor dimers provides a kind of reagent right, this reagent causes the cracking probe of part to comprising the cracking with effective short range, and one or more binding compounds, every kind has one or more molecular labels, connect thereon by the cleavable connector, the described molecular label of different binding compounds has different separation characteristics;

With described cracking probe with at one or more binding compounds and patient's sample mix of every kind of described one or more receptor dimers, make cracking probe and one or more binding compounds specificitys be incorporated on its corresponding receptor dimer, and the cleavable connector of described one or more binding compounds is in cracking to be caused in effective short range partly, and molecular label is released; And

Separate and identify the molecular label that is discharged, thereby whether or amount the existence that is determined at one or more receptor dimers described in described patient's sample.

15. patient's method is selected in the treatment of cancer at one or more dimer drugs with function, this method may further comprise the steps:

Patient's sample that will contain cancer cell separates from the patient;

Directly measure in patient's sample every kind amount in one or more cell surface receptor dimers;

Above-mentioned every kind of amount is compared the school with its respective amount in standard model; And

When no matter when having exceeded in the standard model respectively corresponding amount, select this patient to be used for the treatment of one or more dimer drugs with function from the dimeric amount of one or more cell surface receptors of described patient's sample.

16. the described method of claim 15, wherein said cell surface receptor dimer is that VEGFR dimer and described dimer drugs with function are selected from the group that following material constitutes: PTK787/K222584, ZD6474, SU6668, SU11248, CHR200131, CP547632, AG13736, CEP7055/5214 and KRN633.

17. being PDGFE dimer and described dimer drugs with function, the described method of claim 15, wherein said cell surface receptor dimer be selected from the group that following material constitutes: SU6668, SU11248, AG13736, CHR200131.

18. being FGFR dimer and described dimer drugs with function, the described method of claim 15, wherein said cell surface receptor dimer be selected from the group that following material constitutes: CP547632 and CHR200131.

19. according to claim 15,16,17 or 18 described methods, wherein said one or more cell surface receptor dimers are measured by following steps:

Provide a kind of reagent right for dimeric every kind to described one or more cell surface receptors, this reagent causes the cracking probe of part to comprising the cracking with effective short range, and one or more binding compounds, every kind has one or more molecular labels, connect thereon by the cleavable connector, the described molecular label of different binding compounds has different separation characteristics;

With described cracking probe with at every kind of dimeric one or more binding compounds of described one or more cell surface receptors and patient's sample mix, make cracking probe and one or more binding compounds specificitys be incorporated on its corresponding cell surface receptor dimer, and the cleavable connector of described one or more binding compounds is in cracking to be caused in effective short range partly, and molecular label is released; And

Separate and identify the molecular label that is discharged, thereby be determined at the dimeric existence of one or more cell surface receptors described in described patient's sample whether or amount.

20. the described method of claim 19, wherein said patient's sample are described fixing tissue sample, described freezing tissue sample or circulation epithelial cell.

21. a method of measuring patient's cancerous state, this patient's cancer is characterised in that the unconventionality expression of one or more cell surface receptor compounds, and this method may further comprise the steps:

Directly measure in patient's sample every kind amount in one or more cell surface receptor compounds;

Above-mentioned every kind of amount is compared with its respective amount in standard model; And

The difference of corresponding respectively amount in the amount in patient's sample and the standard model is associated with the disease of patient state;

Wherein said one or more cell surface receptor dimers are measured by following steps:

Organize indicant to provide a kind of reagent right to every kind of described one or more cell surface receptor compounds and one or more, this reagent causes the cracking probe of part to comprising the cracking with effective short range, and one or more binding compounds, every kind has one or more molecular labels, connect thereon by the cleavable connector, the described molecular label of different binding compounds has different separation characteristics;

Organize one or more binding compounds and described patient's sample mix of indicant with described cracking probe with at every kind of described one or more cell surface receptor compounds and one or more, make cracking probe and one or more binding compounds specificitys be incorporated on its corresponding target, and the cleavable connector of described one or more binding compounds is in cracking to be caused in effective short range partly, and molecular label is released; And

Separate and identify the molecular label that is discharged, thereby whether or amount the existence that is determined at one or more cell surface receptor compounds described in described patient's sample.

22. the method for claim 21, wherein said disease are cancer and wherein said patient's sample is fixing organization sample, freezing tissue sample or circulation epithelial cell.

23. the method for claim 22, wherein said morbid state are the reaction of described patient to the treatment of dimer drugs with function.

24. the method for claim 22, wherein said cancer are selected from the group that following illness constitutes: breast cancer, oophoroma, prostate cancer and colorectal cancer.

25. the method for claim 22, wherein said one or more cell surface receptor compounds are selected from the group that following material constitutes: PDGFR α homodimer, PDGFR β homodimer, PDGFR α-PDGFR β heterodimer, the PDGFR-SHC compound, the PDGFR-PI3K compound, the Her1-PDGFR receptor dimer, the Her2-PDGFR receptor dimer, the Her3-PDGFR receptor dimer, the PDGFR-IGF-1R receptor dimer, the VEGFR1 homodimer, the VEGFR2 homodimer, the VEGFR1-VEGFR2 heterodimer, the VEGFR2-VEGFR3 heterodimer, the VEGFR2-SHC compound, and VEGFR3-SHC compound.

26. according to claim 21,22,23,24 or 25 described methods, the wherein said indicant of organizing is tubulin or cytokeratin.

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