CN101490273A - Detecting diastolic heart failure by protease and protease inhibitor plasma profiling - Google Patents

Abstract

The invention discloses methods of detecting and predicting diastolic heart failure and predicting congestive heart failure comprising protease and protease inhibitor profiling.

Description

Utilize proteolytic enzyme and proteinase inhibitor plasma analysis to detect heart failure diastole

The cross reference of related application

The application requires to enjoy the U.S. Provisional Application No.60/798 that submitted on May 9th, 2006, the U.S. Provisional Application No.60/893 that on March 8th, 953 and 2007 submitted to, and 781 rights and interests, the application is included in above-mentioned application all by reference mode in full in.

The relevant statement of federal funding research

The application finishes under the value of veterans affair portion (Depar tment of Veterans Affairs) is appraised the government-funded of summary (Spinale 0001) research service (Merit Review (Spinale 0001) Research Service) VA contract and national heart, lung, PO1-HL48788, RO1-HL-59165 that Blood Research Institute is authorized and MO1-RR-01070-251 contract through discussion.

Background technology

Although at hypertension (hyperpiesia) medicine and thinking that hypertension is to take place to make great progress aspect this understanding of substantial risk factor in heart failure, in the U.S., this disease remains main cardiovascular disorder.The particular problem that identification has the patient that the hypertension risk of heart failure takes place is, lacks a kind of rapid screening and tests and discern the patient who hypertension secondary cardiac muscle altered self occurs.Long-term hypertension makes myocardial mass and size increase, but this phenomenon just can occur up to the late period of this lysis.The critical event of a uniqueness in hypertensive heart disease and the disease process in heart failure is the aggravation of cardiac muscle self fibrosis.The molecular basis of this change is unknown so far.

Summary of the invention

According to purpose of the present invention, as this paper institute particular embodiment and broadly described, the present invention relates to take place the MMP/TIMP pattern of the uniqueness that occurs in patient's body of hypertensive heart failure, this pattern in fact can the predicted anomaly heart function existence, and only may discern this existence before this by method of testing expensive and that be difficult to implement.The MMP/TIMP pattern of this uniqueness is used to discern in the method with the patient of hypertension Secondary cases risk of heart failure taking place and being about to take place the heart failure of hypertension Secondary cases.

Other advantages of the application's disclosed method and composition are explained part by following specification sheets, part obtains by specification sheets understanding, perhaps can learn by implementing the application's disclosed method and composition.The advantage of the application's disclosed method and composition can be by key element and combination realization and the acquisition that particularly points out in the claim of enclosing.Should be understood that the describe, in general terms of preamble and specific descriptions hereinafter all only for exemplary and indicative, there is no restriction to invention required for protection.

Description of drawings

Accompanying drawing is included into this specification sheets and constitutes the part of this specification sheets, and accompanying drawing shows several embodiments of disclosed method and composition, and combines to illustrate the principle of disclosed method and composition with specification sheets.

Fig. 1 shows the detectability that has and do not exist the MMP-13 in the hypertensive reference contrast and has and do not exist detectability among the LVH of chronic heart failure.MMP-13 detectability among the LVH patient significantly reduces.*=and comparing p＜0.05 with no hypertensive reference contrast, #=compares p＜0.05, Δ=compare p＜0.05 with the LVH of no CHF with hypertensive reference contrast is arranged.

Fig. 2 A shows the relation between the ratio of matrix metalloprotease tissue depressant-1 (TIMP-1) and left ventricular volume/quality.Higher TIMP-1 level is relevant with the ratio of lower LV volume/quality, and this shows that proper alignment reconstruct (concentric remodeling) is more obvious, r=-0.56, p＜0.05.Fig. 2 B shows the relation between matrix metalloprotease tissue depressant-1 (TIMP-1) and tissue Doppler imaging (TDI) rapid filling wave (E ').Higher TIMP-1 level is relevant with lower E ' value, and this relaxation rate that shows LV diastole slows down r=-0.41, p＜0.05.

The 26S Proteasome Structure and Function that Fig. 3 shows the heart of normal heart and heart failure diastole compares.

Fig. 4 shows contrast that has and do not exist hypertension (HTN) and the echocardiography result who has and do not exist the ventricular hypertrophy experimenter of congestive heart failure (CGF), and blood plasma MMP-9, MMP-2 and TIMP-1 measuring result.

Fig. 5 shows with respect to the tissue Doppler imaging of TIMP-1 level (TDI) rapid filling wave (E ').

Fig. 6 shows contrast and left ventricular hypertrophy experimenter's blood plasma MMP-13 level.

Fig. 7 shows existence or does not exist congestive heart failure and blood plasma TIMP-1 level to be higher or lower than the left ventricular hypertrophy percentage of patients of 1200ng/ml.

Fig. 8 shows the working curve of MMP-9, MMP-13, TNF-α and the IL-6 of multivariate method mensuration.

Fig. 9 shows by MMP and TIMP level and determines algorithm to hyperpietic's treatment.Fig. 9 A shows and is proved the treatment synoptic diagram of suffering from hypertension and regularly carrying out the patient of non-emergency outpatient service visit.Fig. 9 B shows the treatment synoptic diagram of suffering from new hypertension incidence and carrying out the patient of non-emergency outpatient service visit.Fig. 9 C shows that show may be by the patient's of sign due to the HF or symptom treatment synoptic diagram.

Embodiment

With reference to following to include in herein specific embodiments and the detailed description of embodiment, and with reference to the civilian description in accompanying drawing front and back relevant with it, can the disclosed method and composition of easier understanding.

Disclose can be used for disclosed method and composition, that can use with disclosed method and composition, can be used for preparing disclosed composition and as material, composition and the component of the product of disclosed method and composition.These materials and other materials are disclosed in this article, should be understood that, when disclosing the combination of these materials, subclass, interaction and group etc., although may be open clearly, all considered especially and described for every kind at this about the combination and permutation of every kind of these compounds concrete different individuality and collective.For example, if open and a kind of peptide has been discussed, and the many modifications that can carry out the many molecules that comprise this peptide have been discussed, unless then specialize on the contrary, every kind of peptide and various combination and permutation and possible modification are all considered especially.Therefore, if disclose molecule A, B and C, and disclose the example A-D of molecule D, E and a F and a combination molecule, then even without each particularize, each combination molecule also obtains separately and jointly considering.Therefore, in this example, every kind of above-mentioned each combination of also being paid attention to especially, and think among combination A-E, A-F, B-D, B-E, B-F, C-D, C-E and the C-F is also because A, B and C; D, E and F; And example makes up disclosing and being disclosed of A-D.Equally, any subclass of these molecules or combination are also paid attention to especially and are disclosed.Therefore, for example, the subgroup of A-E, B-F and C-E is also considered especially, and is considered to because A, B and C; D, E and F; And example makes up disclosing and being disclosed of A-D.This notion is applicable to all aspects of the application, includes but not limited to prepare and use the step in the disclosed method for compositions.Therefore, if a plurality of other steps that can implement are arranged, should be understood that then each step of described other steps may be used to any specific embodiments of disclosed method or the combination of embodiment, and every kind of such combination is all paid attention to especially and is considered to and disclosed.

Those skilled in the art only just can recognize or can determine many equivalents of the specific embodiments of methods described herein and composition by normal experiment.Claim hereinafter is intended to contain this class equivalent.

Should be understood that disclosed method and composition is not limited to described concrete grammar, scheme and reagent, they can change.Should also be understood that term used herein is intended to only describe specific embodiment, but not be intended to limit the scope of the invention that scope of the present invention is only limited by the claim of enclosing.

Unless clearly demonstrate in addition, any method as herein described should not be interpreted as, and need implement its step by specific order.Therefore, when a claim to a method is not addressed the order that its step is followed, specify in claim or specification sheets that perhaps this step is limited to particular order, then in office where face should not thought certain order.This point is applicable to any possible unaccounted basis that is used to explain, comprising: step or operating process are provided with relevant logical relation, come from the obvious semanteme of syntactic structure or punctuate, and the number of the embodiment of putting down in writing in the specification sheets and type.More specifically, the MMP of measured content and TIMP can have the described observed value of any amount level.

A. definition

Unless otherwise defined, all technology used herein and scientific terminology all have the identical implication of implication with disclosed method and composition one of ordinary skill in the art common sense.Though can use when implementing or testing method and composition of the present invention and the described method materials similar of the application or any method and the material that are equal to, useful especially method, device and material are as described.The material mode by reference that quote the publication that this paper quoted and they is included this specification sheets particularly in.Any content of this paper all should not be interpreted as admitting that the present invention is not endowed the right that has precedence over this class disclosure of the prior art.Do not admit that any reference constitutes prior art.The content that its author claimed has been stated in discussion in the reference, and the applicant keeps the accuracy of the document that query quotes and the right of dependency.

Unless it should be noted that in the context to spell out in addition, " " of the singulative that uses in this paper and the appended claim book, " a kind of " and " being somebody's turn to do " comprise that plural number refers to object.Therefore, for example, the object that refers to of " a kind of peptide " comprises multiple this class peptide, and the object that refers to of " this peptide " refers to one or more kind peptides and its equivalent well known by persons skilled in the art or the like.

" optional " or " randomly " means incident, situation or the material described later and may or also may not occur or exist, and such description comprises that described incident, situation or material occur or the situation of existence and described incident, situation or material do not occur or non-existent situation.

Scope can be expressed as from " approximately " concrete numerical value herein, and/or to " approximately " another concrete numerical value.When being expressed as this scope, another embodiment comprises from a described concrete numerical value and/or to described another concrete numerical value.Similarly, when by using " approximately " that numeric representation during as approximation, be should be understood that this concrete numerical value constitutes another embodiment in front.Should be understood that further that the end points of each scope is relevant with another end points and all be significant when being independent of another end points.It is to be further understood that many numerical value disclosed herein, and each numerical value also is disclosed as " approximately " this concrete numerical value at this except numerical value itself.For example, if disclose numerical value " 10 ", " about 10 " are disclosed so also.It is to be further understood that when disclosing a numerical value, also disclose " being less than or equal to " this numerical value, possible scope between " more than or equal to this numerical value " and numerical value, this as those skilled in the art appropriate understand.For example, if disclose numerical value " 10 ", also disclose " being less than or equal to 10 " and " more than or equal to 10 ".Should also be understood that provides data with multiple different-format in whole the application, this is data represented end points and starting point, and the scope of any combination of these data points.For example,, should be appreciated that if disclose concrete data point " 10 " and concrete data point 15, greater than, more than or equal to, less than, be less than or equal to, equal 10 and 15, and the numerical value between 10 and 15 is also thought and is disclosed.What will also be understood that is that each unit between two discrete cells also all has been disclosed.For example, if 10 and 15 be disclosed, 11,12,13 and 14 also all be disclosed so.

The application's specification sheets part and claim partly in the whole text in, word " comprises " and the implication of being out of shape (for example " comprising " and " containing ") is " including but not limited to ", and is not intended to for example other additive, component, integer or step of eliminating.

" experimenter " includes but not limited to that animal, plant, bacterium, virus, parasite and any other have the biology or the entity of nucleic acid.Described experimenter can be a vertebrates, more specifically can be Mammals (for example, the mankind, horse, pig, rabbit, dog, sheep, goat, non-human primates, ox, cat, cavy or rodent), fish, bird or Reptilia or Amphibians.Described experimenter can be an invertebrates, more specifically can be arthropods (for example, insect and crustacean).This term does not indicate specific age and sex.Therefore, this term is intended to contain male or female, that grow up and experimenter and fetus new life.The patient is meant the experimenter who suffers from a kind of disease or illness.Term " patient " comprises human or veterinary experimenter.

" sample " of this paper definition is meant any sample that is obtained by a kind of organism.The example of biological specimen comprises body fluid and tissue sample.The source of described sample can be a Physiological Medium, and for example blood, serum, blood plasma, breast milk, purulence, tissue are scraped bits, washing lotion, urine, tissue, for example lymphoglandula or the like.

The application has quoted various publications in the whole text.The disclosure of these publications mode is by reference included the application in full in, so that describe the prior art situation affiliated with the present invention more fully.Rely in the disclosed reference material discussed in the sentence of described reference also by reference mode include this specification sheets individually and especially in.

B. method

1. in heart failure

Congestive heart failure (CHF) also is referred to as congestive heart failure (CCF) or only is called heart failure, and it is a kind of full or pump any structure of ability of competent whole body blood volume or the disease due to the functional cardiac conditions by the infringement heart.Therefore, disclosed method can be used for treating any type of heart failure.

Because not all patient has volume overload when initial or follow-up evaluation, so term " heart failure " is better than term " congestive heart failure " once.The cause of disease of congestive heart failure or short because of comprising following listed (and specifically note still right side (R), left side (L)): CHF heredity family history, ischemic heart disease/myocardial infarction (coronary artery disease), infect, drink, heart worm, anaemia, thyrotoxicosis (hyperthyroidism), irregular pulse, hypertension (L), aortic stenosis (L), aorta [lobe] be narrow/instead flow (L), mitral incompetence (L), cause pulmonary stenosis/pulmonary hypertension/pulmonary infarction of cor pulmonale (R), and mitral disease (L).

Heart failure has multiple different mode classification, any comprise: relate to heart side (left heart failure and right heart failure), unusually whether unusually whether contraction or the diastole by heart causes (in heart failure and heart failure diastole of systole), by low cardiac output or low systemic vascular resistance (low blood discharge amount heart failure and high output heart failure).

Congestive heart failure (CHF) is a cardiac performance---particularly sign due to the potential disease of left ventricle (LV) function and syndrome (promptly be short of breath, hydrops).The cause of disease of CHF has multiple, can mainly be divided into following three classes: the CHF of heart attack after (myocardial infarction), follow the CHF of hypertensive heart disease and follow the CHF of inner muscle disease (being commonly referred to myocardosis).The potential cause of disease of for example identifying the CHF that is caused by hypertensive heart disease is also very difficult, and this becomes the focus of method of the present invention.Particularly, hypertensive heart disease causes the LV muscle growth, and this is referred to as hypertrophy.LV hypertrophy (LVH) self can cause the myocardial function defective by himself or LV hypertrophy, but does not all also quick and precisely discern the blood testing of LVH before this.The application has found effective novel method of a kind of LVH of identification patient.If the LVH process continues or is fully treated, then the patient will to occur mainly be because CHF sign and the symptom that heart failure diastole (DHF) causes.Yet,, also can't discern these patients with blood testing simply fast to all also being difficult to the patient that identification suffers from CHF (mainly being DHF) up to now.The application has found that a kind of identification exists the patient of LVH, existence that effective novel method of the patient of DHF risk takes place, and identification DHF patient's method.Therefore, the invention provides the existence of a kind of LVH of detection, the means that prediction may exist generation high risk patient of DHF and identification DHF patient.By using body fluid sample in a small amount, for example following blood sample, can carry out four kinds of present method (but needn't exclusively) independent utility: screening, prediction/prognosis, diagnosis and treatment are monitored.

Therefore, herein disclosed is the method that a kind of diagnosis suffers from the experimenter of left ventricular hypertrophy (LVH, HCM or HOCM).For example, the method that can a kind ofly detect LVH in the subject is provided, comprise the matrix metalloproteinase (MMP) of discerning described experimenter's body fluid and the spectrum of matrix metalloprotease tissue depressant (TIMP), described herein experimenter is attended by heart failure diastole (DHF).A kind of method of the experimenter's of prediction heart failure diastole also is provided, comprise the matrix metalloproteinase (MMP) of discerning described experimenter's body fluid and the spectrum of matrix metalloprotease tissue depressant (TIMP), described herein experimenter may follow, and heart failure diastole (DHF) takes place.

2.MMP

Matrix metalloproteinase (MMP) is a zinc dependency endopeptidase; Other family member has Viprinex, husky thunder mycoproteinase (serralysin), astacin.MMP belongs to a bigger proteolytic enzyme family that is called as the metzincin superfamily.

Share a kind of total structural domain structure between the MMP.Three kinds of apokoinou construction territories are propetide, catalyst structure domain and the Hemopexin-like C-terminal structural domain that links to each other with catalyst structure domain by the flexible hinge district.

MMP is as the non-activity proenzyme synthetic with preceding peptide domain at first, and described preceding peptide domain must be removed before enzyme activates.Peptide domain is the part of " halfcystine switch " before described, and described " halfcystine switch " comprises and can combine and cut to keep enzyme to be in the conservative property cysteine residues of inactive form with zinc interaction and the prevention substrate in the avtive spot.Among most MMP, described cysteine residues is arranged in conserved sequence PRCGxPD.Some MMP has the part of prohormone convertase cutting cleavage site (furin sample) as this structural domain, and it can activate this enzyme when cutting is cut.MMP-23A and MMP-23B comprise TMD in this structural domain (PMID10945999).

The x-ray crystal structure of the catalyst structure domain of multiple MMP has proved that this structural domain is that observed value is 35 * 30 * 30

(3.5 * 3 * 3nm) oblate sphere.Avtive spot is one and passes 20 of catalyst structure domain

Ditch (2nm).Have very important Zn2+ ion in the catalysis in the part of this avtive spot of formation of described catalyst structure domain, this ion combines with three Histidines in being present in conserved sequence HexxHxxGxxH are amino.Therefore, this sequence is a zinc-binding motif.

This gelatinase (for example MMP-2) contains the II type fibronectin structure function territory (module) that is inserted into the positive anteposition of zinc-binding motif in this catalyst structure domain (PMID 12486137).

Described catalyst structure domain is connected with the C-terminal structural domain by flexible hinge district or connector area.It reaches 75 amino acid longs most and has uncertain structure.

Described C-terminal structural domain has and the similar structure of serum protein Hemopexin.It has four foliated lamellar β propeller structures.β propeller structure provides a big flat surfaces, and this surface is considered to participate in the interaction of protein-protein.This has also determined substrate specificity, and becomes the interactional site with TIMP.There is not Hemopexin (Haemopexin) spline structure territory in MMP-7, MMP-23 and MMP-26 and plant and the nematode.MT-MMP is anchored on the plasma membrane by this structural domain, and some MT-MMP has the cytoplasmic structure territory.

MMP can be segmented with multitude of different ways.By bioinformatics method being used for the primary sequence of comparison MMP, the evolution grouping of following MMP: MMP-19 can be proposed; MMP11,14,15,16 and 17; MMP-2 and MMP-9; Every other MMP.

Separate analysis to each catalyst structure domain shows that when the primary categories differentiation phase, catalyst structure domain also can further develop, and this also is referred to as the substrate specificity of enzyme.(MMP biological study personnel) the most frequently used classification is based in part on the Histological evaluation to the MMP substrate specificity, is based in part on the cellular localization to MMP.These classifications have collagenase, gelatinase, extracellular matrix degrading enzyme and membranous type MMP (MT-MMP).Find that little by little these classification have certain human factor, because also there are many MMP that are unsuitable for assigning to any the General category.

Collagenase can be degraded to triple helical fiber collagen distinguishing 3/4 and 1/4 fragment.These collagens are main components of bone and cartilage, and MMP is the mammalian enzyme of unique known described collagen of degrading.Usually, described collagenase is MMP-1 (small intestine collagenase), MMP-8 (neutrophil collagenase), MMP-13 (3 Collagen Type VI enzyme), MMP-18 (4 Collagen Type VI enzymes, xco14, African toad collagenase.Unknown human forward homologue), MMP-14 (MT1-MMP) but also shown cutting fibre collagen, more controversial is to evidence suggests that MMP-2 can dissolve collagen.

Extracellular matrix degrading enzyme demonstrates the ability of cutting extracellular matrix protein widely, but it can not cut triple helical fiber collagen.Three standard members of this zymoid are: MMP-3 (extracellular matrix degrading enzyme 1), MMP-10 (extracellular matrix degrading enzyme 2) and MMP-11 (extracellular matrix degrading enzyme 3).MMP-11 demonstrates more similar to MT-MMP, and it can be activated by saccharase, but so its secretion usually relevant with saccharase activated MMP.

The stromlysin class comprise MMP-7 (stromlysin, PUMP) and MMP-26 (stromlysin-2, endometase).

The main substrate of gelatinase is IV Collagen Type VI and gelatin, and the characteristics of these enzymes are to be inserted with additional structural domain in catalyst structure domain.The gelatin land is positioned at the zinc-binding motif right on, and forms independently folding unit, and this unit can not destroy the structure of this catalyst structure domain.Two members of this subclass are MMP-2 (72kDa gelatinase, gelatinase-A) and MMP-9 (92kDa gelatinase, gelatinases-B).

Excretory MMP comprises MMP-11 (extracellular matrix degrading enzyme 3), MMP-21 (X-MMP) and MMP-28 (epithelium hydrolysis element (Epilysin)).

Film comprises that in conjunction with MMP the II type is striden film halfcystine array MMP-23, glycosyl-phosphatidyl inositol bonded MMP 17 and 25 (being respectively MT4-MMP and MT6-MMP) and the I type is striden film MMP14,15,16,24 (being respectively MT1-MMP, MT2-MMP, MT3-MMP and MT5-MMP).

6 kinds of MT-MMP all have the furin cleavage site in propetide, MMP-11 also has this feature.

Other MMP comprises MMP-12 (scavenger cell metalloelastase), MMP-19 (RASI-I, sometimes be also referred to as extracellular matrix degrading enzyme-4), enamel dissolution element (Enamelysin) (MMP-20) and MMP-27 (MMP-22, C-MMP), MMP-23A (CA-MMP) and MMP-23B.

3.TIMP

MMP can be suppressed by specific endogenous tissue inhibitor of metalloproteinase (TIMP), and TIMP comprises the family of four kinds of proteinase inhibitor: TIMP-1, TIMP-2, TIMP-3 and TIMP-4.Generally speaking, in a single day all MMP are activated, and all can be suppressed by TIMP, but gelatinase (MMP-2 and MMP-9) can form complex body with TIMP when being in its potential form.The MMP-2 of potential form (MMP-2 proenzyme) can promote that with the complex body of TIMP-2 a kind of film grappling MMP is the MMP-2 proenzyme on MT1-MMP (MMP-14) activating cells surface.

4.MMP/TIMP the ratio

The specific characteristic that the MMP-TIMP of hypertensive heart disease analyzes is, having utilized specific heart TIMP is TIMP-4, and be placed on contain cardiac muscle block with the hypertensive cerebral patient in take place in the environment of MMP of more noticeable change.The ratio of MMP (for example MMP-9 or MMP-13) with TIMP (for example TIMP-1, TIMP-2 or TIMP-4) is also disclosed.Described ratio is used as the diagnosis diagnostic characteristics in this article first and is used to discern the patient with visibly different disease condition.

5. plasma screening

The major advantage of the present invention instruction is, method disclosed herein provides a kind of and had the quicker simple method that experimenter that bad LVH risk takes place and identification are just quickening to take place the patient of this process from tissue or body fluid identification.Therefore, method disclosed herein can comprise MMP and the TIMP that detects in experimenter's body fluid, and described body fluid for example has blood, urine, blood plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous humor or vitreous humor, colostrum, phlegm, amniotic fluid, saliva, anus and vaginal secretions, sweat, seminal fluid, spills liquid, exudate and synovia.

Blood plasma is the liquid component of blood, wherein is suspended with hemocyte.Blood plasma is the topmost single component of blood, and it accounts for about 55% of total blood volume.Serum is meant the blood plasma of removing thrombin (for example scleroproein).Blood plasma contains many important protein matter, comprises fibrinogen, sphaeroprotein and human serum albumin.Sometimes, blood plasma may contain the viral impurity that must extract by virus treated.

6. immunoassay

Have many known or newfound methods that are used for the analyte of test example such as protein (as MMP and TIMP) in this area, they can be used in the method disclosed herein.For example, the available standards immunodetection detects MMP and TIMP.The step of various useful immunodetections has had description in scientific and technical literature, for example, Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays:Heterogeneous andHomogeneous Systems, Handbook of Experimental Immunology, Vol.1:Immunochemistry, 27.1-27.20 (1986), mode by reference is with their full text---particularly it is about instruction of the method for immunodetection---include this paper in.On the simplest direct meaning, immunoassay relate to bonded binding assay between antibody and the antigen.The immunoassay of many types and form all are known, and they all are suitable for detecting disclosed biomarker.The example of immunoassay has that enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmuno-precipitation assay (RIPA), immunobead catch assay, western blotting, dot blotting, gel move that detection, flow cytometry, protein array, many beads array, magnetic are caught, fluorescence recovery/location (FRAP/FLAP) behind in-vivo imaging, FRET (fluorescence resonance energy transfer) (FRET) and the photobleaching.

Generally speaking, immunoassay comprise makes the sample that contains molecules of interest (biomarker for example disclosed herein) under a cloud contact with the antibody of this molecules of interest, the antibody that perhaps makes molecules of interest (for example, the antibody of biomarker disclosed herein) with can be contacted by the molecule of this antibodies, described contact process may be to carry out effectively making it possible to form under the condition of immunocomplex.Under the condition that effectively forms immunocomplex (one-level immunocomplex) and be enough to make in the time period that immunocomplex forms, between the antibody of sample and molecules of interest contact or with can be by contacting between the molecule of the antibodies of molecules of interest, usually described molecule or antibody are contacted with described sample, and, hatch said mixture being enough to make antibody and existing any can formation in the time period of (promptly combining) immunocomplex with the molecule (for example antigen) of this antibodies with it.In the immunoassay of many forms, can wash sample-antibody compositions (for example tissue slice, elisa plate, the some marking or western blotting) subsequently removing the antibody type of any non-specific binding, thereby only allow to exist with those antibody of one-level immunocomplex specificity bonded to be detected.

Immunoassay can comprise the molecules of interest (biomarker for example disclosed herein or their antibody) that is used for detecting sample or to its quantitative methods, and described method generally includes any immunocomplex that detection forms or quantitative to it in cohesive process.Usually, the detection that immunocomplex is formed is well known in the art, and this can use accomplished in many ways.These methods generally are based on and detect a kind of marker or mark, for example any radioactive labels, fluorescence labels, biological label or enzyme label or any other known marker.Referring to for example, United States Patent (USP) 3,817,837,3,850,752,3,939,350,3,996,345,4,277,437,4,275,149 and 4,366,241, mode is by reference included above-mentioned every piece of document full text---particularly relevant with immunologic detection method or mark instruction---in this paper.

The employed mark of this paper can comprise the epi-position label of fluorescence dye, one of them (biological example element/streptavidin) in conjunction with right, metal (for example gold) or can interact with molecular specific ground that can be detected (for example by producing coloured substrate or fluorescence).Be applicable to can detect the proteic substrate of ground mark comprise fluorescence dye (being also referred to as fluorochrome and fluorophore herein) and can with the enzyme of chromogenic substrate (for example horseradish peroxidase) reaction.Implement in the process of the present invention, general preferred use fluorescence dye is because they can be detected when seldom measuring.In addition, when a plurality of antigens and the reaction of single array, each antigen can be by different fluorescent chemicals marks so that detect simultaneously.Can use the gauge point on the photofluorometer detection arrays, just show that when having signal antigen combines with specific antibody.

Fluorophore is luminous compound or a molecule of energy.Usually, fluorophore absorbs electromagnetic energy at a wavelength place, in another wavelength place emission electromagnetic energy.

Mark can directly or indirectly carry out.In direct mark, antibody of detection (antibody of molecules of interest) or the molecule that detects (can by the molecule of the antibodies of molecules of interest) comprise marker.Detect marker and just show the antibody that there is described detection or the molecule of described detection, this shows the antibody that has molecules of interest or have molecules of interest again respectively.In indirect labelling, other molecules or part are contacted with immunocomplex, perhaps generate other molecules or part at the immunocomplex place.For example, can make the molecule or the part (for example enzyme) that produce signal be attached to or be attached on described detection antibody or the described detection molecules.Afterwards, the molecule of generation signal can produce detectable signal at the immunocomplex place.For example, when suitable substrate was provided to a kind of enzyme, this enzyme can produce visible or detectable product at the immunocomplex place.ELISA has used this indirect labelling.

Another example of indirect labelling is, but makes other molecules (can be referred to as wedding agent) of the antibody (one is anti-) of binding purposes molecule or binding purposes molecule---for example anti-ly with an anti-bonded two---contacts with immunocomplex.Described other molecules can have marker or produce the molecule or the part of signal.Described other molecules can be antibody, and it can be called as two anti-thus.Two anti-can form so-called sandwich with described first antibody (or resist) and molecules of interest with anti-a combination.This immunocomplex can contact with resisting through two of mark under the condition that effectively makes the secondary immunocomplex form, and is enough to duration of contact make the secondary immunocomplex to form.Then, this secondary immunocomplex can be washed usually, marker remaining in this secondary immunocomplex can be detected subsequently to remove resisting of any non-specific combination through two of mark.Described other molecules also can be or comprise one of them of a pair of molecule that is bonded to each other or part (biological example element/streptavidin to).In this pattern, described detection antibody or detection molecules should comprise another member in this pairing.

Other indirect labelling pattern comprises by two step methods detection one-level immunocomplex.For example, as mentioned above, a kind of for example a kind of antibody of molecule (it can be called as first wedding agent) that molecules of interest or corresponding antibodies are had a binding affinity can be used for forming the secondary immunocomplex.After the washing, also be effectively to make immunocomplex form under the condition of (forming three grades of immunocomplexs thus), described secondary immunocomplex is contacted in the time that immunocomplex forms to the molecule (it can be called as second wedding agent) that described first wedding agent has binding affinity with another kind being enough to make.Described second wedding agent can be connected with molecule or part that detectable or generation signal are arranged, so that can detect described three grades of immunocomplexs that form thus.This system can make signal amplify.

Relate to the immunoassay that detect substrate (antibody of for example a kind of albumen or a kind of specific protein) and comprise detection in unmarked mensuration, albumen sepn method (being electrophoresis), solid carrier catch assay or the body.Unmarked mensuration is to determine whether to exist in the sample diagnostic means commonly used of the antibody of specific protein or specific protein.The albumen sepn method also is used to estimate proteic physical property, for example molecular size or net charge.Catch assay is more useful during the concentration of the antibody of specific protein or specific protein in the quantitative evaluation sample usually.At last, detect the space expression pattern that can be used for estimating material in the body, promptly where intravital can be present in the patient for this material, and tissue is still in the cell.

If concentration is enough, the molecular complex ([Ab-Ag] n) that antibody-AI produces be macroscopic, but also might be because of its energy scattered beam when measuring still less detected or measure.The formation of complex body shows that two kinds of reactants all exist, and in immune precipitation determination, the reagent antibodies of constant density is used to measure specific antigen ([Ab-Ag] n), and antigen reagent is used to detect specific antibodies ([Ab-Ag] n).If reagent type by bag in advance by on cell (as in hemagglutination is measured) or molecule (as in latex agglutination is measured), bag by particulate " aggegation " when the extremely low concentration all as seen.Various assay methods based on above-mentioned ultimate principle all are common methods, comprise Ouchterlony immunodiffusion(ID) mensuration, rocket immunoelectrophoresis and immune nephelometric analysis and turbidity measurement.The main limitation that this class is measured is to compare with the assay method of applying marking thing, its sensitivity limited (lower detectability), and under some situation, this fact of formation that in fact analyte of high concentration can suppress complex body makes these methods more complicated.During some traced back to discovery antibody in these line assay methods, they all do not have real " marker " (for example Ag-enz).The unmarked immunoassay of other types depend on immunosensor, existing at present many commercially available instruments that can directly detect antibody-AI.Great majority all need produce evanescent wave having on the sensor surface of fixed ligand, thereby can continuous monitoring and the combining of part.Immunosensor makes can be easy to carry out the kinetics repercussion study, and along with the appearance of the professional instrument of low cost makes it can be widely used in the immunoassay in future.

Using immunoassay to detect specific protein can comprise by electrophoretic separation protein.Electrophoresis is meant that the charged molecules in response in the solution moves in electric field.Their mobility depends on net charge, size and the shape of strength of electric field, molecule, also depends on wherein ionic strength, viscosity and the temperature of the medium of swimming molecule.Electrophoresis is a kind of simple, quick and high-sensitive analysis tool.It is used to the character of single charged molecule of analyzing and researching, and also can be used as a kind of isolation technique.

Usually, swimming sample on carrier matrix is for example on paper, cellulose acetate, starch gel, agarose or polyacrylamide gel.Convection current due to described matrix can suppress to generate heat mixes, and the electrophoresis record is provided: promptly when electrophoresis finishes, can and be used for scanning, radioautograph or preservation with matrix dyeing.In addition, the most frequently used carrier matrix---agarose and polyacrylamide---provides the instrument by the molecular size isolated molecule, because they are porous gels.Porous gel can by postpone or some situation under stop macromole to move small molecules is moved freely the effect of playing molecular sieve fully.Because rare sepharose is usually than the harder and easier processing of polyacrylamide of same concentrations, so agarose is used to separate bigger macromole for example nucleic acid, big protein and protein complexes.Polyacrylamide is handled easily and preparation when higher concentration, therefore is used to separate most albumen in the less delayed gel aperture of needs and than small oligonucleotide.

Protein is amphoteric substance, therefore can measure their net charge by the pH of its suspension medium.When pH value of solution is higher than isoelectric points of proteins, the clean negative electricity of this protein band, and anode moves in electric field.When being lower than its iso-electric point, protein band positive electricity moves to negative electrode.In addition, protein with net charge and its molecular size irrelevant, promptly different protein, per unit molecular mass (or length, when protein is linear macromolecule with nucleic acid) institute is electrically charged different.Therefore, under given pH, and under non-sex change condition, determine proteinic electrophoretic separation by bulk of molecule and electric charge simultaneously.

Sodium lauryl sulphate (SDS) is a kind of negatively charged ion denaturing agent, and it can make protein denaturation by " encirclement " polypeptide backbone, and SDS specifically combines with the mass ratio of 1.4:1 with the protein utmost point.Thus, SDS will give polypeptide and the proportional anionic charge of its length.In addition, also need usually proteic disulfide bond reduction (sex change), so after, protein just can be taked to separate required curled configuration at random by molecular size; Reduce available 2 mercapto ethanol or dithiothreitol (DTT) and realize.Therefore, in sex change SDS-PAGE separates, move and can't help the internal charge decision of polypeptide, but determine by molecular weight.

The mensuration of molecular weight can be carried out the SDS-PAGE electrophoresis with albumen to be characterized by the albumen with the known molecular amount and be carried out.There is linear relationship between the logarithm of the polypeptide of SDS sex change or the molecular weight of natural acid and its Rf.The molecular migration distance is Rf with the ratio of standard substance dyestuff forward position migration distance.A kind of simple method by electrophoresis (Mr) mensuration relative molecular weight is to draw the typical curve of the relative log 10MW of known sample migration distance, and measuring sample reads sample after the migration distance on the same gel logMr.

In two dimensional electrophoresis, at first protein fractionation is separated based on physical property, pass through another step fractional separation based on another kind of characteristic then.For example, isoelectric focusing method can be used for first dimension, and this can be undertaken by the tubular type gel easily, and the SDS electrophoresis on the slab gel can be used for second dimension.An example of this method is O ' Farrell, P.H., High Resolution Two-dimensionalElectrophoresis of Proteins, method among the J.Biol.Chem.250:4007-4021 (1975), the document is included this paper in full with regard to the instruction mode by reference of its two dimensional electrophoresis method.Other example includes but not limited to Anderson, L and Anderson, NG, Highresolution two-dimensional electrophoresis of human plasmaproteins, Proc.Natl.Acad.Sci.74:5421-5425 (1977) and Ornstein, L., Disc electrophoresis, method among the L.Ann.N.Y.Acad.Sci.121:321349 (1964), they about the instruction of electrophoresis method all by reference mode include this paper in full in.

Laemmli, U.K., Cleavage of structural proteins during theassembly of the head of bacteriophage T4, disclose a kind of discontinuous system that is used to differentiate the SDS metaprotein among the Nature 227:680 (1970), the document is included this paper in full with regard to the instruction mode by reference of its electrophoresis method.Leading ion in the Laemmli buffering system is a chlorine, and trailing ion is a glycine.Therefore, this resolution gel and stacking gel are with (different concns and pH) Tris-HCl buffer preparation, and the electrophoresis chamber damping fluid is the Tris-glycine.All damping fluids all contain 0.1% SDS.

An example that uses electrophoretic immunoassay of considering in the inventive method is western blot analysis.Western blotting or immunoblotting make can measure proteic molecular mass, and measures the proteic relative quantity that exists in the different samples.Detection method comprises chemoluminescence and color developing detection.The standard method of western blot analysis can be referring to for example D.M.Boltag et al., ProteinMethods (2d edition 1996) and E.Harlow ﹠amp; D.Lane, Antibodies, aLaboratory Manual (1988) and United States Patent (USP) 4,452,901, their each pieces about the instruction of western blotting method all by reference mode include this paper in full in.General with gel electrophoresis (SDS-PAGE commonly used) isolated protein.Protein transduction is moved on on a kind of special trace thin paper of for example nitrocellulose, but also can use the paper or the film of other types.This albumen keeps its clastotype on gel.This trace is hatched, with in conjunction with any remaining adhesive region on the nitrocellulose filter with a kind of total protein of for example milk-protein.Can join in conjunction with the antibody of its specific proteins in this solution then.

Combining of specific antibody and specificity immobilized antigen be can easily show by indirect enzyme immunoassay technique, chromogenic substrate (for example, alkaline phosphatase or horseradish peroxidase) or luminous substrate used usually.Other possible detection methods comprise use fluorescence or radioisotopic tracer (for example, fluorescein, ¹²⁵I).The probe that detects antibodies can be the probe (avidin/streptavidin of for example, puting together) that the anti-immunoglobulin of puting together, the SP of puting together (in conjunction with IgG) or biotinylation one resist.

The validity of this technology is that it can detect this specific protein and its molecular weight simultaneously by the antigenicity of specific protein.At first,, detect specifically by the immunoassay step then according to mass separation protein with SDS-PAGE.Therefore, electrophoresis protein standard substance (molecular weight standard (ladder)) simultaneously is to estimate the molecular weight of target protein in the allos sample.

Gel shift is measured or electrophoretic mobility is measured (EMSA) and be can be used for qualitative and detect interaction between conjugated protein and its homologous dna recognition sequence of DNA quantitatively.Exemplary technology such as Ornstein L., Disc electrophoresis-I:Background and theory, Ann.NY Acad.Sci.121:321-349 (1964) and Matsudiara, PT and DR Burgess, SDS microslab linear gradient polyacrylamide gel electrophoresis, described in the Anal.Biochem.87:386-396 (1987), the relevant teachings that above-mentioned document is measured with regard to its gel shift all by reference mode is included this paper in full in.

In the gel determination of routine, can be (for example with the albumen of purifying or cell crude extract and a kind of mark ³²The P radio-labeled) DNA or rna probe are hatched together, make this complex body and free probe separates by non-denaturing polyacrylamide gel then.The travelling speed that this complex body passes gel is slower than uncombined probe.According to described protein-bonded activity, the probe of mark can be two strands or strand.For the protein-bonded detection of the DNA of for example transcription factor, can use purifying or partially purified albumen or karyocyte extract.For the detection of rna binding protein, can use purifying or partially purified albumen or nuclear or kytoplasm cell extract.DNA or rna binding protein can be determined by the competitiveness experiment for the specificity of inferring binding site, wherein use DNA or RNA fragment or the oligonucleotide that contains the target protein binding site, perhaps other irrelevant sequences.The character of the complex body that forms in the presence of special and non-special competition thing and strength difference make can discern special interaction.Gel shift referring to Promega is measured FAQ, it can obtain from http://www.promega.com/faq/gelshfaq.html (last time was visited on March 25th, 2005), and this content is included this paper in full with regard to its gel shift method relevant teachings mode by reference.

The gel shift method can comprise, for example uses coomassie (Imperial ChemicalsIndustries, Ltd) protein on the blue dyes detection gel (for example polyacrylamide gel electrophoresis gel) of colloidal form.These class methods are at for example Neuhoff et al., Electrophoresis 6:427-448 (1985) and Neuhoff et al, Electrophoresis 9:255-262 has description in (1988), described document with regard to its gel shift method relevant teachings all by reference mode include this paper in full in.Except above-mentioned conventional method of protein measurement, United States Patent (USP) 5,424 has also been described the composition of a kind of combined cleaning and protein staining in 000, and the document is included this paper in full with regard to its gel shift method relevant teachings mode by reference.This solution can comprise phosphoric acid, sulfuric acid and nitric acid and acid violet dyestuff.

Radioimmuno-precipitation assay (RTPA) is a kind of sensitive determination method of using radiolabeled antigen to detect specific antibody in the serum.Make antigen and sero-reaction, use then special reagent for example albumin A agarose (sepharose) bead precipitate.Afterwards, use the described bonded of gel electrophoresis analysis through radiolabeled immunoprecipitation usually.The radioimmunoassay precipitation is measured (RIPA) and is often used as a kind of test of making a definite diagnosis that whether has HIV antibody that is used to diagnose.RIPA also is known as Farr in the art and measures, precipitates mensuration, radioimmuno-precipitation assay; Radioimmunoprecipitation assay; Radioimmunoprecipitation assay and immunoprecipitation analysis.

Though above-mentionedly utilize electrophoretic separation and detect the size that the proteic immunoassay of described specific purpose can assess proteins, they are not too sensitive aspect assess proteins concentration.Yet; also considered a kind of like this immunoassay: wherein said albumen or proteic specific antibody combine with solid carrier (for example pipe, hole, bead or cell) catching described antibody or target protein respectively from sample, and this mensuration is detected described albumen on the carrier or the method for proteic specific antibody combines with a kind of.The example that this para-immunity is measured comprises that radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), flow cytometry, protein array, many beads array and magnetic catches.

Radioimmunoassay (RIA) is a kind of classical quantitative assay that detects antigen-antibody reaction, and it has used radiolabeled material (radioligand), directly or indirectly to measure combining of unmarked material and specific antibody or other receptor systems.Can use radioimmunoassay for example to detect the hormonal readiness of blood, and not need to use bioassay method.Non-immunogenicity material (for example haptens) as with bigger carrier proteins (for example, ox gamma Globulin or the human serum albumin) coupling that can induce antibody to form, then also can measure it.RIA comprises radiolabeled antigen (is easier to because the iodine atom introduced in the proteinic tyrosine residues, so use radio isotope usually ¹²⁵I or ¹³¹I) mix with this antigenic antibody.Generally, this antibody is combined with for example solid carrier of pipe or microballon.Then, add unlabelled antigen or " cold " antigen of known quantity, and measure by the amount of metathetical labelled antigen.At first, described radiolabeled antigen and antibodies.When adding cold antigen, both can compete antibody combining site, and the cold antigen of greater concn can be more and this antibodies, thereby displace this radioactivity variant.Make that unconjugated antigen is separated in bonded antigen and the solution, draw binding curve by each antigenic radioactivity.This technology extremely sensitivity and specificity is very high.

It is a kind of immunoassay that detect proteic specific antibody that enzyme-linked immunosorbent assay (ELISA)---perhaps is referred to as EIA (enzyme immunoassay)---usually.In this was measured, the detectable label that combines with antibodies reagent or antigen binding reagents was a kind of enzyme.When enzyme contacted with its substrate, this enzyme reacts with ad hoc fashion for example can pass through the chemical part that spectrophotometry, fluorometric assay or visual way detect to produce.The enzyme that can be used for detecting the ground mark detection reagent includes but not limited to: horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase enzymes, rnase, urase, catalase, malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha-phosphate glycerol dehydrogenase, triosephosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.About the description of ELISA method referring to Voller, A.et al., J.Clin.Pathol.31:507-520 (1978); Butler, J.E., Meth.Enzymol.73:482-523 (1981); Maggio, E. (ed.), EnzymeImmunoassay, CRC Press, Boca Raton, 1980; Butler, J.E., In:Structure of Antigens, Vol.1 (Van Regenmortel, M., CRC Press, BocaRaton, 1992, pp.209-259; Butler, J.E., In:van Oss, C.J.et al., (eds), Immunochemistry, Marcel Dekker, Inc., New York, 1994, pp.759-803; Butler, J.E. (ed.), Immunochemistry of Solid-PhaseImmunoassay, CRC Press, Boca Raton, 1991); Crowther, ＂ ELISA:Theoryand Practice, ＂ In:Methods in Molecule Biology, Vol.42, HumanaPress; New Jersey, 1995; United States Patent (USP) 4,376,110, the above-mentioned every piece of document instruction of ELISA method---particularly about---all by reference mode are included this paper in full in.

The variation scheme of elisa technique is well known by persons skilled in the art.In a variation scheme, can be with being fixed on the selected surface that demonstrates the albumen affinity, for example in the hole of polystyrene microtiter plates with protein bound antibody.Then, in described hole, add the test composition that suspection contains labelled antigen.In conjunction with and the immunocomplex of flush away non-specific binding after, can detect bonded antigen.Can resist at two of target protein and detect by adding specificity, described two anti-ly link to each other with detectable label.This class ELISA is a kind of simple " sandwich ELISA ".Also can anti-ly have three of binding affinity to resist to detect to described two by adding earlier for two anti-addings again, described three anti-ly link to each other with detectable label.

Another variation scheme is a competitive ELISA.In competitive ELISA, test sample book is competitively in conjunction with the labelled antigen or the antibody of known quantity.Can by with the bag hatched preceding by the hole or the process of hatching in, make sample mix the amount of measuring reactant in the sample with described known marker.But can reduce the amount of the marker of combined hole when having reactant in the sample, weaken final signal therefrom.

No matter adopt which kind of form, ELISA has some common feature, for example bag by, hatch or in conjunction with the material of, flush away non-specific binding and detect the bonded immunocomplex.Antigen or antibody are linked to each other with solid carrier, and sample to be analyzed is added on fixed antigen or the antibody, described solid carrier for example is the form of plate, bead, bar, film or base for post matter.During with antigen or antibody sandwich plate, hatch plate hole with antigen or antibody-solutions usually, and overnight incubation or one period concrete time period.Then, clean plate hole to remove the material of incomplete absorption.Then, use for the test antiserum(antisera) and be any remaining adsorbable hole surface of antigen neutral nonspecific proteins " bag by ".They comprise bovine serum albumin (BSA), casein and milk powder solution.Described bag is caught to seal the lip-deep non-specific adsorption of immobilization site, and therefore reduces the background due to antiserum(antisera) and the surperficial non-specific combination.

In ELISA, also can use secondary or three detection meanss, and without direct method.Therefore, albumen or antibody are combined with the hole, wrap by to reduce background and flush away not after the binding substance with reactionless active substance, immobilization surface or biological specimen clinical with contrast to be measured contacted under the condition of immunocomplex (antigen/antibody) formation can effectively making.Like this, the detection of this immunocomplex just need a kind of mark the secondary binding reagents or with the combination of three grades of binding reagents of a kind of secondary binding reagents and a kind of mark.

" can effectively making under the condition that immunocomplex (antigen/antibody) forms " means this condition and comprises with for example the solution dilution antigen and the antibody of BSA, ox gamma Globulin (BGG) and phosphate buffer soln (PBS)/tween, with the minimizing non-specific binding and improve rational signal to noise ratio.

Appropriate condition also can mean making and hatch under effective bonded temperature and in the enough time.Incubation step can be carried out under about 20 ℃ to 30 ℃ about 1 minute to 12 hours usually, perhaps can about 0 ℃ to about 10 ℃ of following overnight incubation.

In ELISA, carry out all incubation step after, the washing surface in contact to remove not compound material.Washing step can comprise the solution washing with for example PBS/ tween or borate buffer solution.After forming the specific immune complex body between test sample book and the initial bonded material and washing subsequently, can measure even immunocomplex to exist very in a small amount.

For a kind of detection means is provided, as mentioned above, described two is anti-or three anti-ly can have the bonded marker so that detect.It can be a kind of can colorific enzyme after hatching with suitable chromogenic substrate.Therefore, for example, can first or second immunocomplex be contacted with traget antibody and hatch (for example at room temperature, in containing the solution of PBS (for example PBS-tween), hatching 2 hours).

Hatch and subsequently behind the unconjugated material of flush away with traget antibody, can be quantitative to marker, for example with peroxidase during as enzyme labelling, can by with chromogenic substrate for example urea and purpurum bromocresolis or 2,2 '-nitrine-two-(3-ethyl-benzothiazole quinoline-6-sulfonic acid [ABTS] and H ₂O ₂Hatch together quantitatively.Quantitatively can realize, for example use the visible spectrum spectrophotometer by the measurement degree of adding lustre to.

Protein determination is for having utilized surface immobilized proteic solid phase part in conjunction with the mensuration system, and described surface comprises glass, film, microtiter well, mass spectrograph flat board and bead or other particles.These are measured highly parallel (multiple) and usually are (microarray, the protein chips) of miniaturization.The mass data that their advantage comprises fast and automatization, highly sensitive, reagent economy and single experiment can provide.Information biology is supported extremely important; The processing of data needs complicated software and data comparative analysis.Yet, can obtain above-mentioned software to the software correct that is used for the DNA array, as many hardware and detection system.

A kind of main form is a capture array, and in this array, ligand binding agent is used to the molecules of interest in the mixture of test example such as blood plasma or tissue extract, and described ligand binding agent is generally antibody, but also can be the albumen support, peptide or aptamer.In diagnosis, capture array can be used for parallel a plurality of immunoassay of carrying out, and both can measure the multiple analytes in for example single serum, also can measure a plurality of serum samples simultaneously.In proteomics, catch assay is used to the protein level of different health and ill sample is quantitatively also compared, and promptly protein expression is composed.Albumen except that the sepcific ligands binding substances is used in the described array format that is used for external function interaction (for example protein-protein, protein-DNA, protein-medicine, receptor-ligand, enzyme-substrate or the like) screening.Can select capture agent self and screen at multiple protein, this also can realize by the many array formats at the multiplexed protein target.

For making up array, proteinic source comprises, is used for the expression system based on cell of recombinant protein, from the natural origin purifying, by cell free translation system produced in vitro and peptide synthetic method.Many kinds in these methods all can be used for automatic high-throughput production.For capture array and protein function analysis, importantly, albumen should correctly fold and have function; But the fact is not that such was the case with, for example when under the sex change condition when bacterium is extracted recombinant protein.Yet the array of metaprotein can be used for screening cross reactivity, the identification autoantibody of antibody and screens part conjugated protein.

Protein arrays is designed to, uses the fluorescence reading usually and with the method for immunity commonly used (for example ELISA and dot blotting) of the propulsive microminiaturized form of machine, and the high throughput testing system that can parallelly carry out a plurality of mensuration.Physical support commonly used comprises slide glass, silicon, micropore, nitrocellulose or pvdf membrane and magnetic microballon or other microballons.Although modal form is the albumen droplet that is added to plane surface, other structure comprises based on microfluid (microfluidics) development (Gyros, Monmouth Junction, NJ) and the CD centrifugal device of special chip design, the microchannel of designing on the plate (The Living Chip for example for example ^TM, Biotrove, Woburn, MA) and the miniature 3D bar (Zyomyx, Hayward CA) of silicon face.Suspended particle also can be used as the array basis, and condition is to need they are carried out recognition coding; System comprises and is used for microballon (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystal (for example, QDots ^TM, QuantumDot, Hayward, colour coding CA) and be used for bead (UltraPlex ^TM, SmartBeadTechnologies Ltd, Babraham, Cambridge, UK) and many metals microbot (Nanobarcodes for example ^TMParticle, Nanoplex Technologies, Mountain View, bar coding CA).Bead also can be assembled in the planar array on the semi-conductor chip (LEAPStechnology, BioArray Solutions, Warren, NJ).

Proteic immobilization relates to coupling reagent and treats the character on coupling surface.Good protein arrays carrier surface before and after the coupling process all be chemically stable, make produce good some form, show that non-specific binding is minimum, the background that can not increase detection system and with different detection system compatibilities.The process for fixation that uses has circulation ratio, be applicable to have protein of different nature (big or small, hydrophilic, hydrophobic), be suitable for high-throughput and automatization and compatible with the active reservation of complete functional protein.The proteic orientation of surface bonding is considered to this albumen is offered important factor in part or the substrate process with active condition; For capture array, can be the most effective in conjunction with the result by directed capture agent acquisition, described capture agent need carry out the site-specific labeling to albumen usually.

Proteopexy method covalency and non-covalent all can be used, and they all have various relative merits.The surface is passive to be adsorbed on the method very simply, but almost can not carry out quantitatively or directed control; It may change or may not change proteic function specificity, and repeatable and efficient is variable.E provides a kind of stable connection, can be applicable to a variety of protein and has good repeatability; Yet orientation may be variable, chemically derivedly may change protein function and need stable interactive surfaces.Utilize the biological catching method of the label on the albumen that stable connection can be provided, and can be specifically and repeatably being orientated conjugated protein, but at first must described biological reagent is fully fixing, and, this array may need special processing, and its stability also may change.

Be used for the various fixed chemical method and the existing description of label of protein array structure.Be used for covalently bound substrate and comprise the slide glass of using the silane reagent bag quilt that contains amino or aldehyde.At Versalinx ^TMSystem (Prolinx, Bothell, WA) in, the reversible covalent coupling can be by realizing with the interaction between the fixed salicylhydroxamic acid on benzene hypoboric acid deutero-albumen and the carrier surface.The background of this method a little less than the inner fluorescence, and can make fixed albumen reservation function in conjunction with low.Based on three-dimensional polyacrylamide gel, the proteic non-covalent combination of unmodified occurs in for example HydroGel ^TM(PerkinElmer, Wellesley, the inside of vesicular structure MA); It is reported that the background of this substrate on the glass microarray is low especially, its protein capacity is high and can keep protein function.Widely used biological coupling method is to interact by means of vitamin H/streptavidin or six Histidines/Ni, and albumen is done suitably to modify.Can (Zeptosens, Witterswil be on surface Switzerland) on the fixed polylysine skeleton to titanium dioxide (Zyomyx) for example or tantalum pentoxide with biotin-conjugated.

Array manufacturing method comprises that machine connects printing (robotic contact printing), ink-jet, piezoelectricity point seal and photolithography.Many commercialization arrays (for example PackardBiosciences) and manual equipment (V﹠amp; P Scientific) all be obtainable.Can bacterium colony net be downloaded on the pvdf membrane to induce the original position protein expression by machine.

Nano-array is the size of point and the limit of density, when putting to nanometer space level, thousands of reaction can be carried out on less than 1 square millimeter one chip.BioForce Laboratories has developed 85 μ m with 1521 protein spots ²Nano-array, this is equivalent to every square centimeter 25,000,000 points, has reached the limit of optical detection; Its reading method is fluorescent method and atomic force microscopy (AFM).

Fluorescent mark and detection method are widely used.The instrument that is used for the dna microarray reading is equally applicable to protein array.Show for carrying out difference, can be used to catch (for example antibody) array from the fluorescent mark albumen detection of two kinds of different cell states, wherein cell lysate is directly puted together with different fluorophores (for example Cy-3, Cy-5) and described cell lysate is mixed, thereby with the reading of color as the change of target abundance.Can amplify (TSA) by the tyrasamine signal and (PerkinElmerLifesciences) sensitivity of fluorescence reading be amplified 10-100 doubly.Plane guided wave technology (Zeptosens) can carry out the hypersensitive fluoroscopic examination, also has the advantage of not disturbing washing process in addition.Also can realize highly sensitive by suspension bead or particle with serve as a mark thing (Luminex) or utilize the character of semiconductor nanocrystal (Quantum Dot) of phycoerythrin.Particularly in the biotechnology commercial field, many new alternative method of readings are developed.They comprise the transform method of following method: surface plasma resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), roll circular DNA amplification (Molecular Staging, New Haven CT), mass spectrum (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonant light scattering (Genicon Sciences, San Diego, CA) and atomic force microscopy (BioForceLaboratories).

Capture array has constituted can express the diagnosing chip of making spectrum and the basis of array.They by the high-affinity capture agent with the high-throughput mode in conjunction with and detect special target part, described high-affinity capture agent for example is support, peptide or the aptamer of conventional antibody, single structure territory, structure.

Antibody array possesses required specificity character and acceptable background, some antibody array commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St.Louis, MO).The antibody that is used for capture array can pass through routine immunization method (polyclonal serum and hybridoma) preparation, perhaps as behind phage or ribosomal display library screening, making (Cambridge Antibody Technology usually with the recombinant fragment of escherichia coli expression, Cambridge, UK; Biolnvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA).Except conventional antibody, also can in array, use the single V structural domain of Fab and scFv fragment, Camelidae antibody (camelid) or the human equivalent of transformation (Domantis, Waltham, MA).

Term " support " is meant proteic ligand binding domains, and it is transformed into can be in conjunction with the multiple variant with antibody sample specificity and affinity of multiple target molecules.Described variant can produce by the form in hereditary library, and can screen at individual target by phage, bacterium or ribosomal display.This class part comprises " Affibody " (Affibody based on SP in conjunction with support or skeleton, Bromma, Sweden), based on " Trinectin " (Phylos of fibronectin, Lexington, MA) with based on " Anticalin " (PierisProteolab of lipocalin protein structure, Freising-Weihenstephan, Germany).The similar antibody of they use-patterns in capture array, and have the good and advantage that produce easily of wearing quality.

Also can use non-albumen capture molecules in the array, particularly with high specific and affinity and protein ligands bonded single-chain nucleic acid fit (SomaLogic, Boulder, C0).Fit is to pass through Selex ^TMMethod is selected from oligonucleotide library, and they and protein interactions can be enhanced in covalently bound mode by mixing bromination deoxyuridine and UV activatory crosslinked action (light is fit).Because specific space requirement arranged, can reduce fit cross reactivity with the photo-crosslinking of part.Fit stability and the good advantage of wearing quality that has easy by the synthetic production of automatic oligonucleotide, DNA; For the fit array of light, can use general fluorescin staining agent to detect combination.

Can directly carry out with the detection of antibody array bonded analysis of protein thing or be undertaken by two in the sandwich assay is anti-.Directly mark is used for relatively having the different samples of different colours.Therefore if it is right to obtain at the antibody of same protein ligands, sandwich immunoassay then can provide higher specificity and sensitivity, and becomes and select to be used for for example method of cytokine of low-abundance protein; This mensuration can also detect protein modifier.The unmarked detection method that comprises mass spectrum, surface plasma resonance and atomic force microscopy can be avoided the change of part.All require best sensitivity and specificity for any method, and low background is to obtain higher signal to noise ratio.Because the analyte concentration scope is very wide, therefore need sensitivity is done suitably to regulate; Be to the sample serial dilution or use the antibody of different affinities at the solution of this problem.Target protein usually is a for example low expressed products in cytokine or the cell of concentration is very low in body fluid or the extract albumen, therefore just need the pg scope or more low-level on detection.

A replacement scheme of the array of capture molecules is to realize by " molecular imprinting " technology, and wherein (for example from PROTEIN C stub area) peptide is used as template to produce the sequence-specific cave of complementary on the structure in polymerizable matrix; This cave then can be caught (sex change) protein (ProteinPrint with suitable one-level aminoacid sequence specifically ^TM, Aspira Biosystems, Burlingame, CA).

It is ProteinChip that another kind can be used for diagnosing and expressing the method for making spectrum Array (Ciphergen, Fremont, CA), and the protein that has similar electric charge or hydrophobic character in the mixture of solid phase chromatographic surface in conjunction with blood plasma for example or tumor extract wherein, and the SELDI-TOF mass spectrum is used to detect the protein of reservation.

Large-scale functional chip is to make up by fixing a large amount of purifying protein, and is used to measure multiple biochemical function, for example and the protein-interacting between other albumen, medicine-target interaction, enzyme-substrate interaction etc.Usually they need a kind of expression library, are cloned in intestinal bacteria, the yeast etc., then for example by therefrom purifying expressed proteins and the immobilization of His label.For can not be for good representation in the system in bacterium or other bodies proteic synthetic, the albumen that is independent of cell is transcribed/translated be feasible alternative method.

Detection for protein-protein interaction, protein array can become the external replacement scheme of described yeast two-hybrid system based on cell, and when this two-hybrid system defectiveness, for example interact when relating to excretory albumen or having the albumen of disulphide bridges, this array is useful.With existing description of bioactive high throughput analysis that array carries out the various functions (protein-protein and proteolipid interact) of Yeast protein kinases and yeast protein group, wherein the major part in all open reading frames of zymic is all expressed and is fixed on the microarray.Large-scale " protein group chip " have at aspects such as recognition function interaction, drug screenings good application prospects (Proteometrix, Branford, CT).

Two dimension as individual element of volume shows that protein arrays can be used for screening phage or ribosomal display library, thereby filters out the mating partner of specific combination, comprises antibody, synthetic support, peptide and fit.Can carry out the screening in " library is to the library " in this way.The Another application of present method is, from screening of medicaments material standed for a kind of combinatorial chemistry library of array, described array is the protein targets target array that identifies in the genome project.

BD for example ^TMIt is a series ofly to can be used for catching soluble analyte and to the discontinuous particle of its quantitative spectrum that many beads of cytolytic dose bead array are measured.Described analyte detects by fluorescent emission again and flow cytometry is measured.Many beads are measured generation and based on the suitable data of the mensuration of ELISA, still are with a kind of " multiple " or mode simultaneously.Calculate the unknown material concentration of cytolytic dose bead array in the mode identical, promptly by using known standard and unknown material being plotted on the typical curve with the assay method of any sandwich form.In addition, many beads are measured and are made it possible to the soluble analyte of never considering because sample volume limits previous in the sample is carried out quantitatively.Except quantitative data, also can produce the effective visual image that shows unique spectrum or feature, described unique spectrum or feature provide the user only need sweep a glance can obtain additional information.

MMP/TIMP spectrum disclosed herein is based on the measurement to independent MMP or TIMP.Can be by any known a kind of their amount of method measurement that is illustrated in the available sign that has how many MMP or TIMP in the described analyzing samples that provides.The example of measuring method partly provides at embodiment.The quantivative approach of analyte (for example MPP or TIMP) comprises non-existent or with the measurement of the analyte that can not detection limit exists.

The technology and the method that are used to measure MMP and TIMP that constitute basis of the present invention are based on high-sensitive immunoassay.Have multiple during this para-immunity is measured all by this development in laboratory (being the method for masurement that TIMP-4 measures).(ELISA) carries out immunoassay by enzyme-linked immunoassay, these immunoassay by stdn so that the observed value shown in the table 4 to be provided.Yet other sensitiveer methods that is used to measure MMP and TIMP blood levels have fast been implemented in this laboratory, and they comprise the multiple assay system that uses.In this example, can use based on the multiple sandwich immunoassay of bead and measure multiple analytes in the sample (for example blood plasma or other biological sample) of limited bulk.This new technology that is used for multiple analysis is based on the sensitivity of ELISA and fluidic cell is detected on the technology that combines, thus feasible can the measurement specifically less than reaching most 100 kinds different analytes in the single sample of 50 μ l.The feasible multiple MMP and the TIMP that can measure in a small amount of blood sample of this method.These class methods are fit to the monitoring of diagnosis described herein, prognosis, prediction and treatment and use very much.Particularly, be while Measurement and analysis substrate concentration, microballon is hatched with sample (being blood sample), and make described microballon and described specific purpose analyte (being MMP) form complex body.Then, in mixture, add the detection antibody (biotinylated) of specificity at second epi-position on each analyte, and make described detection antibodies to described with analyte bonded microballon on.Then this mixture is hatched with fluorescent reporter molecule (streptavidin-phycoerythrin), and make whole sample by two laser streaming cell detection instrument.Laser detection is used for determining the definite fluorescence of the microballon of specific analyte to be detected, the fluorescence volume of another laser detection reporter molecules, and this amount is directly proportional with the amount of institute's bonded analyte.This method has been used to other analytes that multiple MMP and CHF process may relate to, shown in Fig. 8 and table 1.How this just uses the minute quantity blood sample to measure an example of single or multiple analytes.Other examples of the measurement of having implemented relevant with the MMP/TIMP analyte comprise radioimmunoassay and immunoblotting mensuration.These methods also are based on antibody.

Table 1: be used for the typical curve that calculates is proofreaied and correct the analyte concentration scope of adding up with linear regression

7. antibody

Specificity is known at the antibody of MMP and TIMP, and commercially available.Table 2 has provided the example of antibody.

Table 2:MMP/TIMP antibody

The broad sense use done in term " antibody " herein, and it comprises polyclonal antibody and monoclonal antibody.Except complete immune globulin ultrawhite, term " antibody " also comprises the fragment of described immunoglobulin molecules or immunoglobulin molecules or its fragment of polymer and the mankind or humanization form, as long as come its selection according to them and MMP or the interactional ability of TIMP.Can use external test as herein described or similar approach to come the required activity of test antibody, measure their interior therapeutic and/or prophylactic activity afterwards according to known clinical trial method.

" term " used herein is meant the antibody that obtains from the antibody colony of homogeneous basically, that is, the individual antibody in this colony is identical, except being present in the natural sudden change that may exist in a small amount of antibody molecule.The monoclonal antibody of this paper comprises " chimeric antibody " and fragment thereof especially, as long as they demonstrate required antagonistic activity, in wherein said " chimeric antibody " part of heavy chain and/or light chain with from a kind of antibody of specific species or belong to the identical or homology of corresponding sequence of the antibody of a kind of specific antibodies classification or subclass, and other parts of this chain with from the antibody of another species or belong to the identical or homology of the corresponding sequence of antibody of another antibody classification or subclass (referring to United States Patent (USP) 4,816,567 and Morrison et al., Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984)).

Disclosed monoclonal antibody can prepare with any production monoclonal antibody method.For example, available hybridoma method prepares disclosed monoclonal antibody, Kohler and Milstein for example, and Nature is described in the 256:495 (1975).In the hybridoma method, use immunizing agent immune mouse or other appropriate host animals usually, with energy of initiation produce or produce can with this immunizing agent lymphocyte of bonded antibody specifically.

Also available recombinant DNA legal system is equipped with monoclonal antibody, and for example United States Patent (USP) 4,816, described in 567 (people such as Cabilly).The DNA of the disclosed monoclonal antibody of encoding can use ordinary method (for example, use can be specifically in conjunction with the oligonucleotide probe of the gene of coding rodent antibody heavy chain and light chain) easily to separate and check order.Also can adopt display technique of bacteriophage to produce and screening antibody or the segmental library of active antibody, for example, of people's such as people's such as Burton United States Patent (USP) 5,804,440 and Barbas United States Patent (USP) 6,096,441.

In vitro method also is suitable for preparing univalent antibody.Can use ordinary method digestion antibody known in the art to produce antibody fragment, particularly Fab fragment.For example, can use papoid to digest.The example of papain digestion has description on December 22nd, 1994 in disclosed WO 94/29348 and the United States Patent (USP) 4,342,566.The papain digestion of antibody produces two identical Fabs and remaining Fc fragment usually, and above-mentioned Fab is called the Fab fragment, and each fragment has single antigen binding site.Pepsin can obtain having two antigen binding sites and still can crosslinked antigenic fragment.

No matter whether be connected with other sequences, described fragment also can comprise insertion, disappearance, replacement or other selected modifications of specific region or particular amino acid residue, condition is, compares with the antibody or the antibody fragment of unmodified, significantly do not change or damage the activity of this antibody or antibody fragment.This class is modified can provide some other characteristic, and for example, removals/interpolations can carry out amino acid that disulfide linkage closes, improve its biology life-span, change it secretes feature etc.In any case, described antibody or antibody fragment all must biologically actives, for example with its correlation antigen specific combine.Functional or the active region of described antibody or antibody fragment can be by to the mutagenesis of protein specific region and express subsequently and test polypeptide expressed and discern.These class methods are conspicuous to those skilled in the art, can comprise that the nucleic acid to encoding said antibody or antibody fragment carries out site-specific mutagenesis (Zoller, MJ.Curr.Opin.Biotechnol.3:348-354,1992).

Term as used herein " antibody " also can refer to human antibodies and/or humanized antibody.A lot of non-human antibodies' (for example deriving from the antibody of mouse, rat or rabbit) have antigenicity to the mankind natively, therefore, and when its administration of human time-like can be produced bad immune response.Therefore, in described method, use the mankind or humanized antibody to reduce and give the possibility that human antibody causes adverse immune response.

8. reference point

The existence that can indicate DHF among the experimenter or the MMP that predicts the generation of DHF among the experimenter and/or TIMP spectrum are provided.Among the described indication experimenter among the existence of DHF or the prediction experimenter MMP of the generation of DHF and/or TIMP spectrum can be numerical value with respect to normal value.The normal value of given analyte (MMP or TIMP) can be the reference point that is proved the age-matched experimenter of no obvious cardiovascular disease indication.Therefore, described normal value can be the population value from a large amount of healthy individual.These can obtain by colony's research with reference to normal value.The research of existing large-scale colony for example determine the TIMP-1 of reference group level relatively (Framingham Heart Study, Circulation 2004; 109:2850-2856) be approximately 800ng/mL, this value is consistent with reference control value disclosed herein.

Perhaps, described normal value can be that to be considered to for set experimenter be normal numerical value.For example, can carry out base measurement, and use it for the observed value that obtains from this individuality afterwards and compared, with the process of discerning current disease or developing to hypertensive heart disease to the analyte of interest of healthy individual.

Discontinuous observation (for example MMP-13) is change continuous variable (for example plasma concentration of given analyte) into dichotomic variable.In this specific examples, if be considered to detectable or positive numerical value greater than the value of 10ng/mL, when being considered to negative numerical value less than the value of 10ng/mL, then can with one+/-value gives MMP-13.

For example, provide a kind of method that does not have the LVH relevant in the subject of diagnosing, comprised MMP and/or the TIMP level measured in described experimenter's tissue or the body fluid, and described level and reference point have been compared with hypertensive heart disease.Therefore, the normal value of MMP-2, MMP-9, MMP-7, MMP-13, MMP-8, TIMP-1, TIMP-2 and/or TIMP-4 is the sign that there be not the left ventricular hypertrophy relevant with hypertensive heart disease in expression.

In some aspects, the MMP-2 blood plasma level in the normal range represents not exist the LVH relevant with hypertensive heart disease.In some aspects, the MMP-9 blood plasma level in the normal range represents not exist the LVH relevant with hypertensive heart disease.In some aspects, the MMP-13 blood plasma level in the normal range represents not exist the LVH relevant with hypertensive heart disease.In some aspects, the TIMP-1 blood plasma level in the normal range represents not exist the LVH relevant with hypertensive heart disease.In some aspects, the TIMP-2 blood plasma level in the normal range represents not exist the LVH relevant with hypertensive heart disease.In some aspects, the TIMP-4 blood plasma level in the normal range represents not exist the LVH relevant with hypertensive heart disease.

In some aspects, the MMP-2 blood plasma level is higher than about 1000ng/ml, comprises being higher than about 1000,1100,1200,1300,1400 and during 1500ng/ml, i.e. there be not the LVH relevant with hypertensive heart disease in expression.

In some aspects, the MMP-9 blood plasma level is lower than about 20ng/ml, comprise and be lower than about 20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2 or during 1ng/ml, i.e. there be not the LVH relevant with hypertensive heart disease in expression.

In some aspects, detectable MMP-13 blood plasma level is higher than about 5ng/ml, comprises that there be not the LVH relevant with hypertensive heart disease in i.e. expression when being lower than about 5,6,7,8,9,10,11,12,13,14,15,20ng/ml.

In some aspects, the TIMP-1 blood plasma level is lower than about 1000ng/ml, comprise and be higher than about 1000,900,800,700,600,500,400,300,200,100,50,40,30,20 or during 10ng/ml, i.e. there be not the LVH relevant with hypertensive heart disease in expression.

In some aspects, the TIMP-2 blood plasma level is lower than about 50ng/ml, comprise and be higher than about 50,49,48,47,46,45,44,43,42,41,40,35,30,25,20,15 or during 10ng/ml, i.e. there be not the LVH relevant with hypertensive heart disease in expression.

In some aspects, the TIMP-4 blood plasma level is lower than about 2ng/ml, comprises being higher than about 2.0,1.9,1.8,1.7,1.6,1.5,1.4,1.3,1.2,1.1,1.0,0.5 or during 0.1ng/ml, i.e. there be not the LVH relevant with hypertensive heart disease in expression.

Described method can further comprise the blood plasma level of measuring two or more MMP and/or TIMP.For example, described method can comprise two kinds, three kinds, four kinds, five kinds, six kinds, seven kinds or eight kinds that measure among MMP-2, MMP-9, MMP-7, MMP-13, MMP-8, TIMP-1, TIMP-2 and the TIMP-4.Therefore, described method can comprise measurement MMP-2 and MMP-9 or MMP-2 and MMP-7, MMP-2 and MMP-13, MMP-2 and MMP-8, MMP-2 and TIMP-1, MMP-2 and TIMP-2, MMP-2 and TIMP-4, MMP-9 and MMP-7, MMP-9 and MMP-13, MMP-9 and MMP-8, MMP-9 and TIMP-1, MMP-9 and TIMP-2, MMP-9 and TIMP-4, MMP-7 and MMP-13, MMP-7 and MMP-8, MMP-7 and TIMP-1, MMP-7 and TIMP-2, MMP-7 and TIMP-4, MMP-13 and MMP-8, MMP-13 and TIMP-1, MMP-13 and TIMP-13, MMP-13 and TIMP-4, MMP-8 and TIMP-1, MMP-8 and TIMP-2, MMP-8 and TIMP-4, TIMP-1 and TIMP-2, TIMP-1 and TIMP-4, TIMP-2 and TIMP-4.Therefore, described method can comprise measurement MMP-2, MMP-13 and TIMP-1; MMP-2, MMP-13 and TIMP-2; MMP-2, MMP-13 and TIMP-4; MMP-13, TIMP-1 and TIMP-2; MMP-13, TIMP-1 and TIMP-4; MMP-13, TIMP-2 and TIMP-4.Therefore, described method can comprise measurement MMP-2, MMP-13, TIMP-1 and TIMP-2; MMP-2, MMP-13, TIMP-1 and TIMP-4; MMP-2, MMP-13, TIMP-2 and TIMP-4; MMP-13, TIMP-1, TIMP-2 and TIMP-4; MMP-2, TIMP-1, TIMP-2 and TIMP-4.Therefore, described method can comprise measurement MMP-2, MMP-13, TIMP-1, TIMP-2 and TIMP-4.This paper also considers and discloses other combinations of these analytes.

Described method can further comprise the ratio that calculates one or more MMP or TIMP and other MMP or TIMP, and for example described method can comprise the ratio that calculates MMP-9 and TIMP-1, TIMP-2 or TIMP-4.

For example, in some aspects, the ratio of MMP-9/TIMP-1 blood plasma level is greater than about 7 * 10 ³, comprise greater than about 7 * 10 ³, 8 * 10 ³, 9 * 10 ³, 10 * 10 ³, 11 * 10 ³, 12 * 10 ³, 13 * 10 ³Or 14 * 103 o'clock, i.e. there be not the LVH relevant with hypertensive heart disease in expression.

In some aspects, the ratio of MMP-9/TIMP-2 blood plasma level is greater than about 10 * 10 ⁴, comprise greater than about 10 * 10 ⁴, 20 * 10 ⁴, 30 * 10 ⁴Or 40 * 10 ⁴The time, i.e. there be not the LVH relevant with hypertensive heart disease in expression.

In some aspects, the ratio of MMP-9/TIMP-4 blood plasma level is greater than about 1, comprises that there be not the LVH relevant with hypertensive heart disease in i.e. expression greater than about 1,2,3,4,5,6,7,8 or at 9 o'clock.

Describedly be shown in Table 3 with reference to normal value and the numerical value that to high blood sample patient screening the time, records.In this example, the MMP-2 value of suffering from the hyperpietic of LVH can reduce, but the MMP-7 value is constant.Yet, will carry out discontinuous observation to MMP-13 because it in suffering from the hyperpietic of LVH, detect less than.Therefore, the dividing point that is lower than 10ng/mL is considered to hypertension and Case definition in heart failure.Compare with the reference control value, TIMP-1 and the TIMP-4 level of suffering from the hyperpietic of LVH can be high by 50%.Compare with the reference normal value, the ratio of MMP-9/TIMP-4 of suffering from the hyperpietic of LVH reduces more than 50%.

Table 3:MMP and TIMP data; With reference to normal value and hypertensive heart disease; Diagnosis per-cent dividing point

NC=is with respect to normal no change

* with normal phase ratio, p＜0.05

9.LVH rapid screening

A kind of " be/not " quick result of type that can obtain by the level that to measure a kind of concrete MMP be MMP-13 is provided.Can be with a set point---can according to the change at age and colony's statistics and adjusted---be used as described effective reading.For example, be lower than 1,2,3,4,5,6,7,8,9 or the MMP-13 level of the threshold value setting of 10ng/mL will make need be more deep plasma screening and other cardiovascular imaging research.In other words, this rapid screening test can be applicable to any large-scale colony, can identify those then and can guarantee stricter test and the experimenter who follows up a case by regular visits to.Also not can be used at present discerning LVH patient's rapid screening test.

A kind of method of the experimenter's of prediction heart failure diastole is provided, has comprised the amount of measuring MMP-13 in experimenter's body fluid, amount is less than 1,2,3,4,5,6,7,8,9 or 10ng/mL or detect less than i.e. expression and have LVH and indicate DHF.When in conjunction with the unusual observed value of other analyte of interest disclosed herein, above-mentioned measurement can detect DHF.

Can carry out blood plasma when tentative experiment or medical screening composes.Can carry out above-mentioned screening measurement at one or more MMP and/or TIMP.If described one or more observed value drops on outside the reference point, then can carry out other measurement.For example, available MMP-13 carries out preliminary screening, like this, if detect less than MMP-13, then can carry out another mensuration to this plasma sample.Similarly, also available MMP-9 and TIMP-1, TIMP-2 and/or TIMP-4 carry out preliminary screening, like this, if when the ratio of MMP-9 and TIMP-1, TIMP-2 or TIMP-4 is lower than the NL with definite threshold value, then can carry out another mensuration to plasma sample.Described another mensuration can be used for the whole spectrums shown in the acquisition table 3 or its part.If this spectrum reaches the standard of hypertensive heart disease, then can pass through the more potent described patient of test evaluation, described test can comprise suitable echocardiography, catheterization, nuclear imaging.Also can carry out more potent medical treatment evaluation to the patient.

10. diagnosis

A kind of for example diagnostic method of following a kind of experimenter that can be used for also is provided, and described experimenter shows CHF sign and symptom, but the basic reason of this performance is difficult to determine.This situation takes place extremely continually; When the patient suffers from CHF, be difficult to determine whether to exist LVH and DHF, and whether they can promote the aggravation of CHF process.The application is described a kind ofly " to be determined " or the application of the blood testing that " eliminating " LVH and DHF exist quickly and easily, and this required diagnostic method can be provided.Particularly, can measure MMP-13, MMP-9, MMP-2, TIMP-1 and/or the TIMP-4 of blood sample.Numerical value and the normal reference value disclosed herein that obtains can be compared.If this value departs from the NL that the threshold value determined by this paper is represented, can determine that then the patient has DHF.

For example, provide the method for a kind of experimenter's of diagnosis LVH, comprised MMP and/or the TIMP level measured in described experimenter's tissue or the body fluid, and described level and reference point have been compared.

Representing when in some aspects, the MMP-2 blood plasma level is lower than normal value has hypertensive heart disease.For example, the amount of MMP-2 can represent to have hypertensive heart disease at low about 20% o'clock at least than normal mean value.In some aspects, the MMP-2 blood plasma level is lower than about 1000ng/ml, comprises being lower than about 1000,990,980,970,960,950,940,930,920,920,900,890,880,870,860,850,840,830,820,810,800,790,780,770,760,750,740,730,720,710,700,650,600,550,500,450,400,350,300,250,200,250 or represent that hypertensive heart disease is arranged during 100ng/ml.

Representing when in some aspects, the MMP-9 blood plasma level is higher than normal value has hypertensive heart disease.For example, the amount of MMP-9 is higher at least about 50% o'clock than normal mean value, can represent to have hypertensive heart disease.In some aspects, the MMP-9 blood plasma level is higher than about 20ng/ml, comprises being higher than about 20,21,22,23,24,15,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,55,60,65,70,75,80,85,90,95 or represent that hypertensive heart disease is arranged during 100ng/ml.

In some aspects, the MMP-13 blood plasma level detects not i.e. expression then LVH.In some aspects, the MMP-13 blood plasma level is lower than about 10ng/ml, comprises being lower than about 10,9,8,7,6,5,4,3,2 or represent that LVH is arranged during 1ng/ml.

Representing when in some aspects, the TIMP-1 blood plasma level is higher than normal value has hypertensive heart disease.For example, the amount of TIMP-1 is higher at least about 50% o'clock than normal mean value, can represent to have LVH.In some aspects, the TIMP-1 blood plasma level is higher than about 1000ng/ml, comprises being higher than about 1000,1010,1020,1030,1040,1050,1060,1070,1080,1090,1100,1150,1200,1250,1300,1350,1400 or represent that LVH is arranged during 1500ng/ml.

Representing when in some aspects, the TIMP-2 blood plasma level is higher than normal value has LVH.For example, the amount of TIMP-2 is higher at least about 50% o'clock than normal mean value, can represent to have LVH.In some aspects, the TIMP-2 blood plasma level is higher than about 50ng/ml, comprises being higher than about 50,55,60,65,70,75,80,85,90,95 or represent that LVH is arranged during 100ng/ml.

Representing when in some aspects, the TIMP-4 blood plasma level is higher than normal value has LVH.For example, the amount of TIMP-4 is higher at least about 50% o'clock than normal mean value, can represent to have LVH.In some aspects, the TIMP-4 blood plasma level is higher than about 2ng/ml, comprises being higher than about 2,3,4,5,6,7,8,9 or represent that LVH is arranged during 10ng/ml.

Can represent to have LVH when in some aspects, the MMP-7 blood plasma level is in normal range.Can represent to have LVH when in some aspects, the MMP-8 blood plasma level is in normal range.

Described method can further comprise the blood plasma level of measuring two or more MMP and/or TIMP.For example, described method can comprise two kinds, three kinds, four kinds, five kinds, six kinds, seven kinds or eight kinds that measure among MMP-2, MMP-9, MMP-7, MMP-13, MMP-8, TIMP-1, TIMP-2 and the TIMP-4.Therefore, described method can comprise measurement MMP-2 and MMP-9; MMP-2 and MMP-13; MMP-13 and TIMP-1; MMP-13 and TIMP-2; MMP-13 and TIMP-4; MMP-2, MMP-13 and TIMP-1; MMP-2, MMP-13 and TIMP-2; MMP-2, MMP-13 and TIMP-4; Perhaps MMP-2, MMP-13, TIMP-1, TIMP-2 and TIMP-4.This paper considers and discloses other combinations of above-mentioned analyte.

For example, when TIMP-1 increases (TIMP-1〉1200ng/mL) and MMP-13 level and reduces, can detect DHF.Again for example, when the amount of TIMP-4 is higher than 3ng/mL, and the MMP-13 level reduces, when the TIMP-1 level increases, just showing has LVH and measurable DHF.Therefore, a kind of experimenter of detection LVH also predicts the method for experimenter's heart failure diastole, comprises the MMP-13, the TIMP-1 that measure in experimenter's body fluid and the spectrum of TIMP-4.Spectrum when the amount of MMP-13 detects less than the amount of, TIMP-1 than the amount of high about 50% (or being higher than 1200ng/mL) of normal value and TIMP-4 than high at least about 50% (or being higher than 3ng/mL) of normal value indicates DHF.

Described method can further comprise the ratio that calculates one or more MMP or TIMP and other MMP or TIMP.For example, described method can comprise the ratio that calculates MMP-9 and TIMP-1, TIMP-2 or TIMP-4.

Represent that LVH is arranged when in some aspects, the ratio of MMP-9/TIMP-1 blood plasma level is less than normal value.For example, MMP-9/TIMP-1 compares normal mean value to when young about 50% o'clock, can represent to have LVH.For example, in some aspects, the ratio of MMP-9/TIMP-1 blood plasma level is less than about 7 * 10 ³, comprise less than about 7 * 10 ³, 6 * 10 ³, 5 * 10 ³, 4 * 10 ³, 5 * 10 ³, 6 * 10 ³, 1 * 10 ³The time, i.e. expression has LVH.

Represent that LVH is arranged when in some aspects, the ratio of MMP-9/TIMP-2 blood plasma level is less than normal value.For example, MMP-9/TIMP-2 compares normal mean value to when young about 50% o'clock, can represent to have LVH.In some aspects, the ratio of MMP-9/TIMP-2 blood plasma level is less than about 100 * 10 ³, comprise less than about 100 * 10 ³, 90 * 10 ³, 80 * 10 ³, 70 * 10 ³, 60 * 10 ³, 50 * 10 ³, 40 * 10 ³, 30 * 10 ³, 20 * 10 ³Or 10 * 10 ³The time, i.e. expression has LVH.

Represent that LVH is arranged when in some aspects, the ratio of MMP-9/TIMP-4 blood plasma level is less than normal value.For example, MMP-9/TIMP-4 compares normal mean value to when young about 50% o'clock, can represent to have LVH.In some aspects, the ratio of MMP-9/TIMP-4 blood plasma level is less than about 3, comprises that i.e. expression has LVH less than about 3.0,2.5,2.0,1.5,1.0,0.9,0.8,0.7,0.6,0.5,0.4,0.3,0.2,0.1,0.05 or at 0.01 o'clock.

In some aspects, the ratio of MMP-9/TIMP-1 blood plasma level is less than about 5 * 10 ³, the ratio of MMP-9/TIMP-2 blood plasma level is less than about 100 * 10 ³, and the ratio of MMP-9/TIMP-4 blood plasma level promptly represented to have LVH less than about 1 o'clock.

In some aspects, the MMP-2 blood plasma level is less than about 1000ng/ml, and the MMP-13 blood plasma level is less than about 5ng/ml, and the ratio of MMP-9/TIMP-1 blood plasma level is less than about 5 * 10 ³, the ratio of MMP-9/TIMP-2 blood plasma level is less than about 100 * 10 ³, and the ratio of MMP-9/TIMP-4 blood plasma level promptly represented to have LVH less than about 1 o'clock.

11. prognosis

A kind of method of prognosis that for example can be used for following a kind of experimenter's heart failure diastole also is provided, described be selected to screen and afterwards further the blood plasma experimenter that makes spectrum be proved and suffer from serious LVH and have the risk that DHF takes place.In this case, quantitative to MMP-13 level and TIMP level.The MMP-13 level is lower/detects less than (0-5ng/mL), and that the TIMP level is compared with contrast normal subjects TIMP level is higher (for example TIMP-1〉1200ng/mL, TIMP-2〉700ng/mL and/or TIMP-4〉3ng/mL) time, may draw the significant observation result of reflecting myocardium fibrosis and diastolic dysfunction degree.This just provides the prognosis values about symptom development and hospital care process.Particularly, available hypertension drug is further strengthened treatment to these patients, and can carry out more conventional cardiovascular imaging research to it.

For example, the patient's that the risk that provides a kind of identification that heart failure diastole (DHF) takes place increases method comprises MMP and/or the TIMP level measured in described experimenter's tissue or the body fluid, and described level and reference point are compared.

Represent when in some aspects, the MMP-2 blood plasma level is lower than normal value that the risk that heart failure diastole takes place increases.For example, the amount of MMP-2 was hanged down about 20% o'clock at least than normal mean value, can represent that the risk that heart failure diastole takes place increases.In some aspects, the MMP-2 blood plasma level is lower than about 500ng/ml, comprises being lower than about 500,450,400,350,300,250,200,250 or during 100ng/ml, i.e. the risk that heart failure diastole takes place in expression increases.

In some aspects, the MMP-13 blood plasma level detects not the risk increase that heart failure diastole takes place in i.e. expression then.In some aspects, the MMP-13 blood plasma level is lower than about 10ng/ml, comprises being lower than about 10,9,8,7,6,5,4,3,2 or during 1ng/ml, i.e. the risk that heart failure diastole takes place in expression increases.

Representing to take place the depleted risk of psychology diastole when in some aspects, the TIMP-1 blood plasma level is higher than normal value increases.For example, the amount of TIMP-1 at least about 50% o'clock, can represent that the risk that heart failure diastole takes place increases than normal mean value height.In some aspects, the TIMP-1 blood plasma level is higher than about 1000ng/ml, comprise and be higher than about 1000,1010,1020,1030,1040,1050,1060,1070,1080,1090,1100,1150,1200,1250,1300,1350,1400,1500,1600,1700,1800,1900 or during 2000ng/ml, i.e. the risk that heart failure diastole takes place in expression increases.

Representing to take place the depleted risk of psychology diastole when in some aspects, the TIMP-2 blood plasma level is higher than normal value increases.For example, the amount of TIMP-2 at least about 50% o'clock, can represent that the risk that heart failure diastole takes place increases than normal mean value height.In some aspects, the TIMP-2 blood plasma level is higher than about 50ng/ml, comprise and be higher than about 50,55,60,65,70,75,80,85,90,95,100,110,120,130,140,150,160,170,180,190 or during 200ng/ml, i.e. the risk that heart failure diastole takes place in expression increases.

Representing to take place the depleted risk of psychology diastole when in some aspects, the TIMP-4 blood plasma level is higher than normal value increases.For example, the amount of TIMP-4 at least about 50% o'clock, can represent that the risk that heart failure diastole takes place increases than normal mean value height.In some aspects, the TIMP-4 blood plasma level is higher than about 2ng/ml, comprises being higher than about 2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45 or during 50ng/ml, i.e. the risk that heart failure diastole takes place in expression increases.

Can represent when in some aspects, the MMP-9 blood plasma level is in normal range that the risk that heart failure diastole takes place increases.Can represent when in some aspects, the MMP-7 blood plasma level is in normal range that the risk that heart failure diastole takes place increases.Can represent when in some aspects, the MMP-8 blood plasma level is in normal range that the risk that heart failure diastole takes place increases.

Described method can further comprise the blood plasma level of measuring two or more MMP and/or TIMP.For example, described method can comprise measures MMP-2, MMP-9, MMP-7, MMP-13, MMP-8, TIMP-1, among TIMP-2 and the TIMP-4 two kinds, three kinds, four kinds, five kinds, six kinds, seven kinds or eight kinds.Therefore, described method can comprise measurement MMP-2 and MMP-9, MMP-2 and MMP-7, MMP-2 and MMP-13, MMP-2 and MMP-8, MMP-2 and TIMP-1, MMP-2 and TIMP-2, MMP-2 and TIMP-4, MMP-9 and MMP-7, MMP-9 and MMP-13, MMP-9 and MMP-8, MMP-9 and TIMP-1, MMP-9 and TIMP-2, MMP-9 and TIMP-4, MMP-7 and MMP-13, MMP-7 and MMP-8, MMP-7 and TIMP-1, MMP-7 and TIMP-2, MMP-7 and TIMP-4, MMP-13 and MMP-8, MMP-13 and TIMP-1, MMP-13 and TIMP-13, MMP-13 and TIMP-4, MMP-8 and TIMP-1, MMP-8 and TIMP-2, MMP-8 and TIMP-4, TIMP-1 and TIMP-2, TIMP-1 and TIMP-4, TIMP-2 and TIMP-4.Therefore, described method can comprise measurement MMP-2, MMP-13 and TIMP-1; MMP-2, MMP-13 and TIMP-2; MMP-2, MMP-13 and TIMP-4; MMP-13, TIMP-1 and TIMP-2; MMP-13, TIMP-1 and TIMP-4; MMP-13, TIMP-2 and TIMP-4.Therefore, described method can comprise measurement MMP-2, MMP-13, TIMP-1 and TIMP-2; MMP-2, MMP-13, TIMP-1 and TIMP-4; MMP-2, MMP-13, TIMP-2 and TIMP-4; MMP-13, TIMP-1, TIMP-2, and TIMP-4; MMP-2, TIMP-1, TIMP-2, and TIMP-4.Therefore, described method can comprise measurement MMP-2, MMP-13, TIMP-1, TIMP-2 and TIMP-4.This paper also considers and discloses other combinations of these analytes.

For example, provide the method for a kind of experimenter's of detection heart failure diastole, comprised the amount of measuring MMP-13, TIMP-1, TIMP-4 and MMP-9 in described experimenter's body fluid.The method of a kind of experimenter's of prediction heart failure diastole also is provided, has comprised the amount of measuring MMP-13, TIMP-1, TIMP-4 and MMP-9 in described experimenter's body fluid.In these methods, the amount that can demonstrate MMP-13 detects less than the amount of (or being lower than 10ng/mL), TIMP-1 than normal value high about 50% or the amount that is higher than 1200ng/mL, TIMP-4 than normal value high at least about 50% or be higher than 3ng/mL, and the amount of MMP-9 can detect LVH and DHF than high at least about 50% the spectrum of normal value.

The method of a kind of experimenter's of detection heart failure diastole also is provided, has comprised the MMP-13, the TIMP-1 that measure in described experimenter's body fluid, the amount of TIMP-4 and MMP-2.The method of a kind of experimenter's of prediction heart failure diastole also is provided, has comprised MMP-13, TIMP-1 in described experimenter's body fluid, the amount of TIMP-4 and MMP-2 measured.In these methods, the amount that spectrogram can demonstrate MMP-13 detects higher at least by about 20% than normal value than the amount of high at least about 50% (or being higher than 3ng/mL) of normal value and MMP-2 than the amount of high about 50% (or being higher than 1200ng/mL) of normal value, TIMP-4 less than the amount of (or being lower than 10ng/mL), TIMP-1.

Described method can further comprise the ratio that calculates one or more MMP or TIMP and other MMP or TIMP.For example described method can comprise the ratio that calculates MMP-9 and TIMP-1, TIMP-2 or TIMP-4.

Represent that LVH is arranged when in some aspects, the ratio of MMP-9/TIMP-1 blood plasma level is less than normal value.For example, MMP-9/TIMP-1 compares normal mean value to when young about 50% o'clock, can represent that the risk that heart failure diastole takes place increases.For example, in some aspects, the ratio of MMP-9/TIMP-1 blood plasma level is less than about 7 * 10 ³, comprise less than about 7 * 10 ³, 6 * 10 ³, 5 * 10 ³, 4 * 10 ³, 5 * 10 ³, 6 * 10 ³, 1 * 10 ³, 9 * 10 ², 8 * 10 ², 7 * 10 ², 6 * 10 ², 5 * 10 ², 4 * 10 ², 3 * 10 ², 2 * 10 ²Or 1 * 10 ²The time, i.e. the risk that heart failure diastole takes place in expression increases.

Represent that LVH is arranged when in some aspects, the ratio of MMP-9/TIMP-2 blood plasma level is less than normal value.For example, MMP-9/TIMP-2 compares normal mean value to when young about 50% o'clock, can represent to have LVH.In some aspects, the ratio of MMP-9/TIMP-2 blood plasma level is less than about 100 * 10 ³, comprise less than about 100 * 10 ³, 90 * 10 ³, 80 * 10 ³, 70 * 10 ³, 60 * 10 ³, 50 * 10 ³, 40 * 10 ³, 30 * 10 ³, 20 * 10 ³, 10 * 10 ³, 9 * 10 ³, 8 * 10 ³, 7 * 10 ³, 6 * 10 ³, 5 * 10 ³, 4 * 10 ³, 3 * 10 ³, 2 * 10 ³Or 1 * 10 ³The time, i.e. expression has LVH.

Represent that LVH is arranged when in some aspects, the ratio of MMP-9/TIMP-4 blood plasma level is less than normal value.For example, MMP-9/TIMP-4 compares normal mean value to when young about 100% o'clock, can represent to have LVH.In some aspects, the ratio of MMP-9/TIMP-4 blood plasma level is less than about 1, comprises about 1,0.9,0.8,0.7,0.6,0.5,0.4,0.3,0.2,0.25,0.2,0.15,0.10,0.05 or at 0.01 o'clock, i.e. expression has LVH.

Therefore, described method provides a kind of and has detected or the method for prediction experimenter heart failure diastole, comprises that the normal ratio that provides of the MMP-9 that detects described experimenter's body fluid and TIMP-4 is low frequently.Described method comprises that measuring the normal ratio of this odds ratio hangs down at least about 50%.

In some aspects, the ratio of MMP-9/TIMP-1 blood plasma level is less than about 5 * 10 ³, the ratio of MMP-9/TIMP-2 blood plasma level is less than about 100 * 10 ³, and the ratio of MMP-9/TIMP-4 blood plasma level promptly represented to have LVH less than about 1 o'clock.

In some aspects, the MMP-2 blood plasma level is less than about 1000ng/ml, and the MMP-13 blood plasma level is less than about 5ng/ml, and the ratio of MMP-9/TIMP-1 blood plasma level is less than about 5 * 10 ³, the ratio of MMP-9/TIMP-2 blood plasma level is less than about 100 * 10 ³, and the ratio of MMP-9/TIMP-4 blood plasma level promptly represented to have LVH less than about 1 o'clock.

12. instruct therapeutic intervention

In treatment, the low-level and TIMP-1 high level of MMP-13 can be used as the monitoring index of curative effect of medication.There is multiple height to be suitable for the relevant clinical situation of These parameters.For example, when hyperpietic's blood pressure was in " NL ", the MMP-13 level reduced, and the TIMP level raises.Increase some hypertension drug so that the biomarker of these myocardial fibrosiss and heart failure diastole is normal so can use.The target of this method is continuously measured blood MMP as shown in table 3 and TIMP value, and increases medication so that these are composed in normally with reference to scope.

For being determined the hyperpietic who causes cardiac mass (size) to increase because of hypertension, the appropriateness that the MMP/TIMP spectrum can be used for following the tracks of treatment.Can monitor the particular spectral that these have identified disclosed herein, described MMP/TIMP spectrum can determine that when normal range moves treatment effectively.

The MMP/TIMP spectrum is based on the measurement to single MMP or TIMP.Their amount can be measured by any known method, to provide a kind of acceptable index to indicate their content in the sample of being analyzed.Embodiment has partly provided an example of measuring method.The method of Measurement and analysis thing (for example, MMP or TIMP) amount comprises that analyte does not exist or analyte can not detect the time to the measurement of amount of analyte.Be used to constitute the MMP on this method basis and measuring technology and the method for TIMP measured based on high-sensitivity immunity.Have multiple in these immunoassay by this development in laboratory (that is, TIMP-4 measures method of masurement).

Can be with the method for immunity stdn so that the observed value shown in the table 1 to be provided, (ELISA) implements this method of immunity with enzyme-linked immunoassay.Yet other sensitiveer MMP fast and TIMP blood levels measuring method have been implemented in this laboratory, and they comprise a kind of multiple assay of use system.In this example, can use based on the multiple sandwich immunoassay of bead and measure multiple analytes in the sample (for example blood plasma or other biological sample) of limited bulk.This new technology that is used for multiple analysis is based on the sensitivity of ELISA and fluidic cell is detected on the technology that combines, thus feasible can the measurement specifically less than reaching most 100 kinds different analytes in the single sample of 50 μ l.The feasible multiple MMP and the TIMP that can measure in a small amount of blood sample of this method.These class methods can be used for diagnosis described herein, prognosis, prediction and treatment monitoring and use.Particularly, be while Measurement and analysis substrate concentration, microballon is hatched with sample (being blood sample), and make described microballon and described specific purpose analyte (being MMP) form complex body.Then, in mixture, add the detection antibody (biotinylated) of specificity, and its bead with compound analyte is combined at second epi-position on each analyte.Then, this mixture is hatched with fluorescent reporter molecule (streptavidin-phycoerythrin), and make whole sample by two laser streaming cell detection instrument.Laser detection is used for determining the definite fluorescence of the microballon of specific analyte to be detected, the fluorescence volume of another laser detection reporter molecules, and this amount is directly proportional with the amount of institute's bonded analyte.This method has been used to other analytes that multiple MMP and CHF process may relate to, shown in Figure 16 and table 1.How this just uses the minute quantity blood sample to measure an example of single or multiple analytes.Other examples of the measurement relevant with the MMP/TIMP analyte that has implemented in this laboratory comprise radioimmunoassay and immunoblotting mensuration.These methods also are based on antibody.

13. combination

Method disclosed herein can further comprise other marks that detect heart failure.For example, method disclosed herein can further comprise the NT-proBNP level of measuring in described experimenter's tissue or the body fluid, and described level and reference point are compared.Method disclosed herein can further comprise the level of measuring the troponin-i in described experimenter's tissue or the body fluid, and described level and reference point are compared.

14. measurement opportunity

As mentioned below and other embodiment that are used to screen and treat monitoring are illustrated, measurement opportunity should be in conjunction with specific background.For screening, can carry out whenever carrying out of medical inspection the experimenter.The example can comprise annual physical examination, health care exhibition and by the screening of apparatus for household use.Therefore, disclosed diagnostic method can be used for that diagnostics table reveals CHF sign and symptom but experimenter that the basic reason of this performance is difficult to determine.

There are at least three initial time points that are used for the MMP/TIMP work spectrum of method disclosed herein.Initial measurement can have definite hypertension history and often visit among the patient of outpatient service and carry out.Initial measurement can carry out in the time may causing the health care exhibition of outpatient service visit.Initial measurement can carry out in the patient of the symptom due to showing hypertensive heart disease.Fig. 9 A-C shows in every kind of situation sampling and the diagnosis scheme at each situation.

Therefore, disclosed method of prognosis can be used for identifying whether show the experimenter that hypertension (hyperpiesia) is arranged has LVH or have the risk that DHF takes place.Disclosed method of prognosis can be used for also identifying whether the experimenter who shows CHF sign and symptom has LVH and have the risk that heart failure diastole (DHF) takes place.For example, described method can be used for telling discomfort and the patient who should discomfort conforms to CHF Xiang the doctor.Like this, the doctor can use blood testing to determine whether to exist the MMP/TIMP spectrum that conforms to DHF with LVH.Can instruct the doctor to formulate further diagnostic test and treatment plan thus.

The example on the opportunity of another blood sample collection is to be identified when suffering from definite LVH the patient, can be with the instrument of MMP/TIMP continuous monitoring spectrum as prediction DHF process.These tests can only be used once in any given experimenter as screening implement, and perhaps continuous application repeatedly.

C. test kit

Test kit disclosed herein relates to the reagent that can be used for implementing method disclosed herein.Described test kit can comprise any reagent as herein described or combination of agents, perhaps be considered to implement method disclosed herein required or to its useful reagent or combination of agents.For example, disclose a kind of test kit that the DHF risk takes place the experimenter that is used to assess, component wherein comprises previously described component.For example, the component of MMP/TIMP test kit can comprise relevant purpose MMP and/or TIMP (referring to the tabulation of relevant MMP and TIMP in the table 3) and the compound necessary reagent of a kind of detection reagent.In the example of method of immunity, blood sample can be hatched with the fluorescently-labeled antibody at specific MMP or TIMP, and after washing and non-specific binding removing step, calculate and described purpose MMP or TIMP bonded antibody amount by measuring the fluorescence relative intensity.It can be a kind of test kit of screening of very simply can be used for, or the system of the multiple MMP/TIMP in a kind of more complicated single sample of measurement.Preamble, describe to the ultimate principle of the multi-level method of measuring multiple MMP/TIMP simultaneously from measuring a kind of purpose MMP or TIMP.For screening assay (being MMP-13), a small amount of blood sample can being processed into blood plasma (centrifugal), and this blood plasma is mixed with anti-MMP-13 antibody.The mixture recentrifuge can be read the antibody of specificity in conjunction with MMP-13 by system of fluorescence analysis.This equipment and measuring system can easily be made small suitcase or desktop system.The reading of said system can indicate MMP-13 whether to be below or above (as defined in the preamble) particular measurement threshold.

D. embodiment

1. embodiment 1: the tissue depressant of matrix metalloproteinase/metalloprotease: the relation between the change of the proteolysis factor of matrix components and structure, function and the clinical manifestation of hypertensive heart disease

Method and result's summary: 103 experimenters are divided into following 4 groups: the reference subject (CTL) that a) does not demonstrate cardiovascular disorder; B) hypertension (HTN), blood pressure have been controlled and have not been had a LV hypertrophy; C) hypertension, blood pressure have been controlled and LV hypertrophy (HTN﹠amp have been arranged; But do not have CHF LVH); D) hypertension, blood pressure is controlled, LVH and CHF (HTN﹠amp; LVH﹠amp; CHF), from above-mentioned experimenter, obtained blood plasma MMP-2, MMP-9, MMP-13, TIMP-1 and TIMP-2, and doppler's echo myocardiogram.Compare with CTL, HTN patient's all MMP or TIMP all do not have obvious change.HTN﹠amp; LVH patient's MMP-9 and MMP-13 reduce, and MMP-9 raises.Has only HTN﹠amp; LVH﹠amp; CHF patient's TIMP-1 raises.TIMP-1〉indicated CHF during 1200ng/mL.

Conclusion: the normal hyperpietic of LV 26S Proteasome Structure and Function has normal MMP/TIMP spectrum.The change of MMP spectrum of tending to reduce the ECM degraded is relevant with diastolic dysfunction with the LV hypertrophy.TIMP-1 raises and to have indicated and have CHF.These data show that the variation of MMP/TIMP equilibrated has vital role in structure, function and the clinical manifestation of hypertensive heart disease,

Method

Experimenter: recruited two groups of experimenters in this research: reference contrast and LVH patient.The health care exhibition of subsidizing by the locality and determine the reference contrast from the employee's of South Carolina medical university volunteer.In the reference contrast of being screened, 35% is selected, and 50% meets in the hereinafter listed exclusion standard, and 15% grants participation.By ultrasonic cardiogram research identification LVH patient.In the patient's ultrasonic cardiogram that is screened, 10% is selected, and 75% meets in the hereinafter listed exclusion standard, and 15% grants participation.Two groups have some total exclusion standards:

1) myocardial infarction medical history, 2) regional ventricular wall motion is unusual, 3) coronary reconstruction operation, 4) amyloidosis, sarcoidosis, HIV, hypertrophic obstructive cardiomyopathy, valvular heart disease, 5) ejection fraction＜50%, 6) malignant tumour, 7) serious kidney or hepatic insufficiency, 8) rheumatism, 9) blood pressure〉140/90mmHg.

103 these researchs of subject enrollment are arranged: 53 references contrast experimenter and 50 demonstrate LVH the experimenter [the LV chamber wall thickness〉1.2cm and/or LV quality index 〉=125gm/m ²(table 4)].Be subdivided into two groups based on whether existing hypertension that reference is contrasted the experimenter; The no hypertension history of 39 contrast experimenters (being called " do not have hypertensive reference contrast "), do not demonstrate cardiovascular (CV) disease, no cardiovascular disorder Sx, be not in the mood for vascular drug delivery and the measurement of all ultrasonic cardiograms all in normal range (table 5); 14 patients (being called " hypertensive reference contrast is arranged ") have arterial hypertension medical history, blood pressure to control (through pharmacological agent to reach the JNC7 standard, promptly＜140/90mmHg), no left ventricular hypertrophy (Chobanian AV, et al.2003) and all ultrasonic cardiograms are measured all in normal range (table 5).

Table 4: the demography data of left ventricle structure/function and MMP/TIMP

Abbreviation: data are represented with mean value+SEM, the LV=left ventricle, the patient of LVH=hypertensive cerebral left ventricular hypertrophy, reference contrast=does not demonstrate the experimenter of cardiovascular disorder, the IVRT=isovolumic relaxation time, PCWP=pulmonary capillary wedge pressure, MMP=matrix metalloproteinase, the TIMP=MMP tissue depressant, *=compare p＜0.05 with the reference contrast.

Table 5: hypertension and no hypertensive reference contrast are arranged, the LVH of CHF and no CHF is arranged

Abbreviation: data are represented with mean value+SEM, the LV=left ventricle, the PCWP=pulmonary capillary wedge pressure, the EDV=end diastolic volume, the effective artery rebound degree of Ea=, MMP=matrix metalloproteinase, TIMP=MMP tissue depressant, the patient of LVH=hypertensive cerebral left ventricular hypertrophy, the CHF=morbus cardiacus.With the significant difference between all 4 groups of ANOVA and the check analyses of Tukey multiple comparisons, *=compare p＜0.05 with no hypertensive reference contrast, #=compares p＜0.05, Δ=compare p＜0.05 with the LVH of no CHF with hypertensive reference contrast is arranged.

Based on whether existing CHF that LVH patient is subdivided into two groups.23 blood pressures are controlled and LVF is arranged but the hyperpietic that do not have a CHF is called as " LVH of no CHF " (table 5).Second subgroup is controlled and be made up of the hyperpietic of LVH and CHF by 26 blood pressures, is called as " LVH that CHF is arranged ".All these patients demonstrate and meet Framingham standard (Levy D, et al.1996) Ding Yi CHF, demonstrate diastole unusual (E ' reduce), hardness increases (PCWP increases and PCWP/EDV ratio increases), and 6 minutes current distance reduces significantly that (compare with 1839 ± 60 feet of the LVH group of no CHF, having the LVH of CHF to organize is 979 ± 86 feet, p＜0.05), EF〉50%, therefore, there is heart failure diastole.

By patient's doctor in charge but not the investigator selects and detection is used for the treatment of hypertensive medicine.They comprise hydragog(ue), RAA antagonist (angiotensin-convertion enzyme inhibitor, angiotensin-ii receptor blockers and aldosterone blocker), direct vasodilator (nitrate, hydralazine), alpha-1 adrenergic blocker, central nervous system blocker, acetylsalicylic acid, Beta-3 adrenergic blocker and calcium channel blocker.The average duration of antihypertensive therapy is 6.4 ± 1.5 years.

The echocardiography method

Obtain ultrasonic cardiogram with Sonos 5500 systems that the S-4MHz transverter is housed.Use American Society of Echocardiography's standard to measure (Sahn DJ, et al.1978; Schiller NB, et al.1989).Calculate LV and left atrium volume with disk method (Schiller NB, et al.19).Formula (Devereux RB, et al.1986) with Reichek and Devereux calculates the LV quality.Mitral valve inflow E and A velocity of wave, E/A ratio, E ripple deceleration time and isovolumic relaxation time (IVRT) are carried out Doppler measurement.Mitral valve E ' and A ' velocity of wave are carried out tissue Doppler (side annulus of mitral valve) measurement.Calculate pulmonary capillary wedge pressure (PCWP) with formula 2+1/3E/E ' (Nagueh SF, et al.1998).Use formula: end systolic pressure/whenever the amount of fighting is calculated effective artery rebound degree (Ea).

MMP/TIMP blood plasma is measured: put enzyme-linked immunosorbent assay (ELISA) test kit (Amersham Pharmacia Biotech with dibit, Buckinghamshire UK) detects gelatinase (MMP-2 and MMP-9), collagenase (MMP-13) and MMP tissue depressant (TIMP-1 and TIMP-2).The pre-bag that blood plasma and each MMP standard substance are added the antibody that contains purpose MMP or TIMP is by in the hole, and washing.At 450nm wavelength place to gained react reading (Labsystems Multiskan MCC/340, Helsinki, Finland).Because MMP-13 exists level extremely low in blood plasma, with MMP-13 be divided into detectable and detect less than.

Statistical analysis: in 6 hour time period, measured MMP and TIMP in per 2 hours, calculate between the individual subjects of reference contrast experimenter's subgroup (n=20) and the variation coefficient of the MMP/TIMP observed value in the individual subjects to utilize single factor random effect ANOVA (random effects ANOVA).Square root by individual square error multiply by the described coefficient of 100 calculating.Patient's within variance coefficient=11.2 ± 1.1% of MMP-2, TIMP-1=8.5 ± 2.2%, TIMP-2=14.3 ± 1.7%.The within variance coefficient of determining the amount of variability of described determination techniques own and measuring for all MMP and TIMP all less than 6%.

At first, compare reference contrast and LVH experimenter with bilateral Studentt check.Between all four groups, (there are hypertensive reference contrast, no hypertensive reference to contrast, have the LVH of CHF and the LVH of no CHF) with ANOVA and the check of Tukery multiple comparisons then and analyze comparison.It is significant that p value＜0.05 just is considered to.Use the relation between simple linear regression investigation MMP and TIMP level and the LV 26S Proteasome Structure and Function.With Mantel Hanzel χ ²Relation between the existence of check and experimenter's working curve evaluation MMP-13 and TIMP-1 level and LVH and CHF.At first with single argument, use multivariate regression analysis then, investigate the potential impact of medicine to structure, function or blood plasma data.Structure, function, MMP or TIMP observed value are dependent variables, simultaneously with the medicine of input as pseudo-variable.Investigate single medicine earlier, investigate drug regimen again.

The research approach that this research is used is by audit committee of the mechanism audit approval of South Carolina medical university.Obtained all participators' written informed consent.Complete these data of acquisition of author are also responsible to its integrity.All authors have read and have agreed the original original text of writing.

The result

Reference contrast and LVH

Structure/performance data: reference contrast experimenter's age and sex distribution similar to LVH experimenter (table 3 and 4).Compare with reference contrast, the systolic pressure of LVH is higher, has the obvious proper alignment reconstruct that shows as LV quality index high 60%, end diastolic volume indifference, and the ratio low 40% of LV end diastolic volume and quality.Compare with the reference contrast, LV diastole diastole exponential sum LV hardness number diastole of LVH is all obviously unusual: IVRT increase, increase deceleration time of E ripple, E ' reduce, pulmonary capillary wedge pressure increases, and with reference contrast (0.09 ± 0.01mmHg/mL) compares, the ratio of PCWP and LV end diastolic volume increases (0.16 ± 0.01mmHg/mL among the LVH, p＜0.05), shown that LV action property of the instantaneous diastole end of term stiff (instantaneous end diastolicoperating stiffness) increases.

MMP and TIMP blood plasma spectrum: compare with the reference contrast, MMP-2 reduces among the LVH, and MMP-9 raises.There is significant difference (Fig. 1) in the detectability of MMP-13.But the contrast of 47% reference has the MMP-13 of detection level, and only can detect MMP-13 (χ in 15% LVH experimenter ²=17.89, p＜0.001, odds ratio=0.24).Compare with the reference contrast, the blood plasma TIMP-1 among the LVH significantly raises.TIMP-2 and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratio and the indifference of reference contrast and LVH.

Do not have hypertensive reference contrast and hypertensive reference contrast is arranged

Structure/performance data: will not have hypertensive reference contrast experimenter and be used for the LVH that hypertensive reference contrast, no CHF are arranged and have the LVH of CHF to organize comparing as the reference control group of age and gender matched.Do not have the contrast of hypertensive reference and have between hypertensive reference contrasts, any demography parameter of LV structure or function or any ultrasonic cardiogram observed value all do not have significant difference (table 3 and 4).Do not have the left room maximum volume (LAMV) and the emptying mark (LAEF) (LAMV=40 ± 2ml, LAEF=42 ± 3%) of hypertensive reference contrast and have hypertensive reference contrast (LAMV=42 ± 4ml, LAEF=43 ± 2%) similar.

MMP and TIMP blood plasma spectrum: do not have hypertensive reference contrast experimenter and any MMP or TIMP blood plasma level there are no significant the difference of hypertensive reference between contrasting is arranged.

The LVH of no CHF and the LVH that CHF is arranged

Structure/performance data: the LVH of no CHF and have systolic pressure, LV volume or quality between the LVH experimenter of CHF all not to have significant difference (table 5).But compare with the LVH of no CHF, have the damaged degree of function diastole of LVH of CHF obviously more serious.Compare with the LVH of no CHF, have the diastole diastole index of LVH of CHF slower, diastole hardness number higher and the filling pressure index is higher.Particularly, with no hypertensive reference contrast (10 ± 0.4cm/s, 95%CI=9.3,11) and hypertensive reference contrast (9.8 ± 0.5cm/s arranged, 95%CI=8.1,11) compare, the LVH patient's of no CHF tissue Doppler E ' reduces (8.4 ± 0.4cm/s, 95% fiducial interval (CI) is 7.4,9.3).There is the E ' among the LVH of CHF further to reduce (7.2 ± 0.5cm/s, 95%CI=6.2,8.3).With no hypertensive reference contrast (10 ± 1mmHg, 95%CI=9.3,10.6) and have hypertensive reference contrast (11 ± 1mmHg, 95%CI=9.1,12.2) to compare, the LVH patient's of no CHF PCWP does not change (13 ± 2mmHg, 95%CI=10.5,15.2), but in being arranged, the LVH of CHF increases (17 ± 2mmHg, 95%CI=15.2,17.7).In the LVH patient of no CHF, PCWP is constant with the ratio of LV end diastolic volume, but this ratio significantly raises in the LVH patient of CHF is arranged.Effectively the artery rebound degree raises in the LVH of no CHF, reduces in the LVH of CHF is arranged.LAMV raises in the LVH of no CHF (LAMV=53 ± 4ml compares p＜0.05 with the reference contrast), further raises in the LVH of CHF is arranged (LAMV=70 ± 5ml compares p＜0.05 with the LVH of no CHF).LAEF constant in the LVH of CHF is arranged (LAEF=42 ± 3% is compared p＜0.05 with the reference contrast), but in the LVH of CHF is arranged, increase (LAEF=48 ± 2% is compared with the LVH of no CHF).

MMP and TIMP blood plasma spectrum: compare with the LVH that CHF is arranged, the MMP-2 of the LVH of no CHF, MMP-9, MMP-13, TIMP-2 or MMP/TIMP ratio do not have significant difference (Fig. 1).Yet, compare (1092 ± 77ng/ml, 95%CI=933,1252) with the LVH of no CHF, there is the TIMP-1 of the LVH of CHF significantly to increase (1364 ± 86ng/ml, 95%CI=1185,1543).In fact, TIMP-1 only increases in the experimenter of CHF is arranged.Contrast (1000 ± 42ng/ml, 95%CI=915,1085) and have hypertensive reference contrast (988 ± 76ng/ml, 95%CI=824,1152) to compare with no hypertensive reference, the LVH patient's of no CHF TIMP-1 does not change.

Relation between MMP and TIMP blood plasma spectrum and the LV 26S Proteasome Structure and Function: have obvious relation between persistence between TIMP-1 and the LV reconstruct degree.When TIMP-1 increases, and the increase of LV quality (r=0.30, p=0.005), volume/quality ratio decline (r=-0.56, p=0.001, Fig. 2 A).There is obvious relation between persistence between TIMP-1 and the diastolic dysfunction degree.When TIMP-1 increases, mitral valve E/A ratio descends (r=-0.22, p＜O.027), E ' descends (r=-0.62, p=0.001, Fig. 2 B) and PCWP increase (r=0.28, p=0.013).At last, there is obvious relation between persistence between CHF degree and the TIMP-1 level.NYHA III class has the LVH experimenter's of CHF TIMP-1 mean value to have the LVH experimenter of CHF higher than NYHA II class.The TIMP-1 level〉during 1200ng/ml, be shown the LVH (χ of CHF in advance ²=4.6, p=0.03, specificity=88% and positive predictive value=94%, odds ratio=3.54,95% fiducial intervals=1.08,11.50).Experimenter's performance curve (ROC) area down is 0.71.

Between the use of certain drug and each group between the difference of LV structure, function or blood plasma MMP/TIMP spectrum and onrelevant.Particularly, with any MMP or the horizontal indifference of TIMP between the patient of any medicine or drug regimen grouping.Yet, it should be understood that this research is not sufficient to thoroughly understand fully the influence of medicine to LV structure, function or blood plasma MMP/TIMP spectrum.Therefore, answer Due Diligence ground to explain these data and analysis.

Discuss

This research has the discovery of three uniquenesses: 1) the normal hyperpietic of LV 26S Proteasome Structure and Function has normal MMP/TIMP spectrum, 2) (MMP-2, MMP-13 reduce to tend to reduce the ECM degraded, TIMP-1 increases) MMP and the change of TIMP spectrum with LV is loose and diastolic dysfunction is relevant and 3) the TIMP-1 increase indicated and had CHF.

Though substrate and the effect of MMP and TIMP have pleiotropy, the change of myocardium MMP and TIMP has predictable influence (Spinale, FG.2002 to ECM; Chapman RE, et al.2004).For example, MMP-2 (a kind of gelatinase) degradable basilar membrane albumen, fiber collagen peptide and new synthetic collegen filament.In this research, the MMP-2 among the hypertensive cerebral LVH patient significantly reduces.MMP-9 (a kind of gelatinase) has the identical protein substrate of structure with MMP-2, but the MMP-9 activity level is much lower.Yet, but MMP-9 remarkably influenced important biological albumen/peptide (for example TGF-) and other " short fibrosis " albumen and approach.Can expect that activating short fiber path by the MMP-9 that increases can increase the ECM accumulation.Therefore, reduction of LVH patient's MMP2 level and the rising of MMP-9 level may be the promotion factors that viewed 26S Proteasome Structure and Function changes in hypertensive heart disease in this research.

MMP-13 be a kind of in blood plasma the extremely low collagenase of level, even also be difficult to its accurate quantification with highly sensitive assay method.Therefore, do not report MMP-13 in this research, but the result is divided into two classes with quantitative values.Detectable MMP-13 level in LVH patient's blood plasma significantly descends, and in the patient who suffers from CHF and LVH, this level further descends.The minimizing of this collagenase is estimated to cause, and the turnover of fiber collagen reduces, degraded reduces and the ECM cumulative rises.

Can not only comprise transcriptional regulatory in a plurality of horizontal adjusted MMP activity, also comprise posttranslational modification, for example TIMP combination.TIMP combines with the relation of 1:1 with active MMP, suppresses active important control point (Spinale, the FG.2002 who also constitutes clean ECM proteolytic activity thus of mmp enzyme; Chapman RE, et al.2004; Brew K, et al.2000).Originally studies show that the TIMP-1 blood plasma level with patient of LVH and CHF raises.So the balance between direction change MMP that helps the ECM proteolytic activity to reduce and TIMP has promoted the ECM accumulation thus.Four kinds of known TIMP are arranged, the transcriptional regulatory of these molecules and inconsistent (Brew K, et al.2000).Level inconsistent (Wilson EM, the et al.2002 of each TIMP in animal model in heart failure and cardiomyopathy patient, have been observed; Stroud RE.2005).In this research, observe the LVH patient's of CHF stable rising of TIMP-1.By contrast, CHF is being arranged or do not having a small amount of increase of all only observing TIMP-2 among the LVH patient of CHF.These observed results may have been given prominence to difference in functionality and the adjusting approach of each TIMP in the LV restructuring procedure.A unique discovery of this research is that a kind of TIMP of particular type is that the generation of TIMP-1 and CHF is closely related.For the patient who suffers from CHF and LVH, whether not clear TIMP-1 increases the generation that whether promotes CHF or is the result that CHF takes place.Yet clear, TMMP-1 raises in the patient that LVH and CHF are arranged only, blood plasma TIMP-1 numerical value〉1200ng/ml indicated and had CHF.Therefore, should be in the Case definition of exploitation ejection fraction normal in heart failure (heart failure diastole), and in being designed for the new treatment processing scheme of heart failure diastole, consider this plasma analysis thing.Yet, it should be understood that this cut off value of TIMP-1=1200ng/ml is selected with empirical data (post-poc), rather than selected in the prediction mode.Therefore, the validity of this predictor is made an explanation with answering Due Diligence, and use the research of large-scale prospective series research and design to confirm with other.

The change of the MMP/TIMP that takes place among the hypertensive heart disease patient can influence the growth regulating of extracellular and myocardial cell's compartment, thereby causes the loose and collagen content increase of proper alignment LV together.The collagen stable state is to be determined by the balance between synthetic, posttranslational modification and degraded.For hypertensive heart disease, people such as Diez have proved that with other people collagen content raises to be increased with collagen synthetic plasma markers thing, collagen degradation reduces and collagen turnover minimizing relevant (Diez J, et al.2002; Lopez B, etal.2001a; Lopez B, et al.2001b).The change of the MMP/TIMP spectrum of finding in this research discloses and may change potential mechanism synthetic, that degrade and have enough to meet the need.

Though the LV structural remodeling has many determinatives, blood pressure is wherein of paramount importance a kind of factor.Yet, the data of this research show, even blood pressure is fully controlled, the change of the persistence of MMP and TIMP has indicated, may determine that secular proper alignment reconstruct, LVH and diastole are in heart failure and it is in heart failure relevant with secular proper alignment reconstruct, LVH and diastole to determine.Disappearing of LVH needs suitable ECM reconstruct, comprises the degraded of ECM composition (particularly basilar membrane albumen) and the change of turnover and myocardial cell-matrix interphase interaction.Originally studies show that, (MMP-2 reduces for hypertensive cerebral LVH patient's specific MMP (MMP-2 minimizing) and TIMP (TIMP-1 increase) spectrum persistent anomaly, TIMP-1 increases), estimate that this can be beneficial to the lasting connection of myocardial cell-basilar membrane-matrix, and be unfavorable for for making the LV quality recover required ECM turnover.Therefore, seem that the persistence of studying observed MMP and TIMP at this changes the phenotype and the structural modification that may promote to be present in the hypertensive heart disease.

The representativeness sign that this research changes as the myocardial level of above-mentioned enzyme of reflection and peptide with the blood plasma level of MMP and TIMP.MMP activates and the TIMP combination is to occur in compartmentation process (Spinale, the FG.2002 in the matter between cardiac muscle; Chapman RE, et al.2004).Therefore, blood plasma level must not reflect the clean ECM proteolytic activity in the present cardiac muscle.Difference (Joffs C, the et al.2001 of MMP in this research between observed reference contrast and hypertensive heart disease patient and the possibility of the difference between TIMP blood plasma level reflecting myocardium level; Yarbrough WM, et al.2003; Lindsey ML, et al.2003).Cardiac muscle may not be unique source of MMP and TIMP among the LVH patient.Therefore, the observed value of blood plasma MMP and TIMP level has been represented from heart and the MMP of non-heart source release and the summation of TIMP.Yet the specific exclusion standard that uses in this research helps to eliminate the main non-myocardium considerable change of originating of MMP and TIMP.Yet, need recognize, exist or do not exist variation in other non-heart tissues (for example kidney and vascular system) of the LVH hyperpietic of chronic heart failure to have the MMP of helping and TIMP discharges to blood plasma.The discovery of this research has proved the difference of blood plasma MMP and TIMP level between reference contrast and the LVH patient.

Conclusion: the specific change pattern of ECM proteolysis system is relevant with every kind of structure, function and/or the clinical manifestation of hypertensive heart disease.Blood pressure is fully controlled and the experimenter's of left ventricle non-structure or changing function MMP/TIMP index does not have any variation.Even but LVH patient's blood pressure is fully controlled and is also shown MMP-2 and MMP-13 and reduce.The TIMP-1 increase appears among the patient who suffers from LVH and CHF.Particularly, prediction that the transformation between the generation of hypertensive cerebral LVH and CHF can be by the variation of MMP and TIMP---TIMP-1 for example〉1200ng/ml or do not have MMP-13---.But sample size is limited in this research, employing be transverse design, and do not carry out long-term series of studies.These limitation make that need utilize large-scale prospective series research to relate to further checks and conclusive evidence our observations.Yet, the digital proof of this research, observed MMP/TIMP randomly changing has vital role in the hypertensive heart disease performance.Help to improve diagnosis and treatment for the understanding of this ECM dependency physiopathology to the hypertensive heart disease patient.

Clinical argument: chronic arterial hypertension is the common cause that causes LV proper alignment hypertrophy, relaxation rate reduction and hardness to increase.The change of the 26S Proteasome Structure and Function of caused by hypertension is because the main component of cardiac muscle---myocardial cell's change and particularly causing owing to the change of extracellular matrix (ECM).These LV 26S Proteasome Structure and Functions changes have produced the necessary substrate of generation heart failure diastole (DHF).Yet these variations of not clear ECM are subjected to which kind of controlling factors, simple controlling blood pressure could prevent or reverse these and change, also unclear diagnosis or the treatment that whether understanding of ECM controlling mechanism is helped hypertensive heart disease.Originally studies have shown that the change of specific ECM proteolysis albumen/peptide (MMP and TIMP) pattern is relevant with every kind of structure, function and the clinical manifestation of hypertensive heart disease.Blood pressure is fully controlled and the MMP/TIMP index of not having an experimenter of LV structure or changing function does not have any change.Therefore, can prevent the change of ECM and ECM proteolysis system to hypertensive treatment.Yet, fully being controlled although have the patient's of remaining LVH or resistance LVH blood pressure, their MMP is still unusual.TIMP-1〉increase of 1200ng/ml indicated the generation of DHF.These data show, LVH disappear and the change of blood pressure is not only depended in the prevention of DHF merely, may need to find accurately MMP/TIMP and change and should change normalizing.Help to improve diagnosis and treatment for the understanding of this ECM dependency physiopathology to the hyperpietic.

2. embodiment 2: matrix metalloproteinase/tissue inhibitor of metalloproteinase: the relation between the change of the proteolysis factor of matrix components and structure, function and the clinical manifestation of hypertensive heart disease

Method

The research access: table 6 shows this research access situation.Exclusion standard is that myocardial infarction medical history, myocardosis, valve or ventricular wall motion are unusual, irregular pulse, wetting property heart trouble, EF＜50%, unsteered hypertension (SBP〉140 or DBP 90) or influence the systemic disease that MMP/TIMP blood plasma is composed.The permit standard of contrast and HTN contrast is age of not demonstrating structural cardiovascular disorder at the masculinity and femininity in 18-90 year.LVH and have the permit standard of the LVH of CHF be LV hypertrophy that the ultrasonic cardiogram conclusive evidence is arranged (chamber wall thickness〉1.2cm or LV quality index 125g/m ²), the age is at the 18-90 masculinity and femininity in year.

Table 6: research access

Ultrasonic cardiogram is measured: use dimension ultrasonic cardiogram standard.

Ultrasonic cardiogram calculates: calculate the LV volume with the disk method.Calculate the LV quality with the Penn method.PCWP is by 2+1.3 * (E/Ea) calculating.

MMP/TIMP blood plasma is measured: obtain gelatinase MMP-2 and the TIMP-1 of MMP-9, collagenase MMP-13 and TIMP class and the blood plasma observed value of TIMP-2 by enzyme-linked immunosorbent assay (ELISA) (AmmershamPharmacia Biotech).

The result

Fig. 7-11 shows result of study.

Conclusion

The normal HTN patient of LV 26S Proteasome Structure and Function has normal MMP/TIMP spectrum.The change of MMP/TIMP spectrum of tending to reduce the ECM degraded is relevant with diastolic dysfunction with the LV hypertrophy.The increase of TIMP-1 has indicated the existence of CHF.Can use the variation of the determination of plasma method measurement myocardial cell epimatrix proteolysis system of selected MMP and TIMP.Every kind of performance of the hypertensive heart disease all specific change pattern with ECM proteolysis system is relevant.Feature with hyperpietic of structural remodeling, diastolic dysfunction and/or clinical CHF is that MMP reduces and TIMP increases.

3. embodiment 4: the standard of distinguishing, predict and diagnose hyperpietic's heart failure

Provide in described the range of age and striden the human experimenter's of sex the clearly tabulation of normal value in the table 7.Do not provide as the writing tabulation of the MMP/TIMP normal reference value that the present invention comprised is former, and owing to comprise the experimenter of the age-matched of no cardiovascular disorder, table 7 also provides normal reference range.And, the new stoichiometry ratio that provides MMP/TIMP to compose, provable this spectrum has contained important diagnostic and prognosis information that following table describes in detail.Collecting these data from more than 100 experimenters is also analyzed.

Table 7: normal human subject * reference range

* normal adult age 25-70 year

Table 8 has provided and has been diagnosed with hypertension but the well controlled patient's of blood pressure MMP and TIMP absolute value, MMP/TIMP ratio absolute value and with respect to the variation per-cent based on the normal reference value of absolute value.Described in original application, collect these numerical value.Confirmed a kind of blood plasma spectrum of uniqueness, this spectrum can't be predicted from the past zooscopy report or previously disclosed limited clinical study and draw.This uniqueness spectrum comprises that MMP-2 decline, MMP-9 no change, MMP-13 detect less than (being lower than the sensitivity of the mensuration system of any present use), and the TIMP-1 horizontal stable rises.And provable cardiovascular specific marker thing TIMP-4 increases.These variations of MMP and TIMP spectrum are that the hyperpietic is distinctive, and have confirmed the early stage change that takes place in these patients' heart tissue.This uniqueness, special spectrum can be used for instructing treatment, reducing to minimum with respect to normal subjects's MMP and the variation of TIMP spectrum.And these blood plasma spectrums can be used for the patient colony on the line of screening comprehensively, and identify the patient of the risk with following generation adverse events.

Table 8: the diagnosis of hypertensive heart disease

* be diagnosed with the suitably patient of medical treatment of hypertension and warp

Table 9 shows the blood plasma spectrum of the MMP that occurs among the patient who suffers from the heart failure of hypertensive heart disease secondary and TIMP.These data are provided by the research that provides in the original application.This previously studies show that, can judge whether the hyperpietic exists heart failure by the disappearance of MMP-13 signal, the stable increase of TIMP-1.In fact, before be provided for predicting experimenter's performance curve (ROC) with diagnosis of heart failure.Significantly different with the patient who suffers from the heart failure of myocardial infarction (heart attack) secondary is that the MMP-9 level is normal or be lower than normal.Can be distinguished these two kinds of morbid states, be provided this differentiation result in the following table.And, by using cardiovascular specific marker thing TIMP-4, proved that hypertensive heart disease patient's TIMP-4 increases, and for providing cardiovascular specificity from unproved blood plasma spectrum before described.These data provide the first discriminating spectrum that is used for having by plasma markers thing identification the patient of the heart failure due to the hypertensive heart disease.This is an important problem, because according to the basic cause of disease difference of heart failure, therapeutic modality can be variant.Provide in the original application about how and these data to be used in reference to training is treated and the content of clinical decision.

Table 9: the hyperpietic * that risk of heart failure increases

* be diagnosed with the suitably patient of medical treatment of hypertension and warp

By unique blood plasma feature of developing among the application and in support material, demonstrate, provide the ability of distinguishing the basic cause of disease of heart failure patient first.Particularly, as shown in table 10, have the patient that risk of heart failure takes place, or show the patient of myocardial infarction secondary heart failure, unique and very different blood plasma spectrums has perhaps appearred among the patient of hypertension secondary heart failure.These data are taken from the research on formation the application basis that we finish.Therefore, these spectrums are carried out differential diagnosis, the more important thing is, can consider to have more clinical decision and therapeutic strategy targetedly.The example of the clinical application of this spectrum, and content how to utilize these spectrums to carry out clinical decision provides in original application.

Table 10: the differential diagnosis * that systole (behind the MI) or diastole (hypertensive heart disease) are in heart failure

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Claims (19)

1. a method of predicting experimenter's heart failure diastole comprises the amount of measuring MMP-13 in described experimenter's body fluid, just shows when measuring less than 10ng/mL to have in heart failure or indication heart failure diastole.

2. a method of predicting experimenter's heart failure diastole comprises that the amount that detects TIMP-1 in described experimenter's body fluid is higher than normal value.

3. the method for claim 2, wherein the amount of MMP-2 is higher at least by about 20% than normal value.

4. a method of predicting experimenter's heart failure diastole comprises that the amount that detects TIMP-4 in described experimenter's body fluid is higher than normal value.

5. the method for claim 4, the amount of wherein said TIMP-4 is higher at least by about 50% than normal value.

6. method of predicting experimenter's heart failure diastole comprises the amount of measuring MMP-13, TIMP-1 and TIMP-4 in described experimenter's body fluid.

7. the method for claim 6, the amount of wherein said MMP-13 detect less than, the amount of described TIMP-1 high at least more about 50% than normal value, and the amount of TIMP-4 is higher at least by about 50% than normal value.

8. a method of predicting experimenter's heart failure diastole comprises the amount of measuring MMP-13, TIMP-1, TIMP-2 and TIMP-4 in described experimenter's body fluid.

9. the method for claim 8, the amount of wherein said MMP-13 detect less than, the amount of described TIMP-1 high at least more about 50% than normal value, and the amount of described TIMP-2 is high at least more about 50% than normal value, and the amount of TIMP-4 is higher at least by about 50% than normal value.

10. a method of predicting experimenter's heart failure diastole comprises the amount of measuring MMP-13, TIMP-1, TIMP-4 and MMP-2 in described experimenter's body fluid.

11. it is high at least more about 50% than normal value that the method for claim 10, the amount of wherein said MMP-13 detect less than, the amount of described TIMP-1, the amount of described TIMP-4 is high at least more about 50% than normal value, and the amount of MMP-2 is higher at least by about 20% than normal value.

12. a method of predicting experimenter's heart failure diastole comprises that to detect in described experimenter's body fluid the normal ratio of odds ratio of MMP-9 and TIMP-1 low.

13. the method for claim 12, the normal ratio of wherein said odds ratio hang down about 50% at least.

14. a method of predicting experimenter's heart failure diastole comprises that to detect in described experimenter's body fluid the normal ratio of odds ratio of MMP-9 and TIMP-2 low.

15. the method for claim 14, the normal ratio of wherein said odds ratio hang down about 50% at least.

16. a method of predicting experimenter's heart failure diastole comprises that to detect in described experimenter's body fluid the normal ratio of odds ratio of MMP-9 and TIMP-4 low.

17. the method for claim 16, the normal ratio of wherein said odds ratio hang down about 50% at least.

18. each method of claim 1-17, wherein said body fluid is blood.

19. each method of claim 1-17, wherein said body fluid is blood plasma.

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