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

Abstract

Methods for detecting and predicting diastolic heart failure and predicting congestive heart failure comprising protease and protease inhibitor assays are disclosed herein.

Description

Detection of Diastolic Heart Failure Using Protease and Protease Inhibitor Plasma Analysis

Cross References to Related Applications

This application claims the benefit of U.S. Provisional Application No. 60/798,953, filed May 9, 2006, and U.S. Provisional Application No. 60/893,781, filed March 8, 2007, which are hereby incorporated by reference in their entirety Apply.

Statement on Federally Sponsored Research

This application was awarded under Contract No. VA for the Merit Review (Spinale 0001) Research Service of the Department of Veterans Affairs and the National Heart, Lung, and Blood Institute Completed with government funding under contracts PO1-HL48788, RO1-HL-59165 and MO1-RR-01070-251.

Background technique

Despite strides in the availability of hypertensive (high blood pressure) medications and the recognition that high blood pressure is a major risk factor for developing heart failure, the disease remains the leading cardiovascular disease in the United States. A specific problem in identifying patients at risk of developing hypertensive heart failure is the lack of a rapid screening test to identify patients with intrinsic changes in the myocardium secondary to hypertension. Chronic hypertension increases myocardial mass and size, but this phenomenon does not appear until late in the disease process. A unique key event in the disease process of hypertensive heart disease and heart failure is the increased fibrosis of the myocardium itself. The molecular basis for this change is as yet unknown.

Contents of the invention

In accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention relates to a unique MMP/TIMP pattern present in patients developing hypertensive heart failure that is, in fact, predictive of the presence of abnormal cardiac function, whereas Previously it was only possible to detect this presence with expensive and difficult-to-implement tests. This unique MMP/TIMP pattern is used in a method to identify patients at risk of developing hypertensive secondary heart failure and at impending hypertensive secondary heart failure.

Other advantages of the methods and compositions disclosed herein will be set forth in part and partly understood from the description below, or can be learned by practicing the methods and compositions disclosed herein. The advantages of the methods and compositions disclosed herein may be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

Description of drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions.

Figure 1 shows the detection limit of MMP-13 in reference controls with and without hypertension and in LVH with and without chronic heart failure. The detection limit of MMP-13 was significantly lower in LVH patients. *=p<0.05 compared to reference control without hypertension, #=p<0.05 compared to reference control with hypertension, Δ=p<0.05 compared to LVH without CHF.

Figure 2A shows the relationship between tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and left ventricular volume/mass ratio. Higher TIMP-1 levels were associated with lower LV volume/mass ratios, indicating more pronounced concentric remodeling, r=-0.56, p<0.05. Figure 2B shows the relationship between tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and tissue Doppler imaging (TDI) rapid filling wave (E'). Higher TIMP-1 levels were associated with lower E' values, indicating a slowed relaxation rate during LV diastole, r=-0.41, p<0.05.

Figure 3 shows a comparison of the structure and function of a normal heart and a heart with diastolic heart failure.

Figure 4 shows the results of echocardiographic imaging of controls with and without hypertension (HTN) and subjects with ventricular hypertrophy with and without congestive heart failure (CGF), as well as plasma MMP-9, MMP- 2 and TIMP-1 measurement results.

Figure 5 shows tissue Doppler imaging (TDI) rapid filling waves (E') relative to TIMP-1 levels.

Figure 6 shows plasma MMP-13 levels in controls and subjects with left ventricular hypertrophy.

Figure 7 shows the percentage of left ventricular hypertrophy patients with and without congestive heart failure and plasma TIMP-1 levels above or below 1200 ng/ml.

Figure 8 shows the calibration curves of MMP-9, MMP-13, TNF-[alpha] and IL-6 determined by multivariate analysis.

Figure 9 shows an algorithm for determining treatment for hypertensive patients by MMP and TIMP levels. Figure 9A shows a schematic diagram of the treatment of a patient with confirmed hypertension who has regular non-emergency outpatient visits. Figure 9B shows a schematic diagram of the treatment of a patient with a new episode of hypertension and a non-emergency outpatient visit. Figure 9C shows a schematic diagram of the treatment of a patient exhibiting signs or symptoms that may be due to HF.

Detailed ways

The disclosed methods and compositions can be understood more readily by reference to the following detailed description of specific embodiments and examples incorporated herein, and to the drawings and their associated context.

Disclosed are materials, compositions and components useful in, in conjunction with, in the preparation of, and as products of, the disclosed methods and compositions . These and other substances are disclosed herein, and it is to be understood that when combinations, subsets, interactions, groups, etc. and permutations may not be explicitly disclosed, but each is specifically contemplated and described herein. For example, if a peptide is disclosed and discussed, and the many modifications that can be made to many molecules including that peptide are discussed, then unless specifically indicated to the contrary, every and every combination and permutation of the peptide and possible modifications are receive special consideration. Thus, if a class of molecules A, B, and C is disclosed, and a class of molecules D, E, and F, and examples A-D of a combination of molecules are also disclosed, each of the combination molecules is individually and collectively get considered. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F is also specifically contemplated, and each of the above combinations is considered also because of A, B, and C; D, E and F; and the disclosure of example combinations A-D are disclosed. Likewise, any subset or combination of these molecules is also specifically contemplated and disclosed. Thus, for example, subgroups of A-E, B-F, and C-E are also specifically contemplated and considered disclosed for disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Therefore, if there are multiple additional steps that can be performed, it is understood that each of the additional steps can be used in any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is are specifically considered and considered disclosed.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein. The following claims are intended to cover such equivalents.

It is to be understood that the disclosed methods and compositions are not limited to the particular methodology, protocols and reagents described which may vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments only, and is not intended to limit the scope of the invention, which will be defined only by the appended claims.

Unless clearly stated otherwise, no method described herein should be construed as requiring its steps to be performed in any particular order. Thus, when a method claim does not recite the order in which its steps are to be followed, or does not specifically state that the steps are limited to a particular order in either the claims or the specification, no order should be deemed to exist in any respect. This applies to any possible unstated basis for interpretation, including: logical relationships related to steps or operational flow arrangements, obvious semantics derived from grammatical structure or punctuation, and the number and type of embodiments described in the specification. More specifically, the measured amounts of MMPs and TIMPs may have said measured values in any order of magnitude.

A. Definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of the invention, particularly useful methods, devices, and materials are as described. Publications cited herein, as well as the materials for which they are cited, are specifically incorporated by reference into this specification. Nothing herein should be construed as an admission that the present invention is not entitled to priority over such disclosures in the prior art. No admission is made that any reference constitutes prior art. The discussion in the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents.

It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes reference to a plurality of such peptides, reference to "the peptide" refers to one or more peptides and equivalents thereof known to those skilled in the art, etc. .

"Optional" or "optionally" means that the event, circumstance or substance described thereafter may or may not occur or exist, and such description includes the circumstances in which said event, circumstance or substance occurs or exists and said event The absence or absence of a situation or substance.

Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value recited and/or to the other recited particular value. Similarly, when values are expressed as approximations, by the preceding use of the "about," it will be understood that the particular value forms another embodiment. It should be further understood that the endpoints of each range are meaningful both in relation to the other endpoints and independently of the other endpoints. It is also to be understood that there are a number of values disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It should also be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as properly understood by those skilled in the art. For example, if the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It should also be understood that throughout this application, data is presented in a number of different formats, representing endpoints and starting points, and ranges for any combination of these data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it will be understood that greater than, greater than or equal to, less than, less than or equal to, equal to 10 and 15, and values between 10 and 15 are also deemed to have been disclosed . It is also to be understood that every element between the two specified elements is also disclosed. For example, if 10 and 15 are published, then 11, 12, 13 and 14 are also published.

Throughout the description and claims of this application, the word "comprise" and its variants (such as "comprising" and "comprising") mean "including but not limited to" and are not intended to exclude, for example, other additions substance, component, integer or step.

A "subject" includes, but is not limited to, animals, plants, bacteria, viruses, parasites, and any other organism or entity having nucleic acid. The subject may be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent) , fish, birds or reptiles or amphibians. The subject may be an invertebrate, more specifically an arthropod (eg, insects and crustaceans). The term does not indicate a specific age or gender. Accordingly, the term is intended to encompass male or female, adult and newborn subjects as well as fetuses. Patient refers to a subject suffering from a disease or condition. The term "patient" includes human or veterinary subjects.

A "sample" is defined herein to mean any sample obtained from an organism. Examples of biological samples include bodily fluids and tissue specimens. The source of the sample may be a physiological medium such as blood, serum, plasma, breast milk, pus, tissue scrapings, washings, urine, tissue such as lymph nodes, and the like.

Various publications are referenced throughout this application. The disclosures of these publications in their entirety are incorporated into this application by reference in order to more fully describe the state of the art to which this invention pertains. The material discussed in the sentences in which said reference is relied upon in a disclosed reference is also individually and specifically incorporated by reference into this specification.

B. method

1. Heart failure

Congestive heart failure (CHF), also known as congestive heart failure (CCF) or just heart failure, is any structural or functional disorder that impairs the heart's ability to fill or pump an adequate volume of blood throughout the body. Diseases caused by sexually transmitted heart disease. Thus, the disclosed methods can be used to treat any form of heart failure.

The term "heart failure" is preferred over the former term "congestive heart failure" because not all patients have volume overload on initial or subsequent evaluation. Etiologies or contributors to congestive heart failure include the following (and specify left (L) or right (R)): Inherited family history of CHF, ischemic heart disease/myocardial infarction (coronary artery disease), infection , alcohol consumption, heartworm, anemia, thyrotoxicosis (hyperthyroidism), cardiac arrhythmia, hypertension (L), aortic stenosis (L), aortic [valve] stenosis/regurgitation (L), two Minus regurgitation (L), pulmonary stenosis/pulmonary hypertension/pulmonary embolism leading to cor pulmonale (R), and mitral valve disease (L).

Heart failure is classified in a number of different ways, including: which side of the heart is involved (left heart failure versus right heart failure), whether the abnormality is caused by the contraction or relaxation of the heart (systolic heart failure versus diastolic heart failure), Low cardiac output or low systemic vascular resistance (low-volume heart failure and high-volume heart failure).

Congestive heart failure (CHF) is a cluster of signs and symptoms (ie, shortness of breath, fluid accumulation) resulting from an underlying disorder of cardiac performance, particularly left ventricular (LV) function. There are many causes of CHF, which can be divided into three main categories: CHF following a heart attack (myocardial infarction), CHF associated with hypertensive heart disease, and CHF associated with internal muscle disease (commonly known as cardiomyopathy). The difficulty of identifying the underlying etiology of CHF, for example due to hypertensive heart disease, is the focus of the method of the present invention. Specifically, hypertensive heart disease causes LV muscle growth, which is known as hypertrophy. LV hypertrophy (LVH) by itself or LV hypertrophy itself can lead to myocardial dysfunction, but until now there has been no blood test for the rapid and accurate identification of LVH. The present application discovers an effective new method for identifying patients with LVH. If the LVH process continues or is not adequately treated, the patient will develop signs and symptoms of CHF, primarily due to diastolic heart failure (DHF). However, until now it has been difficult to identify patients with CHF (mainly DHF) or to identify them with a simple and quick blood test. The present application has discovered a novel and efficient method of identifying patients with LVH, patients at risk of developing DHF, and methods of identifying patients with DHF. Thus, the present invention provides a means of detecting the presence of LVH, predicting the possible presence of patients at high risk of developing DHF, and identifying DHF patients. Four (but not necessarily exclusive) separate applications of the method can be performed through the use of small body fluid samples, such as blood samples as described below: screening, prediction/prognosis, diagnosis and treatment monitoring.

Accordingly, disclosed herein is a method of diagnosing a subject with left ventricular hypertrophy (LVH, HCM or HOCM). For example, there is provided a method of detecting LVH in a subject, comprising identifying a matrix metalloproteinase (MMP) and a tissue inhibitor of matrix metalloproteinase (TIMP) profile of a body fluid of said subject, said subject herein With diastolic heart failure (DHF). Also provided is a method of predicting diastolic heart failure in a subject, comprising identifying profiles of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in a body fluid of the subject, the subjects described herein Patients may be accompanied by diastolic heart failure (DHF).

2.MMP

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases; other family members include snake venom protease, serralysin, and astaxanthin. MMPs belong to a larger family of proteases known as the metzincin superfamily.

A common domain structure is shared among MMPs. The three consensus domains are the propeptide, the catalytic domain, and the hemopexin-like C-terminal domain connected to the catalytic domain by a flexible hinge region.

MMPs are initially synthesized as inactive zymogens with a propeptide domain that must be removed prior to enzyme activation. The propeptide domain is part of a "cysteine switch" containing a protein that interacts with zinc in the active site and prevents substrate binding and cleavage to keep the enzyme free. Conserved cysteine residue in the active form. In most MMPs, the cysteine residue is located in the conserved sequence PRCGxPD. Certain MMPs have a prohormone convertase cleavage cleavage site (furin-like) as part of this domain, which activates the enzyme when cleaved. MMP-23A and MMP-23B include a transmembrane segment in this domain (PMID10945999).

(3.5×3×3nm)的扁圆球体。活性位点是一个穿过催化结构域的20

(2nm)的沟。在所述催化结构域的形成该活性位点的部分中存在催化上非常重要的Zn2+离子，该离子与存在于保守序列HexxHxxGxxH中的三个组氨酸氨基结合。因此，该序列为锌结合基序。X-ray crystal structures of the catalytic domains of several MMPs have demonstrated that the domains are 35 × 30 × 30

(3.5×3×3nm) oblate spheroids. The active site is a 20

(2nm) groove. In the part of the catalytic domain that forms the active site there is a catalytically very important Zn2+ ion bound to the three histidine amino groups present in the conserved sequence HexxHxxGxxH. Therefore, this sequence is a zinc binding motif.

The gelatinase (eg MMP-2) contains a type II fibronectin module inserted just before the zinc binding motif in the catalytic domain (PMID 12486137).

The catalytic domain is connected to the C-terminal domain by a flexible hinge or linker region. It is up to 75 amino acids long and has an indeterminate structure.

The C-terminal domain has a similar structure to the serum protein hemopexin. It has four blade-like beta propeller structures. The β-propeller structure provides a large flat surface that is thought to be involved in protein-protein interactions. This also determines the substrate specificity and becomes the site of interaction with TIMP. Haemopexin-like domains are absent in MMP-7, MMP-23 and MMP-26, as well as in plants and nematodes. MT-MMPs are anchored to the plasma membrane by this domain, and some MT-MMPs have a cytoplasmic domain.

MMPs can be subdivided in a number of different ways. By applying bioinformatics methods to compare the primary sequences of MMPs, the following evolutionary groupings of MMPs can be proposed: MMP-19; MMP11, 14, 15, 16 and 17; MMP-2 and MMP-9; all other MMPs.

A separate analysis of each catalytic domain revealed that the catalytic domains further evolved as the main classes diverged, which is also referred to as the substrate specificity of the enzyme. The most commonly used classification (by researchers of MMP biology) is based partly on the histological evaluation of MMP substrate specificity and partly on the cellular localization of the MMP. These classes are collagenases, gelatinases, matrix degrading enzymes and membrane-type MMPs (MT-MMPs). Increasingly, these classifications have been found to be somewhat artificial, as there are many MMPs that do not fit into any conventional category.

Collagenase is capable of degrading triple-helical collagen into distinct 3/4 and 1/4 fragments. These collagens are the major constituents of bone and cartilage, and MMPs are the only known mammalian enzymes capable of degrading them. Generally, the collagenase is MMP-1 (small intestine collagenase), MMP-8 (neutrophil collagenase), MMP-13 (type 3 collagenase), MMP-18 (type 4 collagenase, xco14, Xenopus collagenase. Unknown human ortholog), MMP-14 (MT1-MMP) have also been shown to cleave fibrillar collagen and, more controversially, there is evidence for MMP-2 to dissolve collagen.

Matrix-degrading enzymes show the ability to cleave extracellular matrix proteins broadly, but it cannot cleave triple-helical fibrillar collagen. The three canonical members of this class of enzymes are: MMP-3 (matrix degrading enzyme 1), MMP-10 (matrix degrading enzyme 2) and MMP-11 (matrix degrading enzyme 3). MMP-11 appears to be more similar to MT-MMP in that it can be activated by invertase, so its secretion is usually associated with invertase-activatable MMPs.

The stromelysins include MMP-7 (matrilysin, PUMP) and MMP-26 (matrilysin-2, endometase).

The main substrates of gelatinases are type IV collagen and gelatin, and these enzymes are characterized by an additional domain inserted in the catalytic domain. The gelatin-binding domain is located directly in front of the zinc-binding motif and forms a self-contained folding unit that does not disrupt the structure of the catalytic domain. Two members of this subclass are MMP-2 (72 kDa gelatinase, Gelatinase-A) and MMP-9 (92 kDa gelatinase, Gelatinase-B).

Secreted MMPs include MMP-11 (matrix degrading enzyme 3), MMP-21 (X-MMP) and MMP-28 (Epilysin).

Membrane-bound MMPs include type II transmembrane cysteine array MMP-23, glycosylphosphatidylinositol-binding MMPs 17 and 25 (MT4-MMP and MT6-MMP, respectively), and type I transmembrane MMPs 14, 15, 16 , 24 (MT1-MMP, MT2-MMP, MT3-MMP and MT5-MMP, respectively).

All six MT-MMPs have furin cleavage sites in their propeptides, and MMP-11 also has this feature.

Other MMPs include MMP-12 (macrophage metalloelastase), MMP-19 (RASI-I, sometimes called matrix degrading enzyme-4), amelysin (MMP-20) and MMP-27 (MMP-22, C-MMP), MMP-23A (CA-MMP) and MMP-23B.

3. TIMP

MMPs are inhibited by specific tissue inhibitors of metalloproteinases (TIMPs), which comprise a family of four protease inhibitors: TIMP-1, TIMP-2, TIMP-3 and TIMP-4. In general, all MMPs are inhibited by TIMPs once activated, but gelatinases (MMP-2 and MMP-9) form complexes with TIMPs in their latent forms. The latent form of MMP-2 (proMMP-2 zymogen) in complex with TIMP-2 promotes the activation of proMMP-2 zymogen on the cell surface by a membrane-anchored MMP, MT1-MMP (MMP-14).

4. The ratio of MMP/TIMP

A unique feature of the MMP-TIMP assay in hypertensive heart disease is the utilization of a cardiac-specific TIMP, TIMP-4, and placing it in the context of an MMP that is more significantly altered in myocardial infarction and hypertensive patients middle. Also disclosed are ratios of MMPs (eg MMP-9 or MMP-13) to TIMPs (eg TIMP-1, TIMP-2 or TIMP-4). The ratio is used here for the first time as a diagnostic differential feature and to identify patients with distinct disease states.

5. Plasma Screening

A major advantage of the teachings of the present invention is that the methods disclosed herein provide a faster and simpler method of identifying subjects at risk of developing adverse LVH from tissues or body fluids, as well as identifying patients who are accelerating the process. Accordingly, the methods disclosed herein may comprise detecting MMPs and TIMPs in a body fluid of a subject, such as blood, urine, plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous humor or vitreous humor, Colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, sweat, semen, transudates, transudates, and synovial fluid.

Plasma is the liquid component of blood in which blood cells are suspended. Plasma is the single most important component of blood, accounting for about 55% of the total blood volume. Serum refers to plasma from which coagulation factors such as fibrin have been removed. Plasma contains many important proteins, including fibrinogen, globulins, and human serum albumin. Occasionally, plasma may contain viral impurities that must be extracted by virus processing.

6. Immunoassay

There are many known or newly discovered methods in the art for the detection of analytes such as proteins such as MMPs and TIMPs, which can be used in the methods disclosed herein. For example, MMPs and TIMPs can be detected using standard immunoassays. Various useful immunoassay procedures have been described in the scientific literature, for example, Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), which is incorporated herein by reference in its entirety, in particular for its teaching on methods of immunoassay. In the simplest and most straightforward sense, an immunoassay is a binding assay involving the binding between an antibody and an antigen. Many types and formats of immunoassays are known, all of which are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoprecipitation assay (RIPA), immunobead capture assay, Western blot, dot blot, gel shift assay, flow cytometry, Protein arrays, multi-bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAP/FLAP).

In general, immunoassays involve contacting a sample suspected of containing a molecule of interest (e.g., a biomarker disclosed herein) with an antibody to the molecule of interest, or contacting an antibody to the molecule of interest (e.g., an antibody to a biomarker disclosed herein) with Molecules that are bound by the antibody are contacted, possibly under conditions effective to enable the formation of an immune complex. Contact between a sample and an antibody to a molecule of interest or a molecule that can be bound by an antibody to a molecule of interest under conditions effective to form an immune complex (primary immune complex) and within a period of time sufficient to allow the formation of an immune complex contact between the molecules or antibodies, usually simply by bringing the molecule or antibody into contact with the sample, and in a state sufficient to allow the antibody to form (i.e. bind to) an immunocomplex with any molecule (e.g., an antigen) present that can bind to the antibody Incubate the above mixture for the same period of time as the body. In many forms of immunoassays, the sample-antibody composition (such as tissue sections, ELISA plates, dot blots, or Western blots) can then be washed to remove any nonspecifically bound antibody species, allowing only the primary immunoassay to be detected. Those antibodies to which the complex specifically binds are present.

Immunoassays can include methods for detecting or quantifying molecules of interest (e.g., biomarkers disclosed herein or antibodies thereto) in a sample, which typically include detecting or quantifying any immune complexes formed during binding. Quantitative. In general, detection of immune complex formation is well known in the art and can be accomplished using a variety of methods. These methods are generally based on the detection of a marker or marker, such as any radioactive, fluorescent, biological or enzymatic label or any other known marker. See, eg, US Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference in its entirety, particularly for its teachings relating to immunoassay methods or labels.

As used herein, labels may include fluorescent dyes, one of a binding pair (such as biotin/streptavidin), a metal (such as gold), or a molecule that specifically interacts with a detectable molecule (such as by producing a colored substrate). epitope tagging of objects or fluorescence). Substrates suitable for detectably labeling proteins include fluorescent dyes (also referred to herein as fluorochromes and fluorophores) and enzymes that react with chromogenic substrates such as horseradish peroxidase. In the practice of the present invention, the use of fluorescent dyes is generally preferred because of their ability to be detected in very small amounts. Furthermore, when multiple antigens are reacted with a single array, each antigen can be labeled with a different fluorescent compound for simultaneous detection. The labeled spots on the array can be detected using a fluorometer, and the presence of a signal indicates that the antigen is bound to the specific antibody.

Fluorophores are compounds or molecules that emit light. Typically, fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at another wavelength. Representative fluorophores include, but are not limited to: 1,5-IAEDANS, 1,8-ANS, 4-methylumbelliferone, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5 -FAM), 5-carboxynaphthalene fluorescein, 5-carboxytetramethylrhodamine (5-TAMRA), 5-hydroxytryptamine (5-HAT), 5-ROX (carboxy-X-rhodamine), 6-carboxyrhodamine Ming 6G, 6-CR6G, 6-JOE, 7-amino-4-methylcoumarin, 7-aminoactinomycin D (7-AAD), 7-hydroxyl-4-Imethylcoumarin, 9-amino-6-chloro-2-methoxyacridine (ACMA), ABQ, acid fuchsin, acridine orange, acridine red, acridine yellow, trypanosomal yellow (Acriflavin), trypanosomal yellow Fuergen SITSA (AcriflavinFeulgen SITSA), Aequorin (photoprotein), AFP-autofluorescent protein-(Quantum Biotechnology) (see sgGFP, sgBFP), Alexa Fluor 350 ^TM , Alexa Fluor 430 ^TM , Alexa Fluor 488 ^TM , Alexa Fluor 532 ^TM , Alexa Fluor 546 ^TM , Alexa Fluor568 ^TM , Alexa Fluor 594 ^TM , Alexa Fluor 633 ^TM , Alexa Fluor 647 ^TM , Alexa Fluor 660 ^TM , Alexa Fluor 680 ^TM , alizarin complexone, alizarin red, allophycocyanin ( APC), AMC, AMCA-S, Aminomethylcoumarin (AMCA), AMCA-X, Aminoactinomycin D, Aminocoumarin, Aniline Blue, Anthrocyl stearate, APC-Cy7 , APTRA-BTC, APTS, Astrazon Bright Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7GLL, Atabrine, ATTO-TAG ^TM CBQCA, ATTO-TAG ^TM FQ, Auramine, Aurophosphine G, Aurophosphine, BAO 9 (Bisaminophenyloxadiazole), BCECF (High pH), BCECF (Low pH), Berberine Sulfate, Beta Endo Amidase, BFP Blue-shifted GFP (Y66H), Blue Fluorescent Protein, BFP/GFP FRET, Bimane, Bisbenzamide, Bisbenzamide (Hoechst), BisBTC, Blancofort Blancophor (Blancophor) FFG, Blancophor SV, BOBO ^TM -1, BOBO ^TM -3, fluoroborate 492/515, Fluorine 493/503, Fluoride 500/510, Fluoride 505/515, Fluoride 530/550, Fluoride 542/563, Fluoride 558/568, Fluoride 564/570, Fluoride Boron 576/589, Fluorin 581/591, Fluorin 630/650-X, Fluorin 650/665-X, Fluorin 665/676, Fluorin F1, Fluorin FL ATP, Fluorin Bodipy F1-Ceramide, Bodipy R6GSE, Bodipy TMR, Bodipy TMR-X Conjugate, Bodipy TMR-X SE, Bodipy TR, Bodipy TR ATP, Bodipy TR- X SE, BO-PRO ^TM -1, BO-PRO ^TM -3, Brilliant Sulphoflavin FF, BTC, BTC-5N, Calcein, Calcein Blue, CalciumCrimson-, Calcium Green, Calcium Green-1 Ca ²⁺ Dye, Calcium Green-2Ca ²⁺ , Calcium Green-5N Ca ²⁺ , Calcium Green-C 18 Ca ²⁺ , Calcium Orange, Calcofluor White, Carboxy-X-rhodamine (5-ROX), Cascade Blue ^TM , CascadeYellow, Catecholamine ), CCF2 (GeneBlazer), CFDA, CFP (Cyano Fluorescent Protein), CFP/YFP FRET, Chlorophyll, Chloromyces A, Chromomyces A, CL-NERF, CMFDA, Coelenterazine, Coelenterazine cp, Coelenterazine f, coelenterazine fcp, coelenterazine h, coelenterazine hcp, coelenterazine ip, coelenterazine n, coelenterazine 0, couniarin phalloidin, C-phycocyanin (C-phycocyanine), CPM I methyl coumarin, CTC, CTC formazan (Formazan), Cy2 ^TM , Cy3.18, Cy3.5 ^TM , Cy3 ^TM , Cy5.18, Cy5.5 ^TM , Cy5 ^TM , Cy7 ^TM , cyano-GFP, cAMP Fluorosensor (FiCRhR), 4-(4′-dimethyl-p-aminoazobenzene)benzoic acid (Dabcyl), dansyl, dansylamide, dansylazine Amine, dansyl chloride, dansyl DHPE, dansyl fluoride, DAPI, Dapoxyl, Dapoxyl2, Dapoxyl3′DCFDA, DCFH (dichlorodihydrofluorescein diethyl ester), DDAO, DHR (dihydrorhodamine 123), Di-4-ANEPPS, Di-8-ANEPPS (non-ratio), DiA (4-Di16-ASP), Dichlorodihydrofluorescein Diethyl Ester (DCFH), DiD-lipophilic tracer (Lipophilic Tracer), DiD(DiI1C18(5)), DIDS, Dihydrorhodamine 123(DHR), Di1(Di1C18(3)), I Dinitrophenol, Di0(Di0C18(3)), DiR, DiR (Di1C18(7)), DM-NERF (high pH), DNP, Dopamine, DsRed, DTAF, DY-630-NHS, DY-635-NHS, EBFP, ECFP, EGFP, ELF 97, Eosin, Phycoerythrin, Phycoerythrin ITC, Ethidium Bromide, Ethidium homodimer-1 (EthD-1), Acridine Orange, EukoLight, Europium Chloride (111), EYFP, Fast Blue ), FDA, Fuergen (paramagentin), FIF (formaldehyde-induced fluorescence), FITC, Flazo Orange, Fluo-3, Fluo-4, fluorescein (FITC), fluorescein diethyl ester, fluorescent emerald ( Fluoro-Emerald), Fluoro-Gold (hydroxydiamidino), Fluor-Ruby, FluorX, FM 1-43 ^TM , FM4-46, Fura Red ^TM (high pH), Fura Red ^TM /Fluo-3 , Fura-2, Fura-2/BCECF, Genacryl BrilliantRed B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, GenacrylYellow 5GF, GeneBlazer, (CCF2), GFP (S65T), red-shifted GFP (rsGFP), non-UV-excited wild Type GFP (wtGFP), UV-excited wild-type GFP (wtGFP), GFPuv, Gloxalic Acid, Granular blue, Haematoporphyrin, Hearst 33258, Hearst 33342, Hearst 34580, HPTS, hydroxycoumarin, hydroxydiamidine (fluorescent gold), serotonin, Indo-1 (high calcium), Indo-1 (low calcium), indodicarbocyanine (Indodicarbocyanine) (DiD), indole tricarbonate Indotricarbocyanine (DiR), Intrawhite Cf, JC-1, JO JO-1, JO-PRO-1, LaserPro, Laurod an, LDS 751 (DNA), LDS 751 (RNA), Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine, Lissamine Rhodamine B, Calcein/Ethidium Isotype Dimer, LOLO-1, LO-PRO-1, Lucifer Yellow, Lyso Tracker Blue, Lyso Tracker Blue-White, LysoTracker Green, LysoTracker Red, LysoTracker Yellow, LysoSensor Blue, LysoSensor Green, LysoSensorYellow/Blue, Mag Green , Magdala Red (Phloxin B), Mag-FuraRed, Mag-Fura-2, Mag-Fura-5, Mag-lndo-1, Magnesium Green, Magnesium Orange, Malachite Green, Marina Blue, I MaxilonBrilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, Merocyanin, Methoxycoumarin, Mitotracker Green FM, Mitochondrial Orange Fluorescence Needle (Mitotracker Orange), Mitotracker Red, Mithramycin, Monobromobimane, Monobromobimane (mBBr-GSH), Monochlorobimane, MPS (Methyl GreenPyronine Stilbene), NBD, NBD Amine, Nile Red, Nitrobenzoxedidole, Noradrenaline, Nuclear Fast Red, i Nuclear Yellow, Nylosan Brilliantlavin E8G, Oregon Green ( Oregon Green) ^TM , Oregon Green ^TM 488, Oregon Green ^TM 500, Oregon Green ^TM 514, Pacific Blue (Pacific Blue), Vice Magenta (Fuergen), P BFI, PE-Cy5, PE-Cy7, PerCP, PerCP-Cy5.5, PE-Texas Red (613 Red), Phloxin B (Maitara Red), Phorwite AR, Phorwite BKL, PhorwiteRev, Phorwite RPA, Phosphine 3R, Photoresist (PhotoResist), Phycoerythrin B[PE], Phycoerythrin R[PE], PKH26(Sigma), PKH67, PMIA, Pontochrome Blue Black, POPO-1, POPO-3, PO-PRO-1, PO-I PRO-3, Primuline, Procion Yellow, Propidium Iodide (P1), PyMPO, Pyrene, Pyrene Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, QSY 7, Quinacrine Mustard, Resorufin, RH414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123 , Rhodamine 5GLD, Rhodamine 6G, Rhodamine B, Rhodamine B200, Rhodamine B extra, Rhodamine BB, Rhodamine BG, Rhodamine Green, Rhodamine Phallicidine, Rhodamine Ming, Phalloidine, Rhodamine Red, Rhodamine WT, Rose Bengal, R-phycocyanine, R-phycoerythrin (PE), rsGFP, S65A, S65C, S65L, S65T , Dark Blue GFP, SBFI, 5-HT, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, sgBFP ^TM (Super Luminescent (super glow) BFP), sgGFP ^TM (super luminescent GFP), SITS (primulaline, 1,2-stilbene isothiosulfate), SNAFL calcein, SNAFL-1, SNAFL-2, SNAR calcein, SNARF1, Sodium Green, SpectrumAqua, SpectrumGreen, SpectrumOrange, Spectrum Red, SPQ (6-methoxy-N-(3-sulfopropyl)ammonium quinolate), 1,2-stilbene, sulforhodamine B and C, SulphoRhodamien Extra, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYT O 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61 , SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange, Tetracycline, Tetramethylrhodamine (TRITC) , Texas Red ^TM , Texas Red-X ^TM conjugate, Thiadicarbocyanine (DiSC3), Thiazine Red R (Thiazine Red R), Thiazole Orange, Thioflavin 5 ( Thioflavin), Thioflavin S, Thioflavin TON, Thiolyte, ThiozoleOrange, Tinopol CBS (Calcofluor White), TIER, TO-PRO-1, TO-PRO-3, TO-PRO-5, TOTO-1, TOTO-3, Tricolor(PE-Cy5), TRITC Tetramethylrhodamine Isothiocyanate, True Blue, True Red, Ultralite, Uranine B, Uvitex SFC, wtGFP, WW781, X-Rhodamine, XRITC, Xylene Orange, Y66F, Y66H, Y66W, Yellow GFP, YFP, YO-PRO-1, YO-PRO 3, YOYO-1, YOY0-3, Sybr Green, Thiazole Orange (interchelating dyes), semiconductor nanoparticles such as quantum dots, or caged flurophores (activatable by light or other electromagnetic energy sources), or combinations thereof.

Marking can be done directly or indirectly. In direct labeling, the detection antibody (antibody to the molecule of interest) or the detection molecule (molecule that can be bound by an antibody to the molecule of interest) includes a label. Detection of the marker indicates the presence of the detected antibody or the detected molecule, which in turn indicates the presence of the molecule of interest or the presence of an antibody to the molecule of interest, respectively. In indirect labeling, other molecules or moieties are brought into contact with, or generated at, the immune complex. For example, a signal-generating molecule or moiety (eg, an enzyme) can be attached to or bound to the detection antibody or the detection molecule. The signaling molecule can then generate a detectable signal at the immune complex. For example, an enzyme may produce a visible or detectable product at the immune complex when provided with an appropriate substrate. ELISA employs this indirect labeling.

Another example of indirect labeling is the combination of other molecules (which may be referred to as binding agents) that bind the molecule of interest or an antibody (primary antibody) that binds the molecule of interest—for example, a secondary antibody that binds to the primary antibody—with the immune complex. touch. The other molecules may bear markers or signal-generating molecules or moieties. Said other molecule may be an antibody, whereby it may be referred to as a secondary antibody. The combination of the secondary antibody and the primary antibody can form a so-called sandwich with the primary antibody (or primary antibody) and the molecule of interest. The immune complex can be contacted with the labeled secondary antibody under conditions effective to allow the formation of the secondary immune complex and for a time sufficient to allow the formation of the secondary immune complex. The secondary immune complex can then typically be washed to remove any non-specifically bound labeled secondary antibody, and the remaining label in the secondary immune complex can then be detected. The other molecule may also be or comprise one of a pair of molecules or moieties that bind to each other (eg biotin/streptavidin pair). In this format, the detection antibody or detection molecule should include the other member of the pair.

Other modes of indirect labeling include the detection of primary immune complexes through a two-step approach. For example, a molecule having a binding affinity for the molecule of interest or a corresponding antibody (which can be referred to as a primary binding agent), such as an antibody, can be used to form a secondary immune complex, as described above. After washing, also under conditions effective to allow formation of immune complexes (thereby forming tertiary immune complexes), the secondary immune complexes may be allowed to react with another immune complex for a time sufficient to allow formation of immune complexes. A molecule with a binding affinity for the first binding agent (which may be referred to as a second binding agent) is contacted. The second binding agent may be linked to a detectable label or signal generating molecule or moiety to enable detection of said tertiary immune complex formed thereby. This system can amplify the signal.

Immunoassays involving the detection of a substrate (eg, a protein or an antibody to a specific protein) include label-free assays, protein separation methods (ie, electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are a common diagnostic tool to determine the presence or absence of a specific protein or antibodies to a specific protein in a sample. Protein separation methods are also used to evaluate physical properties of proteins, such as molecular size or net charge. Capture assays are often more useful for quantitatively assessing the concentration of a specific protein or an antibody to a specific protein in a sample. Finally, in vivo assays can be used to assess the spatial expression pattern of a substance, ie where in the patient's body the substance may be present, whether in tissues or cells.

Molecular complexes ([Ab-Ag]n) produced by antibody-antigen interactions are visible to the naked eye if the concentration is sufficient, but may be detected or measured in smaller amounts because they scatter light. Complex formation indicates the presence of both reactants. In an immunoprecipitation assay, a constant concentration of reagent antibody is used to measure a specific antigen ([Ab-Ag]n), and reagent antigen is used to detect a specific antibody ([Ab-Ag]n). Ag]n). If the reagent species is pre-coated on cells (as in hemagglutination assays) or microparticles (as in latex agglutination assays), "clumps" of the coated particles are visible at very low concentrations. Various assays based on the basic principles described above are commonly used, including Ouchterlony immunodiffusion assay, rocket immunoelectrophoresis, and immunoturbidimetric analysis and nephelometry. The main limitations of this type of assay are their limited sensitivity (lower detection limit) compared to assays using labels, and the fact that, in some cases, extremely high concentrations of analyte can actually inhibit complex formation make these methods more complex. Some of these first-line assays date back to when antibodies were discovered, and none of them have a true "marker" (eg, Ag-enz). Other types of label-free immunoassays rely on immunosensors, and there are many commercially available instruments that can directly detect antibody-antigen interactions. Most require evanescent waves to be generated on the sensor surface with immobilized ligands, allowing continuous monitoring of ligand binding. Immunosensors allow the study of kinetic interactions to be easily performed, and with the advent of low-cost specialized instruments, they will be widely used in immunoassays in the future.

Detection of a particular protein using an immunoassay can involve separating the protein by electrophoresis. Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their mobility depends on the strength of the electric field, the net charge, size and shape of the molecule, and also on the ionic strength, viscosity and temperature of the medium in which the molecules are swimming. Electrophoresis is a simple, fast and highly sensitive analytical tool. It is used analytically to study the properties of single charged molecules and also as a separation technique.

Typically, samples are run on a carrier matrix, such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits heat-induced convective mixing and provides electrophoretic documentation: at the end of electrophoresis, the matrix can be stained and used for scanning, autoradiography or storage. Furthermore, the most commonly used support matrices—agarose and polyacrylamide—provide the means to separate molecules by size because they are porous gels. Porous gels can act as molecular sieves by delaying or in some cases completely preventing the movement of large molecules while allowing small molecules to move freely. Since dilute agarose gels are generally harder and easier to handle than polyacrylamide at the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins, and protein complexes. Polyacrylamide is easy to handle and prepare at higher concentrations and is therefore used to separate most proteins and smaller oligonucleotides that require smaller retardation gel pore sizes.

Proteins are amphoteric, so their net charge can be determined by the pH of the medium in which they are suspended. When the solution pH is above the protein's isoelectric point, the protein is net negatively charged and moves towards the anode in the electric field. Below its isoelectric point, the protein is positively charged and moves towards the cathode. In addition, the net charge of a protein has nothing to do with its molecular size, that is, different proteins have different charges per unit molecular mass (or length, when proteins and nucleic acids are linear macromolecules). Thus, at a given pH, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both the size and the charge of the molecules.

Sodium dodecyl sulfate (SDS) is an anionic denaturant that can denature proteins by "surrounding" the polypeptide backbone. SDS binds to proteins very specifically at a mass ratio of 1.4:1. Thus, SDS will impart a negative charge on the polypeptide proportional to its length. In addition, it is often necessary to reduce (denature) the disulfide bonds of the protein so that the protein adopts the random coil configuration required for separation by molecular size; reduction can be achieved with 2-mercaptoethanol or dithiothreitol. Therefore, in denaturing SDS-PAGE separations, migration is not determined by the internal charge of the polypeptide, but by molecular weight.

The molecular weight can be determined by SDS-PAGE electrophoresis with the protein of known molecular weight and the protein to be characterized. There is a linear relationship between the logarithm of the molecular weight of a polypeptide or natural nucleic acid denatured by SDS and its Rf. The ratio of the molecular migration distance to the migration distance of the standard dye front is Rf. A simple method of determining relative molecular weight (Mr) by electrophoresis is to plot a standard curve of known sample migration distance versus log 10 MW and read the logMr of the sample after measuring the migration distance of the sample on the same gel.

In 2D electrophoresis, proteins are first fractionated based on a physical property and then fractionated in another step based on another property. For example, isoelectric focusing can be used in the first dimension, which is conveniently performed on tubular gels, while SDS electrophoresis on slab gels can be used in the second dimension. An example of this method is that of O'Farrell, P.H., High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), which is incorporated by reference for its teaching of two-dimensional electrophoresis methods method is included in this article in its entirety. Other examples include but are not limited to Anderson, L and Anderson, NG, High resolution two-dimensional electrophoresis of human plasmaproteins, Proc.Natl.Acad.Sci.74:5421-5425 (1977) and Ornstein, L., Disc electrophoresis, L. Methods in Ann. N. Y. Acad. Sci. 121:321349 (1964), which are incorporated herein by reference in their entirety for their teachings on electrophoretic methods.

Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227: 680 (1970) discloses a discontinuous system for resolving SDS denatured proteins, which is cited by reference for its electrophoretic teaching method is included in this article in its entirety. The leading ion in the Laemmli buffer system is chloride and the trailing ion is glycine. Therefore, the resolving and stacking gels were prepared with Tris-HCl buffer (different concentrations and pH), while the electrophoresis tank buffer was Tris-glycine. All buffers contain 0.1% SDS.

One example of an immunoassay using electrophoresis contemplated in the methods of the invention is Western blot analysis. Western or immunoblotting allows the determination of the molecular mass of proteins, as well as the measurement of the relative amounts of proteins present in different samples. Detection methods include chemiluminescent and chromogenic detection. Standard methods for Western blot analysis can be found, for example, in D.M.Boltag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988) and U.S. Patent 4,452,901, each of which deals with Western blot methods The teachings of are incorporated herein by reference in their entirety. Proteins are generally separated by gel electrophoresis (commonly SDS-PAGE). The protein is transferred to a special blotting tissue such as nitrocellulose, but other types of paper or membranes can also be used. The protein retains its separation pattern on the gel. The blot is incubated with a total protein such as milk protein to bind any remaining sticky areas on the nitrocellulose membrane. An antibody capable of binding its specific protein is then added to the solution.

Binding of specific antibodies to specific immobilized antigens is readily demonstrated by indirect enzyme immunoassay techniques, typically using chromogenic (eg, alkaline phosphatase or horseradish peroxidase) or luminescent substrates. Other possible detection methods include the use of fluorescent or radioisotopic labels (eg, fluorescein, ¹²⁵ I). Probes to detect antibody binding can be probes of conjugated anti-immunoglobulin, conjugated staphylococcal protein A (binding IgG), or biotinylated primary antibodies (e.g., conjugated avidin/streptavidin ).

The effectiveness of this technique lies in its ability to simultaneously detect a specific protein and its molecular weight through its antigenicity. First, proteins are separated by mass by SDS-PAGE and then specifically detected by an immunoassay step. Therefore, protein standards (ladders) can be electrophoresed simultaneously to estimate the molecular weight of a protein of interest in a heterogeneous sample.

Gel shift assays or electrophoretic mobility assays (EMSA) can be used to qualitatively and quantitatively detect interactions between DNA-binding proteins and their cognate DNA recognition sequences. Exemplary techniques 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, Anal . Biochem. 87: 386-396 (1987), which are hereby incorporated by reference in their entirety for their teachings on gel shift assays.

In conventional gel assays, purified proteins or crude cell extracts are incubated with a labeled (e.g. ³² P radiolabeled) DNA or RNA probe, and the complexes are then run through a native polyacrylamide gel. The body is separated from the free probe. The complex migrates more slowly through the gel than the unbound probe. Labeled probes can be double-stranded or single-stranded, depending on the activity of the binding protein. For the detection of DNA binding proteins such as transcription factors, purified or partially purified proteins or nuclear cell extracts can be used. For the detection of RNA binding proteins, purified or partially purified proteins or nuclear or cytoplasmic cell extracts can be used. The specificity of a DNA or RNA binding protein for a putative binding site can be determined by competition experiments using DNA or RNA fragments or oligonucleotides containing the binding site for the protein of interest, or other unrelated sequences. Differences in the nature and strength of complexes formed in the presence of specific and non-specific competitors allow identification of specific interactions. See Promega's Gel Shift Assay FAQ, available at http://www.promega.com/faq/gelshfaq.html (last accessed March 25, 2005), which pertains to its gel shift assay The teachings are incorporated herein by reference in their entirety.

Gel shift methods can include, for example, detection of proteins on a gel (eg, polyacrylamide electrophoresis gel) using Coomassie (Imperial Chemicals Industries, Ltd) blue dye in colloidal form. Such methods are described, for example, in Neuhoff et al., Electrophoresis 6: 427-448 (1985) and Neuhoff et al, Electrophoresis 9: 255-262 (1988), both of which have passed The way of citation is incorporated in this article in its entirety. In addition to the conventional protein assay methods described above, a combined washing and protein staining composition is also described in US Pat. No. 5,424,000, which is incorporated herein by reference in its entirety for its teachings on the gel migration method. The solution may include phosphoric, sulfuric, and nitric acids and acid violet dye.

Radioimmunoprecipitation assay (RTPA) is a sensitive assay that uses radiolabeled antigens to detect specific antibodies in serum. Antigens are reacted with serum and then precipitated with special reagents such as protein A sepharose beads. Thereafter, the bound radiolabeled immunoprecipitates are typically analyzed by gel electrophoresis. Radioimmunoprecipitation assay (RIPA) is often used as a confirmatory test for the presence of HIV antibodies. RIPA is also known in the art as Farr assay, precipitation assay, radioimmunoprecipitation assay; radioimmunoprecipitation assay; radioimmunoprecipitation assay and immunoprecipitation assay.

While the above-described immunoassays using electrophoretic separation and detection of the specific protein of interest can assess protein size, they are less sensitive in assessing protein concentration. However, an immunoassay in which the protein or an antibody specific for the protein is bound to a solid support (such as a tube, well, bead or cell) to capture the antibody or protein of interest, respectively, from the sample is also contemplated, and This assay is combined with a method of detecting the protein or antibodies specific for the protein on the support. Examples of such immunoassays include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), flow cytometry, protein arrays, multi-bead arrays, and magnetic capture.

Radioimmunoassay (RIA) is a classical quantitative assay for the detection of antigen-antibody reactions that uses radioactively labeled substances (radioligands) to directly or indirectly measure the interaction of unlabeled substances with specific antibodies or other receptor systems combination. Instead of bioassays, radioimmunoassays, for example, can be used to detect hormone levels in blood. Non-immunogenic substances (eg, haptens) can also be assayed if coupled to larger carrier proteins (eg, bovine gamma globulin or human serum albumin) that induce antibody formation. RIA involves mixing a radioactive antigen (the radioactive isotopes ¹²⁵ I or ¹³¹ I are usually used because of the ease of introducing iodine atoms into tyrosine residues in proteins) with antibodies to that antigen. Typically, the antibody is bound to a solid support such as a tube or beads. Then, a known amount of unlabeled or "cold" antigen is added, and the amount of displaced labeled antigen is measured. Initially, the radioactive antigen is bound to the antibody. When cold antigen is added, the two compete for antibody binding sites, and higher concentrations of cold antigen bind more to the antibody, displacing the radioactive variant. The bound antigen is separated from the unbound antigen in solution, and the binding curve is drawn by the radioactivity of each antigen. This technique is extremely sensitive and highly specific.

Enzyme-linked immunosorbent assay (ELISA)—or more commonly known as EIA (enzyme immunoassay)—is an immunoassay that detects specific antibodies against a protein. In this assay, the detectable label bound to the antibody-binding or antigen-binding reagent is an enzyme. When an enzyme is contacted with its substrate, the enzyme reacts in a specific manner to produce a chemical moiety that can be detected, for example, spectrophotometrically, fluorometrically, or visually. Enzymes that can be used to detectably label detection reagents include, but are not limited to: horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, apple Acid dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine enzyme. See Voller, A.et al., J.Clin.Pathol.31:507-520 (1978) about the description of ELISA method; 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, Boca Raton, 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-Phase Immunoassay, CRC Press, Boca Raton, 1991); Crowther, "ELISA: Theory and Practice," In: Methods in Molecule Biology, Vol.42, Humana Press; New Jersey, 1995; teachings - are incorporated herein by reference in their entirety.

Variations of ELISA techniques are known to those skilled in the art. In one variation, antibodies capable of binding a protein can be immobilized on a selected surface that exhibits an affinity for the protein, for example in the wells of a polystyrene microtiter plate. A test composition suspected of containing the labeled antigen is then added to the wells. After binding and washing away non-specifically bound immune complexes, bound antigen can be detected. Detection can be performed by adding a secondary antibody specific for the target protein linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection can also be performed by adding a secondary antibody followed by a tertiary antibody that has binding affinity for the secondary antibody, the tertiary antibody being linked to a detectable label.

Another variation is the competition ELISA. In a competition ELISA, a test sample competitively binds a known amount of labeled antigen or antibody. The amount of the reactant in the sample can be determined by mixing the sample with the known marker prior to or during incubation with the coated wells. The presence of reactants in the sample reduces the amount of label that can bind to the wells, thereby reducing the resulting signal.

Regardless of the format used, ELISAs share certain common features, such as coating, incubation or binding, washing away non-specifically bound substances, and detection of bound immune complexes. The antigen or antibody can be attached to a solid support, for example in the form of a plate, bead, rod, membrane or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody. When coating plates with antigen or antibody, the wells are typically incubated with the antigen or antibody solution and incubated overnight or for a specified period of time. Then, wash the wells to remove incompletely adsorbed material. Any remaining adsorbable well surfaces are then "coated" with a non-specific protein that is antigenically neutral to the test antiserum. They include solutions of bovine serum albumin (BSA), casein and milk powder. The coating makes it possible to block non-specific adsorption sites on the immobilization surface and thus reduce the background caused by non-specific binding of antisera to the surface.

In ELISA, secondary or tertiary detection means can also be used instead of direct methods. Therefore, after the protein or antibody is bound to the well, coated with non-reactive substances to reduce the background and wash away the unbound substances, the immobilized surface and the control clinical or biological sample to be tested can effectively make the immune complex (Antigen/antibody) form contact under conditions. Thus, detection of the immune complex requires a labeled secondary binding reagent or a combination of a secondary binding reagent and a labeled tertiary binding reagent.

"Under conditions effective to allow the formation of an immune complex (antigen/antibody)" means that the conditions include dilution of the antigen and Antibodies to reduce nonspecific binding and improve reasonable signal-to-noise ratios.

Suitable conditions can also mean incubation at a temperature and for a sufficient time to allow effective binding. The incubation step may typically be performed at about 20°C to 30°C for about 1 minute to 12 hours, or may be incubated overnight at about 0°C to about 10°C.

In ELISA, after all incubation steps, the contact surfaces are washed to remove uncomplexed material. Washing steps may include washing with a solution such as PBS/Tween or borate buffer. After the formation of specific immune complexes between the test sample and the initially bound substances and subsequent washing, the immune complexes present even in small amounts can be determined.

In order to provide a means of detection, as described above, the secondary or tertiary antibody may have a bound label for detection. It may be an enzyme that produces color upon incubation with a suitable chromogenic substrate. Thus, for example, the first or second immune complex can be contacted and incubated with the labeled antibody under conditions and for a period of time favorable to further formation of the immune complex (for example, at room temperature, in a solution containing PBS (for example, PBS -Tween) for 2 hours).

After incubation with labeled antibody and subsequent washing of unbound material, the label can be quantified, for example, by reacting with chromogenic substrates such as urea and bromocresol violet or 2, in the case of peroxidase as the enzyme label. 2′-Azide-bis-(3-ethyl-benzothiazoline-6-sulfonic acid [ABTS] is quantified by incubation with H ₂ O ₂ . Quantification can be achieved by measuring the degree of chromogenicity, e.g. using visible spectroscopy spectrophotometer.

Protein assays are solid-phase ligand-binding assay systems that utilize immobilized proteins on surfaces including glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. These assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Their advantages include rapidity and automation, high sensitivity, economical reagents, and large amounts of data that can be given from a single experiment. Bioinformatics support is very important; processing of data requires sophisticated software and comparative analysis of data. However, the software for DNA arrays can be modified to obtain the above software, as can many hardware and detection systems.

One major format is the capture array, in which a ligand-binding agent, usually an antibody but can also be a protein, is used to detect a molecule of interest in a mixture such as plasma or tissue extracts Scaffolds, peptides or aptamers. In diagnostics, capture arrays can be used to perform multiple immunoassays in parallel, either for multiple analytes in, for example, a single serum, or multiple serum samples simultaneously. In proteomics, capture assays are used to quantify and compare protein levels in different healthy and diseased samples, ie, protein expression profiling. Proteins other than specific ligand binders are used in the described assays for in vitro functional interaction (e.g., protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc.) screening in array form. The capture reagents themselves can be selected and screened against multiple proteins, which can also be achieved in multiple array formats against multiple protein targets.

For constructing arrays, sources of protein include cell-based expression systems for recombinant proteins, purification from natural sources, in vitro production by cell-free translation systems, and peptide synthesis methods. Many of these methods can be used for automated high-throughput production. For capture arrays and protein functional analysis, it is important that the protein is correctly folded and functional; this is not always the case, for example when recombinant proteins are extracted from bacteria under denaturing conditions. However, arrays of denatured proteins can be used to screen antibodies for cross-reactivity, identify autoantibodies, and screen for ligand-binding proteins.

Protein arrays have been designed, often using fluorescent readouts and machine-propelled miniaturized versions of common immunoassay methods (such as ELISA and dot blots), as well as high-throughput detection systems capable of performing multiple assays in parallel. Commonly used physical supports include glass slides, silica, microwells, nitrocellulose or PVDF membranes, and magnetic or other microbeads. Although the most common format is protein droplets applied to a planar surface, other configurations include CD centrifugation devices based on microfluidics development (Gyros, Monmouth Junction, NJ) and special chip designs, such as microchannels designed on board. (eg The Living Chip ^™ , Biotrove, Woburn, MA) and micro 3D rods on a silicon surface (Zyomyx, Hayward CA). Suspended particles can also be used as the basis for arrays, provided they are identified; systems include microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDots ^™ , QuantumDot, Hayward, CA). ) and bar coding for beads (UltraPlex ^™ , SmartBead Technologies Ltd, Babraham, Cambridge, UK) and polymetallic microrods (eg Nanobarcodes ^™ particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPStechnology, BioArray Solutions, Warren, NJ).

Immobilization of proteins involves coupling reagents and the nature of the surface to be coupled. A good protein array support surface is chemically stable before and after the coupling process, results in good spot morphology, exhibits minimal non-specific binding, does not increase the background of the detection system, and is compatible with different detection systems. The immobilization method used is reproducible, adaptable to proteins with different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation and compatible with the retention of fully functional protein activity. The orientation of a surface-bound protein is considered to be an important factor in the process of presenting the protein in an active state to a ligand or substrate; for capture arrays, the most efficient binding results are obtained with oriented capture reagents that Often site-specific labeling of proteins is desired.

Both covalent and non-covalent methods of protein immobilization are available, each with various advantages and disadvantages. Passive surface adsorption is methodologically simple but offers little quantitative or directional control; it may or may not alter the functional specificity of the protein, and reproducibility and efficiency are variable. Covalent conjugation provides a stable linkage that is applicable to a wide variety of proteins with good reproducibility; however, orientation may be variable, chemical derivatization may alter protein function and requires stable interaction surfaces. Biocapture methods using tags on proteins can provide stable linkages and can bind proteins in a specific and reproducible orientation, but first the biological reagents must be sufficiently immobilized, and the array may require special handling to stabilize it. Sex can also vary.

Various immobilization chemistries and labels for protein array construction have been described. Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents. In the Versalinx ^™ system (Prolinx, Bothell, WA), reversible covalent coupling can be achieved by the interaction of proteins derivatized with benzenediboronic acid and salicylhydroxamic acid immobilized on the support surface. The method has low background binding, weak internal fluorescence, and allows immobilized proteins to retain function. Based on three-dimensional polyacrylamide gels, non-covalent binding of unmodified proteins occurs inside porous structures such as HydroGel ^™ (PerkinElmer, Wellesley, MA); Low, its protein capacity is high and protein function can be preserved. Widely used bioconjugation methods are via biotin/streptavidin or hexahistidine/Ni interactions with appropriate protein modifications. Biotin can be conjugated to a polylysine backbone immobilized on the surface of eg titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).

Array fabrication methods include robotic contact printing, inkjet, piezoelectric dot printing, and photolithography. Many commercial arrays (eg Packard Biosciences) as well as manual devices (V&P Scientific) are available. Bacterial colonies can be netted onto PVDF membranes by robotics to induce protein expression in situ.

Nanoarrays are the limit of spot size and density. When the spot is at the nanoscale space level, thousands of reactions can be carried out on a single chip less than 1 square millimeter. BioForce Laboratories has developed an ⁸⁵ μm nanoarray with 1521 protein spots, which corresponds to 25 million spots per square centimeter, reaching the limit of optical detection; the readout methods are fluorescence and atomic force microscopy surgery (AFM).

Fluorescent labeling and detection methods are widely used. The same instrumentation used for DNA microarray readout is applicable to protein arrays. For differential visualization, a capture (e.g. antibody) array can be probed with fluorescently labeled proteins from two different cell states, where cell lysates are directly conjugated to different fluorophores (e.g. Cy-3, Cy-5) and the Cell lysates are mixed so that the color serves as a readout for changes in target abundance. Fluorescence readout sensitivity can be amplified 10-100-fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar guided wave technology (Zeptosens) enables ultrasensitive fluorescence detection and has the additional advantage of not interfering with the washing process. High sensitivity can also be achieved by suspending beads or particles with phycoerythrin as a marker (Luminex) or by exploiting the properties of semiconductor nanocrystals (Quantum Dot). Especially in the biotechnology commercial field, many new alternative readout methods have been developed. They include variations of the following methods: surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), Resonant light scattering (Genicon Sciences, San Diego, CA) and atomic force microscopy (BioForce Laboratories).

Capture arrays form the basis of diagnostic chips and arrays that enable expression profiling. They bind and detect specific target ligands in a high-throughput manner through high-affinity capture reagents, such as conventional antibodies, single domains, constructed scaffolds, peptides or nucleic acid aptamers.

Antibody arrays possess the desired specificity properties and acceptable background, and some antibody arrays are commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays can be prepared by conventional immunization methods (polyclonal sera and hybridomas) or as recombinant fragments usually expressed in E. coli after screening from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; Biolnvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to conventional antibodies, Fab and scFv fragments, single V domains of camelids, or engineered human equivalents (Domantis, Waltham, MA) can also be used in arrays.

The term "scaffold" refers to the ligand-binding domain of a protein that has been engineered to bind multiple variants of target molecules with antibody-like specificity and affinity. The variants can be generated in the form of genetic libraries and screened against individual targets by phage, bacterial or ribosomal display. Such ligand-binding scaffolds or scaffolds include the staphylococcal protein A-based "Affibody" (Affibody, Bromma, Sweden), the fibronectin-based "Trinectin" (Phylos, Lexington, MA) and the lipocalin-based "Affibody". Anticalin" (Pieris Proteolab, Freising-Weihenstephan, Germany). They are used in capture arrays in a manner similar to antibodies and have the advantage of being robust and easy to produce.

Non-protein capture molecules, particularly single-stranded nucleic acid aptamers (SomaLogic, Boulder, CO), which bind protein ligands with high specificity and affinity, can also be used in the array. Aptamers are selected from oligonucleotide libraries by the Selex ^TM method, and their interaction with proteins can be obtained by covalent linkage through the incorporation of bromodeoxyuridine and UV-activated cross-linking (photoaptamers) enhanced. Photocrosslinking with ligands reduces aptamer cross-reactivity due to specific steric requirements. Aptamers have the advantages of easy production by automated oligonucleotide synthesis, DNA stability, and robustness; for photoaptamer arrays, universal fluorescent protein stains can be used to detect binding.

Detection of protein analytes bound to antibody arrays can be performed directly or via secondary antibodies in sandwich assays. Direct markers are used to compare different samples with different colors. If antibody pairs directed against the same protein ligand are available, sandwich immunoassays offer high specificity and sensitivity and are therefore the method of choice for low-abundance proteins such as cytokines; the assay also detects protein modifications . Label-free detection methods including mass spectrometry, surface plasmon resonance, and atomic force microscopy avoid ligand modification. Optimum sensitivity and specificity, as well as low background for high signal-to-noise ratio, are required for any method. Due to the wide range of analyte concentrations, appropriate adjustments to the sensitivity are required; solutions to this problem are serial dilutions of the sample or the use of antibodies of different affinities. The target protein is often a protein with a very low concentration in body fluid or extract, such as a cytokine or a product with low expression in cells, so detection in the pg range or lower is required.

An alternative to arrays of capture molecules is achieved by "molecular imprinting" techniques, in which peptides (e.g., from the C-terminal region of a protein) are used as templates to create structurally complementary sequence-specific cavities in a polymerizable matrix; the cavities The (denatured) protein can then be specifically captured with the appropriate primary amino acid sequence (ProteinPrint ^™ , Aspira Biosystems, Burlingame, CA).

Another method that can be used for diagnosis and expression profiling is ProteinChip array (Ciphergen, Fremont, CA) in which a solid phase chromatographic surface binds proteins with similar charge or hydrophobicity characteristics in a mixture such as plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to detect the retained proteins.

Large-scale functional chips are constructed by immobilizing a large number of purified proteins and used to measure various biochemical functions, such as protein interactions with other proteins, drug-target interactions, enzyme-substrate interactions, etc. Usually they require an expression library, cloned into E. coli, yeast, etc., from which the expressed protein is purified and immobilized, for example via a His-tag. Cell-independent protein transcription/translation is a viable alternative for the synthesis of proteins that cannot be expressed well in bacteria or other in vivo systems.

Protein arrays can be an in vitro alternative to the cell-based yeast two-hybrid system for the detection of protein-protein interactions, and when the two-hybrid system is deficient, such as when the interaction involves secreted proteins or proteins with disulfide bridges. This array is useful for proteins. High-throughput analysis of the biological activity of yeast protein kinases and various functions of the yeast proteome (protein-protein and proteolipid interactions) using arrays has been described, where most of all translatable frames of yeast Expressed and immobilized on microarrays. Large "proteome chips" hold promise for identifying functional interactions, drug screening, and more (Proteometrix, Branford, CT).

As a two-dimensional display of individual elements, protein arrays can be used to screen phage or ribosome display libraries for specific binding partners, including antibodies, synthetic scaffolds, peptides, and aptamers. "Library-to-library" screening can be performed in this manner. Another application of this method is the screening of drug candidates from combinatorial chemical libraries against an array of protein targets identified in genomic projects.

A multi-bead assay such as the BD ^™ cytometric bead array is a series of spectrally discrete particles that can be used to capture and quantify soluble analytes. The analytes are then determined by fluorescence emission detection and flow cytometric analysis. Multiple bead assays generate comparable data to ELISA-based assays, but in a "multiplex" or simultaneous fashion. Unknown concentrations for cytometric bead arrays were calculated in the same manner as for any sandwich format assay, ie by using known standards and plotting the unknowns onto a standard curve. In addition, multi-bead assays enable the quantification of soluble analytes in samples that were previously never considered due to sample volume limitations. In addition to quantitative data, effective visual images can be produced that display unique spectra or features that provide additional information at a glance for the user.

The MMP/TIMP spectra disclosed herein are based on measurements of MMPs or TIMPs alone. Their amount can be measured by any method known to provide a usable indication of how many MMPs or TIMPs are present in the analyzed sample. Examples of measurement methods are given in the Examples section. Methods of quantification of an analyte (eg, MPP or TIMP) include the measurement of the analyte when it is absent or present in undetectable amounts.

The technique and method for measuring MMPs and TIMPs which form the basis of the present invention is based on highly sensitive immunoassays. Several of these immunoassays were developed in this laboratory (ie, the measure of the TIMP-4 assay). Immunoassays were performed by enzyme-linked immunoassay (ELISA) standardized to provide the measurements shown in Table 4. However, other, more sensitive and rapid methods for measuring blood levels of MMPs and TIMPs have been implemented in our laboratory, which include the use of multiplex assay systems. In this example, a bead-based multiplex sandwich immunoassay can be used to measure multiple analytes in a limited volume of sample, such as plasma or other biological sample. This new technology for multiplexing is based on combining the sensitivity of ELISA with flow cytometry, allowing the specific measurement of up to 100 different analytes in a single sample of less than 50 μl. This method allows the measurement of multiple MMPs and TIMPs in small blood samples. Such methods are well suited for the diagnostic, prognostic, prognostic and therapeutic monitoring applications described herein. Specifically, for simultaneous measurement of analyte concentration, microbeads are incubated with a sample (ie, blood sample), and the microbeads are complexed with the specific analyte of interest (ie, MMP). A detection antibody (biotinylated) specific for a second epitope on each analyte is then added to the mixture and allowed to bind to the analyte-bound beads. This mixture was then incubated with a fluorescent reporter molecule (streptavidin-phycoerythrin) and the entire sample was passed through a dual laser flow cytometer. One laser detects the exact fluorescence of the beads for the specific analyte being detected, and the other detects the amount of fluorescence of the reporter molecule, which is proportional to the amount of bound analyte. This method has been used for a variety of MMPs, as well as other analytes that may be involved in the CHF process, as shown in Figure 8 and Table 1. This is just one example of how single or multiple analytes can be measured with very small blood samples. Other examples of measurements related to MMP/TIMP analytes that have been performed include radioimmunoassays and immunoblot assays. These methods are also antibody-based.

Table 1: Analyte concentration ranges used for calibration and linear regression statistics for calculated standard curves

7. Antibodies

Antibodies specific for MMPs and TIMPs are known and commercially available. Table 2 gives examples of antibodies.

Table 2: MMP/TIMP Antibodies

The term "antibody" is used herein in a broad sense and includes polyclonal antibodies and monoclonal antibodies. In addition to intact immunoglobulins, the term "antibody" also includes fragments or polymers of said immunoglobulin molecules and human or humanized forms of immunoglobulin molecules or fragments thereof, as long as they are based on their interaction with MMPs or TIMPs. The ability to use it can be used to select it. Antibodies can be tested for the desired activity using the in vitro assays described herein, or similar methods, after which their in vivo therapeutic and/or prophylactic activity is determined according to known clinical testing procedures.

The "term" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for possible natural mutations that may be present in a small number of antibody molecules. The monoclonal antibodies herein specifically include "chimeric antibodies" and fragments thereof, as long as they show the desired antagonistic activity, wherein a part of the heavy chain and/or light chain in the "chimeric antibodies" is derived from a specific Species of antibodies or antibodies belonging to a specific antibody class or subclass of the corresponding sequence is identical or homologous to the corresponding sequence of antibodies from another species or antibodies belonging to another antibody class or subclass, and the rest of the chain Identical or homologous (see US Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be prepared by any method for producing monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using the hybridoma method, e.g., as described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma approach, a mouse or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or produce antibodies that specifically bind the immunizing agent.

Monoclonal antibodies can also be prepared using recombinant DNA methods, such as described in US Patent 4,816,567 (Cabilly et al.). DNA encoding the disclosed monoclonal antibodies is readily isolated and sequenced by conventional methods (eg, using oligonucleotide probes that bind specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display technology, eg, as described in US Patent No. 5,804,440 to Burton et al. and US Patent No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies. Antibodies can be digested using routine methods known in the art to produce antibody fragments, particularly Fab fragments. For example, papain can be used for digestion. Examples of papain digestion are described in WO 94/29348, published December 22, 1994, and U.S. Patent 4,342,566. Papain digestion of antibodies typically yields two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, as well as a remaining Fc fragment. Pepsin treatment yields a fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

Such fragments may also contain insertions, deletions, substitutions or other selected modifications of specific regions or of specific amino acid residues, whether or not linked to other sequences, provided that they are not significantly modified compared to the unmodified antibody or antibody fragment. Alter or impair the activity of the antibody or antibody fragment. Such modifications may confer certain other properties, eg, removal/addition of amino acids capable of disulfide bonding, increase of its biological longevity, alteration of its secretion profile, etc. In any event, the antibody or antibody fragment must have biological activity, eg, specifically bind to its cognate antigen. Functional or active regions of the antibody or antibody fragment can be identified by mutagenizing specific regions of the protein followed by expression and testing of the expressed polypeptide. Such methods will be apparent to those skilled in the art and may include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment (Zoller, MJ. Curr. Opin. Biotechnol. 3:348-354, 1992).

The term "antibody" as used herein may also refer to human antibodies and/or humanized antibodies. Many non-human antibodies (such as those derived from mice, rats, or rabbits) are naturally antigenic to humans and, therefore, produce an adverse immune response when administered to humans. Thus, the use of human or humanized antibodies in the methods reduces the likelihood that the antibodies administered to humans will elicit an adverse immune response.

8. Reference value

MMP and/or TIMP profiles indicative of the presence of DHF in a subject or predictive of the occurrence of DHF in a subject are provided. The MMP and/or TIMP profile indicative of the presence of DHF in a subject or predictive of the occurrence of DHF in a subject may be a value relative to normal values. A normal value for a given analyte (MMP or TIMP) may be the reference value for an age-matched subject who has been shown to have no overt evidence of cardiovascular disease. Thus, the normal value may be a population value from a large number of healthy individuals. These reference normal values can be obtained through population studies. For example, a large population study has determined that the relative level of TIMP-1 in the reference group (Framingham Heart Study, Circulation 2004; 109: 2850-2856) is about 800 ng/mL, which is consistent with the reference control value disclosed herein.

Alternatively, the normal value may be a value considered normal for a given subject. For example, a baseline measurement of an analyte of interest in a healthy individual can be taken and used for comparison with subsequent measurements obtained from that individual to identify current disease or progression towards hypertensive heart disease.

Discontinuous observations (such as for MMP-13) convert continuous variables (such as plasma concentration of a given analyte) into dichotomous variables. In this particular example, a +/- value can be assigned to MMP-13 if a value greater than 10 ng/mL is considered a detectable or positive value and a value less than 10 ng/mL is considered a negative value.

For example, a method for diagnosing the absence of LVH associated with hypertensive heart disease in a subject is provided, comprising measuring the levels of MMP and/or TIMP in tissues or body fluids of the subject, and comparing the levels with Compare with the reference value. Therefore, normal values of MMP-2, MMP-9, MMP-7, MMP-13, MMP-8, TIMP-1, TIMP-2, and/or TIMP-4 indicate the absence of hypertensive heart disease-related sign of left ventricular hypertrophy.

In certain aspects, MMP-2 plasma levels within the normal range indicate the absence of LVH associated with hypertensive heart disease. In certain aspects, MMP-9 plasma levels within the normal range indicate the absence of LVH associated with hypertensive heart disease. In certain aspects, MMP-13 plasma levels within the normal range indicate the absence of LVH associated with hypertensive heart disease. In certain aspects, TIMP-1 plasma levels within the normal range indicate the absence of LVH associated with hypertensive heart disease. In certain aspects, TIMP-2 plasma levels within the normal range indicate the absence of LVH associated with hypertensive heart disease. In certain aspects, TIMP-4 plasma levels within the normal range indicate the absence of LVH associated with hypertensive heart disease.

In certain aspects, MMP-2 plasma levels above about 1000 ng/ml, including above about 1000, 1100, 1200, 1300, 1400, and 1500 ng/ml, are indicative of the absence of LVH associated with hypertensive heart disease.

In certain aspects, the MMP-9 plasma level is less than about 20 ng/ml, including less than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 , 5, 4, 3, 2 or 1ng/ml, it means that there is no LVH associated with hypertensive heart disease.

In certain aspects, the detectable plasma level of MMP-13 is above about 5 ng/ml, including below about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 ng/ml , which means the absence of LVH associated with hypertensive heart disease.

In certain aspects, the plasma level of TIMP-1 is less than about 1000 ng/ml, including greater than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, or 10 ng /ml, it means that there is no LVH associated with hypertensive heart disease.

In certain aspects, the plasma level of TIMP-2 is less than about 50 ng/ml, including greater than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 35, 30, 25, 20 , 15 or 10ng/ml, it means that there is no LVH associated with hypertensive heart disease.

In certain aspects, the plasma level of TIMP-4 is less than about 2 ng/ml, including greater 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 0.1 ng/ml , which means the absence of LVH associated with hypertensive heart disease.

The method may further comprise measuring plasma levels of two or more MMPs and/or TIMPs. For example, the method may comprise measuring two, three, four, Five, six, seven or eight. Thus, the method may comprise measuring 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. Accordingly, the method may comprise measuring 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. Accordingly, the method may comprise measuring 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. Thus, the method may comprise measuring MMP-2, MMP-13, TIMP-1, TIMP-2 and TIMP-4. Other combinations of these analytes are also contemplated and disclosed herein.

The method may further comprise calculating a ratio of one or more MMPs or TIMPs to other MMPs or TIMPs, for example the method may comprise calculating a ratio of MMP-9 to TIMP-1, TIMP-2 or TIMP-4.

For example, in certain aspects, the ratio of MMP-9/TIMP-1 plasma levels is greater than about 7×10 ³ , including greater than about 7×10 ³ , 8×10 ³ , 9×10 ³ , 10×10 ³ , 11× When it is 10 ³ , 12×10 ³ , 13×10 ³ or 14×103 , it means that there is no LVH related to hypertensive heart disease.

In certain aspects, a ratio of MMP-9/TIMP-2 plasma levels greater than about 10×10 ⁴ , including greater than about 10×10 ⁴ , 20×10 ⁴ , 30×10 ⁴ or 40×10 ⁴ , is not Presence of LVH associated with hypertensive heart disease.

In certain aspects, a ratio of MMP-9/TIMP-4 plasma levels greater than about 1, including greater than about 1, 2, 3, 4, 5, 6, 7, 8, or 9, indicates the absence of hypertensive Heart disease-associated LVH.

The reference normal values and values measured during the screening of patients with high blood samples are shown in Table 3. In this example, hypertensive patients with LVH will have decreased MMP-2 values but no MMP-7 values. However, MMP-13 will be discontinuously observed because it is undetectable in hypertensive patients with LVH. Therefore, a cut-off point below 10 ng/mL is considered a diagnostic criterion for hypertension and heart failure. TIMP-1 and TIMP-4 levels can be up to 50% higher in hypertensive patients with LVH compared to reference control values. The MMP-9/TIMP-4 ratio is reduced by more than 50% in hypertensive patients with LVH compared to reference normal values.

Table 3: MMP and TIMP Data; Reference Normal Values and Hypertensive Heart Disease; Diagnosis Percent Cutpoints

NC = no change from normal

*p<0.05 compared to normal

9. LVH rapid screening

Provides a quick "yes/no" type result obtainable by measuring the level of one specific MMP, MMP-13. A set point - which can be adjusted for changes in age and population demographics - can be used as the valid readout. For example, MMP-13 levels below a threshold setting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mL would necessitate more intensive plasma screening and other cardiovascular imaging studies. In other words, this rapid screening test can be applied to any large cohort and can then identify those subjects who warrant more rigorous testing and follow-up. There are currently no rapid screening tests available to identify patients with LVH.

Provided is a method for predicting diastolic heart failure in a subject, comprising measuring the amount of MMP-13 in the body fluid of the subject, the amount being less than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ng/ mL or undetectable indicates the presence of LVH and predicts DHF. When combined with abnormal measurements of other relevant analytes disclosed herein, the above measurements can detect DHF.

Plasma profiling can be performed at the time of preliminary testing or medical screening. The screening measurements described above can be performed for one or more MMPs and/or TIMPs. If the one or more measurements fall outside the reference values, additional measurements may be made. For example, an initial screen can be performed with MMP-13 so that if MMP-13 is not detected, another assay can be performed on the plasma sample. Similarly, MMP-9 and TIMP-1, TIMP-2 and/or TIMP-4 can also be used for initial screening, such that if the ratio of MMP-9 to TIMP-1, TIMP-2 or TIMP-4 is lower than When the normal limits of the threshold are reached, another assay can be performed on the plasma sample. The other assay can be used to obtain the entire spectrum shown in Table 3 or a part thereof. If the spectrum meets the criteria for hypertensive heart disease, the patient can be evaluated by more robust tests, which can include echocardiographic imaging, catheterization, nuclear imaging as appropriate. Patients may also be evaluated for more intensive medical treatments.

10. Diagnosis

Also provided is a diagnostic method useful, for example, in a subject exhibiting signs and symptoms of CHF, but the underlying cause of the manifestations is difficult to determine. This occurs extremely frequently; when a patient has CHF, it is difficult to determine whether LVH and DHF are present and whether they contribute to the exacerbation of the CHF process. The application of a simple and rapid blood test to "confirm" or "rule out" the presence of LVH and DHF as described in this application can provide this needed diagnostic method. In particular, the blood sample can be measured for MMP-13, MMP-9, MMP-2, TIMP-1 and/or TIMP-4. The values obtained can be compared with normal reference values disclosed herein. If this value deviates from the normal limits represented by the thresholds established herein, the patient can be determined to have DHF.

For example, a method of diagnosing LVH in a subject is provided, comprising measuring the levels of MMPs and/or TIMPs in tissues or body fluids of the subject, and comparing the levels with reference values.

In certain aspects, hypertensive heart disease is indicated by lower than normal plasma levels of MMP-2. For example, an amount of MMP-2 that is at least about 20% lower than the normal mean may indicate hypertensive heart disease. In certain aspects, the MMP-2 plasma level is less than about 1000 ng/ml, including less 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 100ng/ml, that means hypertensive heart disease.

In certain aspects, hypertensive heart disease is indicated by plasma levels of MMP-9 above normal. For example, an amount of MMP-9 that is at least about 50% above the normal mean may indicate hypertensive heart disease. In certain aspects, the MMP-9 plasma level is greater than about 20 ng/ml, including greater 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 100ng/ml, it means hypertensive heart disease.

In certain aspects, LVH is indicated by undetectable plasma levels of MMP-13. In certain aspects, LVH is indicated by a plasma level of MMP-13 below about 10 ng/ml, including below about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/ml.

In certain aspects, elevated plasma levels of TIMP-1 are indicative of hypertensive heart disease. For example, an amount of TIMP-1 that is at least about 50% above the normal mean may indicate LVH. In certain aspects, the plasma level of TIMP-1 is greater than about 1000 ng/ml, including greater than about 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1150, 1200, 1250, 1300 , 1350, 1400 or 1500ng/ml when there is LVH.

In certain aspects, LVH is indicated by plasma levels of TIMP-2 above normal. For example, an amount of TIMP-2 that is at least about 50% above the normal mean may indicate LVH. In certain aspects, TIMP-2 plasma levels above about 50 ng/ml, including above about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng/ml, are indicative of LVH.

In certain aspects, LVH is indicated by plasma levels of TIMP-4 above normal. For example, an amount of TIMP-4 that is at least about 50% above the normal mean may indicate LVH. In certain aspects, TIMP-4 plasma levels above about 2 ng/ml, including above about 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/ml, are indicative of LVH.

In certain aspects, MMP-7 plasma levels within the normal range can be indicative of LVH. In certain aspects, MMP-8 plasma levels within the normal range can be indicative of LVH.

The method may further comprise measuring plasma levels of two or more MMPs and/or TIMPs. For example, the method may comprise measuring two, three, four, Five, six, seven or eight. Thus, the method may comprise measuring 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; 2. MMP-13 and TIMP-1; MMP-2, MMP-13 and TIMP-2; MMP-2, MMP-13 and TIMP-4; or MMP-2, MMP-13, TIMP-1, TIMP-2 and TIMP-4. Other combinations of the above analytes are contemplated and disclosed herein.

For example, DHF can be detected when TIMP-1 is increased (TIMP-1 >1200 ng/mL) and MMP-13 levels are decreased. As another example, when the amount of TIMP-4 is higher than 3 ng/mL, and the level of MMP-13 decreases and the level of TIMP-1 increases, it indicates LVH and can predict DHF. Accordingly, a method of detecting LVH in a subject and predicting diastolic heart failure in the subject comprises measuring the profiles of MMP-13, TIMP-1 and TIMP-4 in a body fluid of the subject. The amount of MMP-13 is undetectable, the amount of TIMP-1 is about 50% higher than normal (or higher than 1200 ng/mL), and the amount of TIMP-4 is at least about 50% higher than normal (or higher than 3 ng/mL) ) when the spectrum predicts DHF.

The method may further comprise calculating a ratio of one or more MMPs or TIMPs to other MMPs or TIMPs. For example, the method can comprise calculating the ratio of MMP-9 to TIMP-1, TIMP-2 or TIMP-4.

In certain aspects, LVH is indicated when the ratio of MMP-9/TIMP-1 plasma levels is less than normal. For example, an MMP-9/TIMP-1 ratio that is at least about 50% less than the normal mean may indicate LVH. For example, in certain aspects, the ratio of MMP-9/TIMP-1 plasma levels is less than about 7×10 ³ , including less than about 7×10 ³ , 6×10 ³ , 5×10 ³ , 4×10 ³ , 5×10 3 When it is 10 ³ , 6×10 ³ , or 1×10 ³ , it means that there is LVH.

In certain aspects, LVH is indicated when the ratio of MMP-9/TIMP-2 plasma levels is less than normal. For example, an MMP-9/TIMP-2 ratio that is at least about 50% less than the normal mean may indicate LVH. In certain aspects, the ratio of MMP-9/TIMP-2 plasma levels is less than about 100×10 ³ , including 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 or 10×10 ³ , that means there is LVH.

In certain aspects, LVH is indicated when the ratio of MMP-9/TIMP-4 plasma levels is less than normal. For example, an MMP-9/TIMP-4 ratio that is at least about 50% less than the normal mean may indicate LVH. In certain aspects, the ratio of MMP-9/TIMP-4 plasma levels is less than about 3, including 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 0.01, that means there is LVH.

In certain aspects, the ratio of MMP-9/TIMP-1 plasma levels is less than about 5×10 ³ , the ratio of MMP-9/TIMP-2 plasma levels is less than about 100×10 ³ , and the ratio of MMP-9/TIMP-4 plasma levels A ratio of levels less than about 1 indicates LVH.

In certain aspects, the MMP-2 plasma level is less than about 1000 ng/ml, the MMP-13 plasma level is less than about 5 ng/ml, the MMP-9/TIMP-1 plasma level ratio is less than about 5×10 ³ , the MMP-9/TIMP -2 LVH is indicated when the ratio of plasma levels is less than about 100 x 10 ³ and the ratio of MMP-9/TIMP-4 plasma levels is less than about 1.

11. Prognosis

Also provided is a prognostic method useful for, for example, diastolic heart failure in a subject selected for screening and subsequent plasma profiling who is confirmed to have severe LVH and has developed DHF risk. In this case, MMP-13 levels as well as TIMP levels were quantified. MMP-13 levels were low/undetectable (0-5 ng/mL), and TIMP levels were high compared to control normal subject TIMP levels (e.g., TIMP-1 >1200 ng/mL, TIMP-2 >700 ng/mL, and and/or TIMP-4 >3 ng/mL), important observations reflecting the degree of myocardial fibrosis and diastolic dysfunction may be made. This provides prognostic value regarding the development of symptoms and the course of hospitalization. Specifically, these patients could be further intensively treated with hypertension drugs and more routinely studied with cardiovascular imaging.

For example, there is provided a method of identifying patients at increased risk of developing diastolic heart failure (DHF), comprising measuring the levels of MMPs and/or TIMPs in tissues or body fluids of said subjects, and comparing said levels to a reference value Compare.

In certain aspects, lower than normal plasma levels of MMP-2 are associated with an increased risk of developing diastolic heart failure. For example, an amount of MMP-2 that is at least about 20% lower than the normal mean may indicate an increased risk of developing diastolic heart failure. In certain aspects, a plasma level of MMP-2 of less than about 500 ng/ml, including less than about 500, 450, 400, 350, 300, 250, 200, 250, or 100 ng/ml, is indicative of diastolic heart failure Increased risk.

In certain aspects, undetectable plasma levels of MMP-13 indicate an increased risk of developing diastolic heart failure. In certain aspects, a plasma level of MMP-13 of less than about 10 ng/ml, including less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/ml, is indicative of diastolic cardiac stress Increased risk of failure.

In certain aspects, higher than normal plasma levels of TIMP-1 are associated with an increased risk of diastolic failure. For example, an amount of TIMP-1 that is at least about 50% higher than the normal mean may indicate an increased risk of developing diastolic heart failure. In certain aspects, the plasma level of TIMP-1 is greater than about 1000 ng/ml, including greater 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 2000ng/ml, it means that the risk of diastolic heart failure increases.

In certain aspects, higher than normal plasma levels of TIMP-2 are associated with an increased risk of diastolic failure. For example, an amount of TIMP-2 that is at least about 50% higher than the normal mean may indicate an increased risk of developing diastolic heart failure. In certain aspects, the plasma level of TIMP-2 is greater than about 50 ng/ml, including greater than about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140 , 150, 160, 170, 180, 190 or 200 ng/ml, that means an increased risk of diastolic heart failure.

In certain aspects, higher than normal plasma levels of TIMP-4 are associated with an increased risk of diastolic collapse. For example, an amount of TIMP-4 that is at least about 50% higher than the normal mean may indicate an increased risk of developing diastolic heart failure. In certain aspects, TIMP-4 plasma levels are greater than about 2 ng/ml, including greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 , 45 or 50ng/ml, that means an increased risk of diastolic heart failure.

In certain aspects, MMP-9 plasma levels within the normal range can be indicative of an increased risk of developing diastolic heart failure. In certain aspects, MMP-7 plasma levels within the normal range can be indicative of an increased risk of developing diastolic heart failure. In certain aspects, MMP-8 plasma levels within the normal range may indicate an increased risk of developing diastolic heart failure.

The method may further comprise measuring plasma levels of two or more MMPs and/or TIMPs. For example, the method may comprise measuring two, three, four, Five, six, seven or eight. Thus, the method may comprise measuring 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. Accordingly, the method may comprise measuring 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. Accordingly, the method may comprise measuring 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. Thus, the method may comprise measuring MMP-2, MMP-13, TIMP-1, TIMP-2 and TIMP-4. Other combinations of these analytes are also contemplated and disclosed herein.

For example, there is provided a method of detecting diastolic heart failure in a subject comprising measuring the amount of MMP-13, TIMP-1, TIMP-4, and MMP-9 in a body fluid of the subject. Also provided is a method of predicting diastolic heart failure in a subject comprising measuring the amount of MMP-13, TIMP-1, TIMP-4, and MMP-9 in a body fluid of the subject. In these methods, it can be shown that the amount of MMP-13 is undetectable (or less than 10 ng/mL), the amount of TIMP-1 is about 50% higher than normal or higher than 1200 ng/mL, and the amount of TIMP-4 is higher than A profile at least about 50% above normal or greater than 3 ng/mL and an amount of MMP-9 at least about 50% above normal can detect LVH and DHF.

Also provided is a method of detecting diastolic heart failure in a subject comprising measuring the amount of MMP-13, TIMP-1, TIMP-4, and MMP-2 in a body fluid of the subject. Also provided is a method of predicting diastolic heart failure in a subject comprising measuring the amount of MMP-13, TIMP-1, TIMP-4, and MMP-2 in a body fluid of the subject. In these methods, the profile may show an undetectable amount of MMP-13 (or less than 10 ng/mL), an amount of TIMP-1 approximately 50% higher than normal (or greater than 1200 ng/mL), TIMP-1 The amount of 4 is at least about 50% higher than normal (or greater than 3 ng/mL), and the amount of MMP-2 is at least about 20% higher than normal.

The method may further comprise calculating a ratio of one or more MMPs or TIMPs to other MMPs or TIMPs. For example, the method may comprise calculating the ratio of MMP-9 to TIMP-1, TIMP-2 or TIMP-4.

In certain aspects, LVH is indicated when the ratio of MMP-9/TIMP-1 plasma levels is less than normal. For example, an MMP-9/TIMP-1 ratio of at least about 50% less than the normal mean may indicate an increased risk of developing diastolic heart failure. For example, in certain aspects, the ratio of MMP-9/TIMP-1 plasma levels is less than about 7×10 ³ , including less than about 7×10 ³ , 6×10 ³ , 5×10 ³ , 4×10 ³ , 5×10 3 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 ² or 1×10 ² , it means that the risk of diastolic heart failure increases.

In certain aspects, LVH is indicated when the ratio of MMP-9/TIMP-2 plasma levels is less than normal. For example, an MMP-9/TIMP-2 ratio that is at least about 50% less than the normal mean may indicate LVH. In certain aspects, the ratio of MMP-9/TIMP-2 plasma levels is less than about 100×10 ³ , including 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 ³ or 1×10 ³ , that means there is LVH.

In certain aspects, LVH is indicated when the ratio of MMP-9/TIMP-4 plasma levels is less than normal. For example, an MMP-9/TIMP-4 ratio that is at least about 100% less than the normal mean may indicate LVH. In certain aspects, the ratio of MMP-9/TIMP-4 plasma levels is less than about 1, including 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 When it is 0.01, it means that there is LVH.

Accordingly, the method provides a method for detecting or predicting diastolic heart failure in a subject comprising detecting that the ratio of MMP-9 to TIMP-4 in a body fluid of the subject is lower than the normal ratio provided. The method includes measuring that the ratio is at least about 50% lower than the normal ratio.

In certain aspects, the ratio of MMP-9/TIMP-1 plasma levels is less than about 5×10 ³ , the ratio of MMP-9/TIMP-2 plasma levels is less than about 100×10 ³ , and the ratio of MMP-9/TIMP-4 plasma levels A ratio of levels less than about 1 indicates LVH.

In certain aspects, the MMP-2 plasma level is less than about 1000 ng/ml, the MMP-13 plasma level is less than about 5 ng/ml, the MMP-9/TIMP-1 plasma level ratio is less than about 5×10 ³ , the MMP-9/TIMP -2 LVH is indicated when the ratio of plasma levels is less than about 100 x 10 ³ and the ratio of MMP-9/TIMP-4 plasma levels is less than about 1.

12. Guide therapeutic interventions

During treatment, low levels of MMP-13 and high levels of TIMP-1 can be used as monitoring indicators for drug efficacy. There are a variety of relevant clinical conditions for which the above indicators are highly appropriate. For example, when blood pressure is within "normal limits" in hypertensive patients, MMP-13 levels decrease and TIMP levels increase. The addition of certain hypertension drugs can then be used to normalize these biomarkers of myocardial fibrosis and diastolic heart failure. The goal of this method is to continuously measure the blood MMP and TIMP values shown in Table 3, and to increase the medication to bring these profiles within the normal reference range.

For hypertensive patients identified as having increased heart mass (size) due to hypertension, MMP/TIMP profiles can be used to track the adequacy of treatment. These identified specific profiles disclosed herein can be monitored and a shift of the MMP/TIMP profile towards the normal range can be used to determine that the treatment is effective.

MMP/TIMP profiles are based on measurements of individual MMPs or TIMPs. Their amounts can be measured by any known method to provide an acceptable indicator of their presence in the sample being analyzed. An example of the measurement method is given in the Examples section. Methods of measuring the amount of an analyte (eg, MMP or TIMP) include measurement of the amount of analyte in the absence or in the absence of analyte. The measurement techniques and methods used for MMPs and TIMPs forming the basis of this method are based on highly sensitive immunoassays. Several of these immunoassays were developed in this laboratory (ie, the TIMP-4 assay measurement).

The immunoassay, which was performed with an enzyme-linked immunoassay (ELISA), can be standardized to provide the measurements shown in Table 1. However, other, more sensitive and rapid methods of measuring blood levels of MMPs and TIMPs have been implemented in our laboratory, including the use of a multiplex assay system. In this example, a bead-based multiplex sandwich immunoassay can be used to measure multiple analytes in a limited volume of sample, such as plasma or other biological sample. This new technology for multiplexing is based on combining the sensitivity of ELISA with flow cytometry, allowing the specific measurement of up to 100 different analytes in a single sample of less than 50 μl. This method allows the measurement of multiple MMPs and TIMPs in small blood samples. Such methods find use in the diagnostic, prognostic, prognostic, and therapy monitoring applications described herein. Specifically, for simultaneous measurement of analyte concentration, microbeads are incubated with a sample (ie, blood sample) and allowed to form complexes with the specific analyte of interest (ie, MMP). A detection antibody (biotinylated) specific for a second epitope on each analyte is then added to the mixture and allowed to bind to the analyte-complexed beads. This mixture was then incubated with a fluorescent reporter molecule (streptavidin-phycoerythrin) and the entire sample was passed through a dual laser flow cytometer. One laser detects the exact fluorescence of the beads for the specific analyte being detected, and the other detects the amount of fluorescence of the reporter molecule, which is proportional to the amount of bound analyte. This method has been used for a variety of MMPs, as well as other analytes that may be involved in the CHF process, as shown in Figure 16 and Table 1. This is just one example of how single or multiple analytes can be measured with very small blood samples. Other examples of measurements related to MMP/TIMP analytes that have been performed in our laboratory include radioimmunoassays and immunoblot assays. These methods are also antibody-based.

13. Combination

The methods disclosed herein may further comprise detecting other markers of heart failure. For example, the methods disclosed herein can further comprise measuring the level of NT-proBNP in a tissue or bodily fluid of the subject, and comparing the level to a reference value. The methods disclosed herein may further comprise measuring the level of Troponin-I in the subject's tissue or body fluid, and comparing said level to a reference value.

14. Measurement timing

As described below and illustrated in other examples for screening and treatment monitoring, the timing of measurement should be context-specific. For screening, it can be done at any time when the subject is undergoing medical examination. Examples may include annual medical exams, health fairs, and screening by means of home devices. Thus, the disclosed diagnostic methods are useful for diagnosing subjects exhibiting signs and symptoms of CHF, but the underlying cause of the manifestations is difficult to ascertain.

There are at least three initial time points for MMP/TIMP profiling for the methods disclosed herein. Initial measurements can be performed in patients with an established history of hypertension who visit the clinic frequently. Initial measurements may be taken at health fairs that may lead to outpatient visits. Initial measurements may be performed in patients exhibiting symptoms due to hypertensive heart disease. Figures 9A-C show the sampling and diagnostic schemes for each case in each case.

Thus, the disclosed prognostic methods can be used to identify whether a subject exhibiting hypertension (high blood pressure) has LVH or is at risk of developing DHF. The disclosed prognostic methods can also be used to identify whether subjects exhibiting signs and symptoms of CHF have LVH and are at risk of developing diastolic heart failure (DHF). For example, the method can be used in a patient who complains to a physician of a disorder consistent with CHF. In this way, a doctor can use a blood test to determine if there is a MMP/TIMP profile consistent with LVH and DHF. This can guide physicians in developing further diagnostic tests and treatment plans.

Another example of the timing of blood sampling is when a patient is identified as having established LVH, the MMP/TIMP continuum can be used as a tool to predict the progression of DHF. These tests can be used as screening tools only once in any given subject, or multiple times in succession.

C. Kit

The kits disclosed herein relate to reagents that can be used to practice the methods disclosed herein. The kits may include any reagent or combination of reagents described herein or deemed necessary or beneficial for the practice of the methods disclosed herein. For example, a kit for assessing the risk of developing DHF in a subject is disclosed, wherein the components include the components described above. For example, the components of an MMP/TIMP kit may include the reagents necessary to complex the relevant MMP and/or TIMP of interest (see Table 3 for a list of relevant MMPs and TIMPs) with a detection reagent. In an example of an immunoassay method, a blood sample can be incubated with a fluorescently-labeled antibody directed against a specific MMP or TIMP, and after washing and non-specific binding clearance steps, the relative intensity of fluorescence to the target MMP or TIMP can be calculated by measuring the relative intensity of the fluorescence. The amount of antibody bound. It can be a very simple kit that can be used for screening, or a more complex system that measures multiple MMPs/TIMPs in a single sample. The rationale for a multilevel approach from measuring one MMP or TIMP of interest to simultaneously measuring multiple MMPs/TIMPs has been described above. For screening assays (ie, MMP-13), a small blood sample can be processed into plasma (centrifuged) and the plasma mixed with anti-MMP-13 antibodies. The mixture can be centrifuged again and the antibodies that specifically bind MMP-13 can be read by a fluorescence analysis system. The device and measurement system can easily be made into a small suitcase or tabletop system. The readings of the above system can indicate whether MMP-13 is below or above (as defined hereinabove) a particular measurement threshold.

D. Example

1. Example 1: Tissue Inhibitors of Matrix Metalloproteinases/Metalloproteinases: Changes in Proteolytic Factors of Matrix Components and the Relationship Between Structure, Function and Clinical Manifestations of Hypertensive Cardiac Diseases

Brief description of methods and results: 103 subjects were divided into 4 groups as follows: a) reference subjects (CTL) without evidence of cardiovascular disease; b) hypertensive (HTN), controlled blood pressure and no LV hypertrophy ; c) Hypertension, blood pressure controlled and LV hypertrophy (HTN&LVH), but no CHF; d) Hypertension, blood pressure controlled, LVH and CHF (HTN&LVH&CHF), plasma MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2, and Doppler echocardiography. All MMPs or TIMPs were not significantly altered in HTN patients compared with CTL. In HTN&LVH patients, MMP-9 and MMP-13 decreased, while MMP-9 increased. TIMP-1 was only elevated in HTN&LVH&CHF patients. TIMP-1>1200ng/mL predicted CHF.

Conclusions: Hypertensive patients with normal LV structure and function have normal MMP/TIMP profiles. Alterations in MMP profiles that tend to reduce ECM degradation are associated with LV hypertrophy and diastolic dysfunction. Elevated TIMP-1 indicates the presence of CHF. These data suggest that changes in the MMP/TIMP balance have an important role in the structure, function, and clinical manifestations of hypertensive heart disease,

method

Subjects: Two groups of subjects were recruited in this study: reference controls and LVH patients. Reference controls were identified through locally funded health fairs and volunteers from Medical University of South Carolina staff. Of the reference controls screened, 35% were included, 50% met one of the exclusion criteria listed below, and 15% were not allowed to participate. Identify patients with LVH by echocardiographic studies. Of the patients screened for echocardiography, 10% were included, 75% met one of the exclusion criteria listed below, and 15% were not cleared for participation. The two groups shared some exclusion criteria:

1) history of myocardial infarction, 2) regional wall motion abnormalities, 3) coronary artery reconstruction surgery, 4) amyloidosis, sarcoidosis, HIV, hypertrophic embolic cardiomyopathy, valvular heart disease, 5) ejection Score <50%, 6) malignancy, 7) severe renal or hepatic dysfunction, 8) rheumatic disease, 9) blood pressure >140/90mmHg.

103 subjects were enrolled in this study: 53 reference control subjects and 50 subjects who demonstrated LVH [LV wall thickness > 1.2 cm and/or LV mass index ≥ 125 gm/ ^m2 (Table 4 )]. Reference control subjects were subdivided into two groups based on the presence or absence of hypertension; 39 control subjects (referred to as "reference controls without hypertension") had no history of hypertension and no evidence of cardiovascular (CV) disease, no symptoms or signs of cardiovascular disease, no cardiovascular medications, and all echocardiographic measurements were within normal limits (Table 5); 14 patients (referred to as "reference controls with hypertension") had high arterial pressure Medical history, blood pressure controlled (medicated to reach JNC7 criteria, ie <140/90mmHg), no left ventricular hypertrophy (Chobanian AV, et al. 2003) and all echocardiographic measurements within normal ranges (Table 5).

Table 4: Demographic Data for Left Ventricular Structure/Function and MMP/TIMP

Abbreviations: Data expressed as mean + SEM, LV = left ventricle, LVH = hypertensive left ventricular hypertrophy, reference control = subjects without evidence of cardiovascular disease, IVRT = isovolumic relaxation time, PCWP = Pulmonary capillary wedge pressure, MMP = matrix metalloproteinase, TIMP = tissue inhibitor of MMP, * = p<0.05 compared to reference control.

Table 5: Reference Controls with and without Hypertension, LVH with and without CHF

Abbreviations: Data expressed as mean + SEM, LV = left ventricle, PCWP = pulmonary capillary wedge pressure, EDV = end-diastolic volume, Ea = effective arterial elastance, MMP = matrix metalloproteinase, TIMP = tissue inhibitor of MMP, LVH = hypertensive left ventricular hypertrophy, CHF = chronic heart disease. Significant differences among all 4 groups were analyzed by ANOVA and Tukey's multiple comparison test, *=p<0.05 compared with reference control without hypertension, #=p<0.05 compared with reference control with hypertension, Δ = p<0.05 compared to LVH without CHF.

LVH patients were subdivided into two groups based on the presence or absence of CHF. Twenty-three hypertensive patients with controlled blood pressure and LVF but no CHF were referred to as "LVH without CHF" (Table 5). The second subgroup consisted of 26 hypertensive patients with controlled blood pressure who had both LVH and CHF and was termed "LVH with CHF". All of these patients exhibited CHF as defined by the Framingham criteria (Levy D, et al. 1996), exhibiting diastolic abnormalities (decreased E'), increased stiffness (increased PCWP and increased PCWP/EDV ratio), and 6-minute travel distance Significantly reduced (979±86 feet in the LVH group with CHF compared to 1839±60 feet in the LVH group without CHF, p<0.05), EF >50%, therefore diastolic heart failure was present.

Medications for hypertension were selected and tested by the patients' attending physicians, not by the investigators. They include diuretics, renin-angiotensin-aldosterone antagonists (angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and aldosterone blockers), direct vasodilators (nitrates, hydralazine ), alpha adrenergic blockers, central nervous system blockers, aspirin, beta adrenergic blockers, and calcium channel blockers. The mean duration of antihypertensive treatment was 6.4±1.5 years.

echocardiography

Echocardiograms were acquired with a Sonos 5500 system equipped with an S-4MHz transducer. Measurements were performed using American Society of Echocardiography criteria (Sahn DJ, et al. 1978; Schiller NB, et al. 1989). LV and left atrial volumes were calculated using the disc method (Schiller NB, et al. 19). LV mass was calculated using the formula of Reichek and Devereux (Devereux RB, et al. 1986). Doppler measurements of mitral inflow E and A wave velocities, E/A ratio, E wave deceleration time, and isovolumic relaxation time (IVRT) were performed. Tissue Doppler (lateral mitral annulus) measurements of mitral E' and A' wave velocities. Pulmonary capillary wedge pressure (PCWP) was calculated using the formula 2+1/3E/E' (Nagueh SF, et al. 1998). Use the formula: end-systolic pressure/stroke volume to calculate the effective arterial rebound rate (Ea).

MMP/TIMP plasma measurement: gelatinase (MMP-2 and MMP-9), collagenase (MMP-13) and MMP tissue inhibitors (TIMP-1 and TIMP-2). Plasma and respective MMP standards were added to precoated wells containing antibodies to the MMP or TIMP of interest and washed. The resulting reactions were read at a wavelength of 450 nm (Labsystems Multiskan MCC/340, Helsinki, Finland). Due to the extremely low levels of MMP-13 present in plasma, MMP-13 was classified as detectable or undetectable.

Statistical analysis: MMP and TIMP were measured every 2 hours over a 6-hour period to calculate individual subjects for a subgroup (n=20) of reference control subjects using a one-way random effects ANOVA Coefficient of variation of MMP/TIMP measurements between and within individual subjects. The coefficients were calculated by multiplying the square root of the individual mean square error by 100. Intra-patient coefficient of variation for MMP-2 = 11.2 ± 1.1%, TIMP-1 = 8.5 ± 2.2%, TIMP-2 = 14.3 ± 1.7%. The internal coefficients of variation determined to determine the amount of variability inherent to the assay technique were less than 6% for all MMPs and TIMPs.

Initially, reference controls and LVH subjects were compared with a two-sided Student's t test. Comparisons were then analyzed between all four groups (reference control with hypertension, reference control without hypertension, LVH with CHF and LVH without CHF) using ANOVA and Tukery's multiple comparison test. A p value <0.05 was considered significant. Simple linear regression was used to examine the relationship between MMP and TIMP levels and LV structure and function. The relationship between MMP-13 and TIMP-1 levels and the presence of LVH and CHF was evaluated using the Mantel Hanzel ^χ2 test and receiver operating curves. Potential drug effects on structural, functional, or plasma data were examined first with univariate and then with multivariate regression analyses. Structural, functional, MMP or TIMP measurements were the dependent variables, while input drug was used as a dummy variable. Look at individual drugs first, then drug combinations.

The study protocol used in this study was approved by the Institutional Review Board of the Medical University of South Carolina. Written informed consent was obtained from all participants. The authors have obtained these data in their entirety and are responsible for their integrity. All authors have read and agreed to the original written manuscript.

result

Reference control vs. LVH

Structural/functional data : The age and sex distribution of reference control subjects was similar to that of LVH subjects (Tables 3 and 4). Compared with reference controls, LVH had higher systolic blood pressure, marked concentric remodeling manifested by a 60% higher LV mass index, no difference in end-diastolic volume, and a 40% lower LV end-diastolic volume-to-mass ratio. LV diastolic diastolic index and LV diastolic stiffness index were significantly abnormal in LVH compared with reference control: increased IVRT, increased E wave deceleration time, decreased E', increased pulmonary capillary wedge pressure, and compared with reference control ( 0.09±0.01mmHg/mL), the ratio of PCWP to LV end-diastolic volume was increased (0.16±0.01mmHg/mL in LVH, p<0.05), indicating LV instantaneous end diastolic operating stiffness Increase.

MMP and TIMP plasma profiles : MMP-2 decreased and MMP-9 increased in LVH compared to reference controls. There was a marked difference in the detectability of MMP-13 (Figure 1). 47% of reference controls had detectable levels of MMP-13, whereas MMP-13 was detectable in only 15% of LVH subjects ( ^χ2 = 17.89, p<0.001, odds ratio = 0.24). Plasma TIMP-1 was significantly elevated in LVH compared with reference controls. TIMP-2 and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios did not differ between reference controls and LVH.

Reference Controls Without Hypertension vs. Reference Controls With Hypertension

Structural/Functional Data : Reference control subjects without hypertension were used as age- and sex-matched reference controls for comparison with the reference controls with hypertension, LVH without CHF, and LVH with CHF groups. There were no significant differences in any demographic parameter of LV structure or function or in any echocardiographic measurement between reference controls without hypertension and those with hypertension (Tables 3 and 4). The left atrial maximum volume (LAMV) and emptying fraction (LAEF) (LAMV = 40 ± 2ml, LAEF = 42 ± 3%) of the reference control without hypertension were compared with the reference control with hypertension (LAMV = 42 ± 4ml , LAEF=43±2%) similarly.

MMP and TIMP Plasma Profiles : There were no significant differences in any MMP or TIMP plasma levels between reference control subjects without hypertension and reference controls with hypertension.

LVH without CHF vs. LVH with CHF

Structural/Functional Data : There were no significant differences in systolic blood pressure, LV volume, or mass between subjects with LVH without CHF and LVH with CHF (Table 5). However, diastolic function was significantly more impaired in LVH with CHF than in LVH without CHF. Compared with LVH without CHF, LVH with CHF had a slower diastolic diastolic index, a higher diastolic stiffness index, and a higher filling pressure index. Specifically, compared with reference controls without hypertension (10±0.4cm/s, 95%CI=9.3, 11) and reference controls with hypertension (9.8±0.5cm/s, 95%CI=8.1, 11) Compared to LVH patients without CHF, tissue Doppler E' was decreased (8.4 ± 0.4 cm/s, 95% confidence interval (CI) 7.4, 9.3). E' was further decreased in LVH with CHF (7.2 ± 0.5 cm/s, 95% CI = 6.2, 8.3). LVH patients without CHF PCWP was unchanged (13±2 mmHg, 95% CI=10.5, 15.2), but increased in LVH with CHF (17±2 mmHg, 95% CI=15.2, 17.7). The ratio of PCWP to LV end-diastolic volume was unchanged in LVH patients without CHF, but was significantly increased in LVH patients with CHF. Effective arterial recoil was increased in LVH without CHF and decreased in LVH with CHF. LAMV was elevated in LVH without CHF (LAMV=53±4ml, p<0.05 compared to reference control) and further increased in LVH with CHF (LAMV=70±5ml, compared to LVH without CHF p<0.05). LAEF was unchanged in LVH with CHF (LAEF=42±3%, p<0.05 compared with reference control), but increased in LVH with CHF (LAEF=48±2%, compared with LVH without CHF Compare).

MMP and TIMP plasma profiles : There were no significant differences in MMP-2, MMP-9, MMP-13, TIMP-2, or the MMP/TIMP ratio in LVH without CHF compared with LVH with CHF (Figure 1). However, TIMP-1 was significantly increased in LVH with CHF (1364±86 ng/ml, 95% CI=1185, 1543) compared to LVH without CHF (1092±77 ng/ml, 95% CI=933, 1252) . In fact, TIMP-1 was only increased in subjects with CHF. Compared with reference controls without hypertension (1000±42ng/ml, 95%CI=915,1085) and reference controls with hypertension (988±76ng/ml, 95%CI=824,1152), no TIMP-1 was unchanged in LVH patients with CHF.

Relationship between MMP and TIMP plasma profiles and LV structure and function: There is a clear association between TIMP-1 and the degree of LV remodeling. When TIMP-1 was increased, LV mass increased (r=0.30, p=0.005) and volume/mass ratio decreased (r=-0.56, p=0.001, Figure 2A). There was a clear association between TIMP-1 and the degree of diastolic dysfunction. When TIMP-1 increased, mitral valve E/A ratio decreased (r=-0.22, p<O.027), E' decreased (r=-0.62, p=0.001, Fig. 2B) and PCWP increased (r =0.28, p=0.013). Finally, there was a clear association between the degree of CHF and TIMP-1 levels. NYHA class III LVH subjects with CHF had higher mean TIMP-1 than NYHA class II LVH subjects with CHF. TIMP-1 levels >1200 ng/ml are predictive of LVH with CHF (χ ² =4.6, p=0.03, specificity=88% and positive predictive value=94%, odds ratio=3.54, 95% confidence interval=1.08 , 11.50). The area under the receiver operating characteristic curve (ROC) was 0.71.

There was no association between specific drug use and differences in LV structure, function, or plasma MMP/TIMP profiles between groups. Specifically, there were no differences in any MMP or TIMP levels between patients grouped by any drug or drug combination. However, it should be recognized that this study is not sufficient to fully understand the effects of drugs on LV structure, function, or plasma MMP/TIMP profiles. Accordingly, these data and analyzes should be interpreted with due care.

discuss

This study has three unique findings: 1) hypertensive patients with normal LV structure and function have a normal MMP/TIMP profile, 2) those with a tendency to reduce ECM degradation (decreased MMP-2, MMP-13, increased TIMP-1) Alterations in MMP and TIMP profiles are associated with LV hypertrophy and diastolic dysfunction, and 3) TIMP-1 increases predict the presence of CHF.

Although the substrates and actions of MMPs and TIMPs are pleiotropic, alterations in myocardial MMPs and TIMPs have predictable effects on the ECM (Spinale, FG. 2002; Chapman RE, et al. 2004). For example, MMP-2, a gelatinase, degrades basement membrane proteins, fibrillar collagen peptides, and newly synthesized collagen fibers. In the present study, MMP-2 was significantly decreased in hypertensive LVH patients. MMP-9, a gelatinase, has the same structural protein substrate as MMP-2, but the level of MMP-9 activity is much lower. However, MMP-9 can significantly affect important bioactive proteins/peptides (such as TGF-) as well as other "pro-fibrotic" proteins and pathways. Activation of pro-fibrotic pathways through increased MMP-9 would be expected to increase ECM accumulation. Therefore, the reduced MMP2 levels and elevated MMP-9 levels in LVH patients in this study may be a factor contributing to the structural and functional changes observed in hypertensive heart disease.

MMP-13 is a collagenase that is found at extremely low levels in plasma, making it difficult to quantify accurately even with highly sensitive assays. Therefore, MMP-13 is not reported as a quantitative value in this study, but the results are divided into two categories. Detectable levels of MMP-13 in the plasma of LVH patients were significantly reduced, and in patients with CHF and LVH, the levels were further reduced. This decrease in collagenase is expected to result in decreased fibrillar collagen turnover, decreased degradation and increased ECM accumulation.

MMP activity can be regulated at multiple levels, including not only transcriptional regulation, but also post-translational modifications such as TIMP binding. TIMPs bind active MMPs in a 1:1 relationship, inhibit MMP enzymatic activity and thus constitute an important control point for net ECM proteolytic activity (Spinale, FG. 2002; Chapman RE, et al. 2004; Brew K, et al. .2000). The present study showed that patients with LVH and CHF had elevated plasma levels of TIMP-1. Therefore, the balance between MMPs and TIMPs is changed in a direction that contributes to the reduction of ECM proteolytic activity, thereby promoting ECM accumulation. There are four known TIMPs, and the regulation of transcription by these molecules is not consistent (Brew K, et al. 2000). Inconsistent levels of TIMPs have been observed in animal models of heart failure and in patients with myocardial disease (Wilson EM, et al. 2002; Stroud RE. 2005). In the present study, a stable increase in TIMP-1 was observed in LVH patients with CHF. In contrast, only small increases in TIMP-2 were observed in LVH patients with or without CHF. These observations may highlight distinct functions and regulatory pathways for individual TIMPs during LV remodeling. A unique finding of this study is that a specific type of TIMP, TIMP-1, is strongly associated with the development of CHF. For patients with CHF and LVH, it is unclear whether increased TIMP-1 contributes to the development of CHF or is a consequence of developing CHF. However, it is clear that TMMP-1 is elevated only in patients with LVH and CHF, with plasma TIMP-1 values >1200 ng/ml predicting the presence of CHF. Therefore, this plasma analyte should be considered in the development of diagnostic criteria for heart failure with normal ejection fraction (diastolic heart failure), as well as in the design of new therapeutic management regimens for diastolic heart failure. However, it should be recognized that the cut-off value of TIMP-1 = 1200 ng/ml was selected post-poc rather than prospectively. Therefore, the validity of this predictive value should be interpreted with due caution and confirmed by other studies using a large prospective series design.

Alterations in MMP/TIMP that occur in patients with hypertensive heart disease can affect the growth regulation of extracellular and cardiomyocyte compartments, which together lead to concentric LV hypertrophy and increased collagen content. Collagen homeostasis is determined by the balance between synthesis, post-translational modification, and degradation. In hypertensive heart disease, Diez et al. and others have shown that elevated collagen content is associated with increased plasma markers of collagen synthesis, decreased collagen degradation, and decreased collagen turnover (Diez J, et al. 2002; Lopez B, et al. .2001a; Lopez B, et al. 2001b). The alterations in the MMP/TIMP profile discovered in this study disclose underlying mechanisms that may alter synthesis, degradation, and turnover.

Although there are many determinants of LV structural remodeling, blood pressure is one of the most important. However, data from this study suggest that, even when blood pressure is adequately controlled, persistent changes in MMPs and TIMPs predict, possibly determine, long-term concentric remodeling, LVH, and diastolic heart failure and are definitively associated with long-term concentric remodeling. , LVH, and diastolic heart failure. Resolution of LVH requires proper ECM remodeling, including degradation and turnover of ECM components (especially basement membrane proteins) and alterations in cardiomyocyte-matrix interactions. This study demonstrates that patients with hypertensive LVH have persistent abnormalities in specific MMP (decreased MMP-2) and TIMP (increased TIMP-1) profiles (decreased MMP-2, increased TIMP-1), which is expected to favor cardiomyocyte-basement membrane -Continuous attachment of the stroma, detrimental to the ECM turnover needed to restore LV mass. Thus, it appears that the persistent alterations in MMPs and TIMPs observed in this study may contribute to the phenotypic and structural changes present in hypertensive heart disease.

In this study, the plasma levels of MMP and TIMP were used as representative markers reflecting changes in the myocardial levels of the above enzymes and peptides. MMP activation and TIMP binding are compartmentalized processes that occur in the myocardial interstitium (Spinale, FG. 2002; Chapman RE, et al. 2004). Therefore, plasma levels do not necessarily reflect the net ECM proteolytic activity present in the myocardium. The observed differences in MMP and TIMP plasma levels between reference controls and patients with hypertensive heart disease in this study may reflect differences in myocardial levels (Joffs C, et al. 2001; Yarbrough WM, et al. 2003; Lindsey ML, et al. 2003). Myocardium may not be the only source of MMPs and TIMPs in LVH patients. Therefore, measurements of plasma MMP and TIMP levels represent the sum of MMP and TIMP released from cardiac as well as non-cardiac sources. However, the specific exclusion criteria used in this study helped to eliminate apparent changes in the major non-cardiac sources of MMPs and TIMPs. However, it is recognized that changes in other noncardiac tissues (eg, kidney and vasculature) in LVH hypertensive patients with or without chronic heart failure may contribute to the release of MMPs and TIMPs to plasma. The findings of the present study demonstrate differences in plasma MMP and TIMP levels between reference controls and LVH patients.

Conclusions: Specific altered patterns of ECM proteolytic systems are associated with each structural, functional and/or clinical manifestation of hypertensive heart disease. Subjects with adequate blood pressure control and no structural or functional changes in the left ventricle had no changes in MMP/TIMP indices. However, LVH patients showed decreased MMP-2 and MMP-13 even when blood pressure was adequately controlled. Increased TIMP-1 occurs in patients with LVH and CHF. Specifically, transitions between the development of hypertensive LVH and CHF were predicted by changes in MMPs and TIMPs, eg TIMP-1 >1200 ng/ml or the absence of MMP-13. However, the sample size in this study was limited, a cross-sectional design was used, and a long-term series was not conducted. These limitations necessitate further testing and corroboration of our observations using large prospective series. However, the data in this study demonstrate that the observed stochastic alterations in MMP/TIMP play an important role in the manifestation of hypertensive heart disease. An understanding of this ECM-dependent pathophysiology could help improve the diagnosis and treatment of patients with hypertensive heart disease.

Clinical Argument: Chronic arterial hypertension is a common cause of LV concentric hypertrophy, decreased relaxation rate, and increased stiffness. The structural and functional changes caused by hypertension are due to changes in the main components of the heart muscle, the cardiomyocytes, and in particular to changes in the extracellular matrix (ECM). These LV structural and functional changes generate substrates necessary for the development of diastolic heart failure (DHF). However, it remains unclear which factors control these changes in the ECM, whether blood pressure control alone can prevent or reverse these changes, and whether understanding the mechanisms of ECM control is helpful for the diagnosis or treatment of hypertensive heart disease. This study demonstrates that alterations in specific ECM proteolytic protein/peptide (MMP and TIMP) patterns are associated with each of the structural, functional and clinical manifestations of hypertensive heart disease. Subjects with adequate blood pressure control and no changes in LV structure or function had no changes in MMP/TIMP indices. Thus, treatment of hypertension prevents changes in the ECM and ECM proteolytic systems. However, patients with residual LVH or resistant LVH have abnormal MMPs despite adequate blood pressure control. The increase of TIMP-1>1200ng/ml predicted the occurrence of DHF. These data suggest that resolution of LVH and prevention of DHF do not depend solely on changes in blood pressure, but may require targeting and normalizing changes in MMP/TIMP. An understanding of this ECM-dependent pathophysiology could help improve the diagnosis and treatment of hypertensive patients.

2. Example 2: Matrix metalloproteinases/tissue inhibitors of metalloproteinases: the relationship between changes in the proteolytic factors of matrix components and the structure, function and clinical manifestations of hypertensive heart disease

method

Study Access: Table 6 shows the study access. Exclusion criteria were history of myocardial infarction, cardiomyopathy, abnormal valve or ventricular wall motion, arrhythmia, invasive heart disease, EF<50%, uncontrolled hypertension (SBP>140 or DBP>90) or affecting MMP/TIMP plasma spectrum of systemic disease. Inclusion criteria for controls and HTN controls were men and women aged 18-90 years without evidence of structural cardiovascular disease. Inclusion criteria for LVH and LVH with CHF were echocardiographically confirmed LV hypertrophy (wall thickness >1.2 cm or LV mass index >125 g/m ² ), males and females aged 18-90 years.

Table 6: Study Access

Echocardiographic measurements: Dimensional echocardiographic criteria were used.

Echocardiographic calculations: LV volumes were calculated using the disk method. LV mass was calculated by Penn's method. PCWP is calculated by 2+1.3×(E/Ea).

MMP/TIMP plasma measurements: Plasma measurements of gelatinase MMP-2 and MMP-9, collagenase MMP-13, and TIMP-like TIMP-1 and TIMP-2 were obtained by enzyme-linked immunosorbent assay (ELISA) (AmmershamPharmacia Biotech) .

result

Figures 7-11 show the results of the study.

in conclusion

HTN patients with normal LV structure and function have normal MMP/TIMP profiles. Alterations in the MMP/TIMP profile that tend to reduce ECM degradation are associated with LV hypertrophy and diastolic dysfunction. An increase in TIMP-1 predicted the presence of CHF. Changes in the cardiomyocyte extracellular matrix proteolytic system can be measured using plasma assays of selected MMPs and TIMPs. Each manifestation of hypertensive heart disease is associated with a specific pattern of alterations in the ECM proteolytic system. Hypertensive patients with structural remodeling, diastolic dysfunction, and/or overt CHF are characterized by decreased MMPs and increased TIMPs.

3. Example 4: Criteria for distinguishing, predicting and diagnosing heart failure in hypertensive patients

A definitive list of normal values for human subjects within the stated age ranges and across genders is provided in Table 7. A compiled list of normal reference values for MMP/TIMP as encompassed by the present invention has not been provided before, and since age-matched subjects without cardiovascular disease were included, Table 7 also provides normal reference ranges. Furthermore, new stoichiometric ratios of the MMP/TIMP profile are provided, demonstrating that this profile covers important diagnostic and prognostic information as detailed in the table below. These data were collected and analyzed from more than 100 subjects.

Table 7: Normal Human* Reference Ranges

*Normal adult age is 25-70 years old

Table 8 presents the absolute values of MMP and TIMP, the absolute value of the MMP/TIMP ratio, and the percent change from a normal reference value based on absolute values in patients diagnosed with hypertension but well-controlled. These values were collected as described in the original application. A unique plasma profile was demonstrated that could not have been predicted from previous animal study reports or previously published limited clinical studies. This unique profile includes a decrease in MMP-2, no change in MMP-9, undetectable MMP-13 (below the sensitivity of any currently used assay system), and a steady increase in TIMP-1 levels. Furthermore, an increase in the cardiovascular-specific marker TIMP-4 could be demonstrated. These changes in the MMP and TIMP profiles are characteristic of hypertensive patients and demonstrate early changes in the cardiac tissue of these patients. This unique, specific profile can be used to guide therapy to minimize changes in MMP and TIMP profiles relative to normal subjects. Moreover, these plasma profiles can be used to comprehensively screen at-risk patient populations and identify patients at risk for future adverse events.

Table 8: Diagnosis of hypertensive heart disease

*Patients diagnosed with hypertension and treated with appropriate medical treatment

Table 9 shows the plasma profiles of MMPs and TIMPs present in patients with hypertensive heart disease secondary to heart failure. These data were taken from the studies provided in the original application. This previous study showed that the absence of MMP-13 signaling and the steady increase of TIMP-1 can be used to judge the presence of heart failure in hypertensive patients. Indeed, receiver operating characteristic curves (ROC) for predicting and diagnosing heart failure have been provided previously. In marked contrast to patients with heart failure secondary to myocardial infarction (heart attack), MMP-9 levels were normal or below normal. A distinction can be made between these two disease states, the results of which are given in the table below. Furthermore, by using the cardiovascular-specific marker TIMP-4, it was demonstrated that TIMP-4 is increased in patients with hypertensive heart disease and provides cardiovascular specificity for this previously unproven plasma profile. These data provide the first differential profile for identifying patients with heart failure due to hypertensive heart disease by plasma markers. This is an important question because treatment approaches vary depending on the underlying cause of heart failure. How these data will be used to guide treatment and clinical decision-making is provided in the original application.

Table 9: Hypertensive Patients at Increased Risk of Heart Failure*

*Patients diagnosed with hypertension and treated with appropriate medical treatment

The unique plasma signature developed by this application and demonstrated in the supporting material provides for the first time the ability to distinguish the underlying etiology in patients with heart failure. Specifically, as shown in Table 10, unique and very different plasma profiles emerged in patients at risk of developing heart failure, or exhibiting heart failure secondary to myocardial infarction, or heart failure secondary to hypertension. These data are taken from the research we have done which forms the basis of this application. Therefore, a differential diagnosis of these spectrums, and more importantly, allows for more targeted clinical decision-making and treatment strategies. Examples of clinical applications of the profiles, and how they can be used for clinical decision-making, are given in the original application.

Table 10: Differential diagnosis of systolic (post-MI) or diastolic (hypertensive heart disease) heart failure*

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Claims (19)

1. A method for predicting diastolic heart failure in a subject, comprising measuring the amount of MMP-13 in the body fluid of the subject, and when the amount is less than 10 ng/mL, it indicates the presence of diastolic heart failure or predicts heart failure. 2. A method for predicting diastolic heart failure in a subject, comprising detecting that the amount of TIMP-1 in the body fluid of the subject is higher than normal. 3. The method of claim 2, wherein the amount of MMP-2 is at least about 20% above normal. 4. A method for predicting diastolic heart failure in a subject, comprising detecting that the amount of TIMP-4 in the body fluid of the subject is higher than normal. 5. The method of claim 4, wherein the amount of TIMP-4 is at least about 50% greater than normal. 6. A method of predicting diastolic heart failure in a subject comprising measuring the amount of MMP-13, TIMP-1 and TIMP-4 in a body fluid of said subject. 7. The method of claim 6, wherein the amount of MMP-13 is undetectable, the amount of TIMP-1 is at least about 50% higher than normal, and the amount of TIMP-4 is at least about 50% higher than normal . 8. A method of predicting diastolic heart failure in a subject comprising measuring the amount of MMP-13, TIMP-1, TIMP-2 and TIMP-4 in a body fluid of said subject. 9. The method of claim 8, wherein said amount of MMP-13 is undetectable, said amount of TIMP-1 is at least about 50% higher than normal, and said amount of TIMP-2 is at least about 50% higher than normal %, and the amount of TIMP-4 is at least about 50% higher than normal. 10. A method of predicting diastolic heart failure in a subject comprising measuring the amount of MMP-13, TIMP-1, TIMP-4, and MMP-2 in a body fluid of said subject. 11. The method of claim 10, wherein said amount of MMP-13 is undetectable, said amount of TIMP-1 is at least about 50% higher than normal, and said amount of TIMP-4 is at least about 50% higher than normal %, and the amount of MMP-2 is at least about 20% higher than normal. 12. A method of predicting diastolic heart failure in a subject comprising detecting a lower than normal ratio of MMP-9 to TIMP-1 in a body fluid of said subject. 13. The method of claim 12, wherein the ratio is at least about 50% lower than the normal ratio. 14. A method of predicting diastolic heart failure in a subject comprising detecting a lower than normal ratio of MMP-9 to TIMP-2 in a body fluid of said subject. 15. The method of claim 14, wherein the ratio is at least about 50% lower than the normal ratio. 16. A method of predicting diastolic heart failure in a subject comprising detecting a lower than normal ratio of MMP-9 to TIMP-4 in a body fluid of said subject. 17. The method of claim 16, wherein the ratio is at least about 50% lower than the normal ratio. 18. The method of any one of claims 1-17, wherein the bodily fluid is blood. 19. The method of any one of claims 1-17, wherein the body fluid is plasma.

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