WO2007014434A1

WO2007014434A1 - Biomarker for cardiovascular disease

Info

Publication number: WO2007014434A1
Application number: PCT/AU2006/001108
Authority: WO
Inventors: Carolyn L. Geczy
Original assignee: NewSouth Innovations Pty Ltd
Current assignee: NewSouth Innovations Pty Ltd
Priority date: 2005-08-04
Filing date: 2006-08-04
Publication date: 2007-02-08
Anticipated expiration: 2008-02-04

Abstract

The present invention relates to methods for determining the level of S100A12 in a subject, the method comprising the steps of obtaining a biological sample from the subject, and analysing the biological sample to determine the amount of S100A12 present wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease or a predisposition thereto.

Description

remain low in patients with stable angina (SA)₁ a condition which often leads to heart attack even though it is generally significantly raised in patients with unstable angina (UA) and upon acute myocardial infarction (2). Elevated plasma levels of CRP are associated with poor prognosis and predict future coronary events in population studies (3, 4). It is therefore clear that there remains a need for an improved marker of early cardiovascular risk.

The S100 Ca²⁺-binding protein family consists of 21 structurally-similar proteins that regulate key cellular processes. Some have extracellular cytokine-like functions including regulation of the cytoskeleton, proliferation, cell migration, adhesion, and host defense.

Murine (m) A8 is a potent chemoattractant and mA8-elicited macrophages exhibit a "pro- atherogenic phenotype", expressing high levels of CD11b/CD18, Fc and scavenger receptors which accumulate acetylated LDL in vitro and in vivo, with cholesterol ester profiles similar to those in human plaque and thus may contribute to potential foam cell (FC) development, and thus to the pathogenesis of atherosclerosis (5).

S100A12 is the likely functional homologue of mA8 (6) and has been demonstrated to recruit monocytes (7). It is also known as calgranulin C, and extracellular newly-identified RAGE-binding protein (EN-RAGE), and binds the receptor for advanced glycosylate end-products (RAGE).

Furthermore, ligation of S100A12 on endothelial cells (EC), monocytes and lymphocytes induces some genes (8) that may promote chronic inflammation.

The present invention is predicated on the surprising and unexpected finding by the inventor that serum levels of S100A12 are positively associated with cardiovascular disease in human subjects.

Summary of the Invention

According to a first aspect of the present invention there is provided a method for determining the level of S100A12 in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of S100A12 present, wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease or a predisposition thereto. Typically the method is used in conjunction with assessment of clinical symptoms and with determining the level of at least one other biomarker in the subject, wherein the amount of the at least one other biomarker is indicative of cardiovascular disease or a predisposition thereto. The at least one other biomarker may be selected from the group comprising CRP, troponin I, creatine kinase, creatine kinase MB, a cardiac index, myoglobin or interleukin-6. The sample may comprise whole blood, blood serum, blood plasma, urine or other bodily fluids. The sample may comprise blood leukocytes,

The step of analysing the sample to determine the amount of S100A12 may comprise measuring S100A12 polypeptide levels, for example using at least one anti-S100A12 antibody. The S100A12 may be measured using an immunoassay. The immunoassay may comprise an enzyme-linked immunoassay, a radioimmunoassay, or an immunoassay comprising a biosensor, typically comprising use of at least one anti-S100A12 antibody. The at least one anti- Si 00A12 antibody may be monoclonal or polyclonal or a combination thereof.

The cardiovascular disease may comprise coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina, deep vein thrombosis, stroke, congestive heart failure or arrhythmia. The angina may be stable angina or unstable angina.

The method may include comparing the level of S100A12 in the sample obtained from the subject with the level of S100A12 from one or more control samples. Typically, a control sample may comprise a sample from a subject with normal levels of S100A12 and/or known not to have the cardiovascular disease or a predisposition thereto.

According to a second aspect of the present invention there is provided a method for diagnosing cardiovascular disease or a predisposition thereto in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and (b) analysing the biological sample to determine the amount of S100A12 present wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease or a predisposition thereto.

Typically the method is used in conjunction with assessment of clinical symptoms and with determining the level of at least one other biomarker in the subject, wherein the amount of the at least one other biomarker is indicative of cardiovascular disease or a predisposition thereto. The at least one other biomarker may be selected from the group comprising CRP, troponin I, creatine kinase, creatine kinase MB, a cardiac index, myoglobin or interleukin-6.

The sample may comprise whole blood, blood serum, blood plasma, urine or other bodily fluids. The sample may comprise blood leukocytes. The step of analysing the sample to determine the amount of S100A12 may comprise measuring S100A12 polypeptide levels, for example using at least one anti-S100A12 antibody.

The S100A12 may be measured using an immunoassay. The immunoassay may comprise an enzyme-linked immunoassay, a radioimmunoassay, or an immunoassay comprising a biosensor, typically comprising use of at least one anti-S100A12 antibody. The at least one anti- S100A12 antibody may be monoclonal or polyclonal or a combination thereof. The cardiovascular disease may comprise coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina, deep vein thrombosis, stroke, congestive heart failure or arrhythmia. The angina may be stable angina or unstable angina.

The method may include comparing the level of S100A12 in the sample obtained from the subject with the level of S100A12 from one or more control samples, Typically a control sample may comprise a sample from a subject with normal levels of S100A12 and/or known not to have the cardiovascular disease or a predisposition thereto.

Elevated levels of S100A12 compared to controls may be indicative of cardiovascular disease or a predisposition thereto. The method may further comprise determining levels of at least one of S100A8 or S100A9 and comparing these levels with determined levels of S100A12.

The S100A8 and/or S100A9 may be measured using an immunoassay. The immunoassay may comprise an enzyme-linked immunoassay, a radioimmunoassay, or an immunoassay comprising a biosensor, typically comprising use of at least one anti-S100A8 antibody and/or at least one anti-S100A9 antibody. The at least one anti-S100A8 antibody and/or at least one anti- Si 00A9 antibody may be monoclonal or polyclonal or a combination thereof.

Elevated levels of S100A12 compared to controls and equivalent levels of at least one of S100A8 or S100A9 compared to controls may be indicative of cardiovascular disease or a predisposition thereto. Alternatively, elevated levels of both S100A12 and S100A8 compared to controls may be indicative of myocardial infarction or a predisposition thereto.

According to a third aspect of the present invention there is provided a method for diagnosing cardiovascular disease or a predisposition thereto in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of S100A12 and at least one of S100A8 or S100A9 present, wherein the amount of S100A12 and at least one of S100A8 or S100A9 present in the sample is indicative of cardiovascular disease or a predisposition thereto.

According to a fourth aspect of the present invention there is provided a method for diagnosing in a subject the over-expression of S100A12, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and (b) analysing the biological sample to determine the amount of S100A12 present. wherein over-expression of S100A12 in the sample is indicative of cardiovascular disease or a predisposition thereto.

The sample may comprise whole blood, blood serum, blood plasma, urine or other bodily fluids. The sample may comprise blood leukocytes. The S100A12 may be measured using an immunoassay. The immunoassay may comprise an enzyme-linked immunoassay, a radioimmunoassay, or an immunoassay comprising a biosensor, typically comprising use of at least one anti-S100A12 antibody. The at least one anti- Si 00A12 antibody may be monoclonal or polyclonal or a combination thereof.

The method may include comparing the level of S100A12 in the sample obtained from the subject with the level of S100A12 from one or more control samples. Typically a control sample may comprise a sample from a subject with normal levels of S100A12 and/or known not to have the cardiovascular disease or a predisposition thereto.

According to a fifth aspect of the present invention there is provided a diagnostic kit for use in determining the level of S100A12 in a subject, the kit comprising at least one agent for measuring S100A12 in a biological sample, wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease or a predisposition thereto. The agent may comprise an antibody that recognises and binds S100A12,

Accordingly, in one embodiment a kit may comprise a first container containing an antibody raised against S100A12 and a second container containing a conjugate comprising a binding partner of the antibody, together with a detectable label.

According to a sixth aspect of the present invention there is provided a diagnostic kit for use in diagnosing in a subject the over-expression of S100A12, the kit comprising at least one agent for measuring S100A12 in a biological sample, wherein the over-expression of S100A12 in the sample is indicative of cardiovascular disease or a predisposition thereto.

According to a seventh aspect of the present invention there is provided a diagnostic kit for use in diagnosing cardiovascular disease in a subject, the kit comprising at least one agent for measuring S100A12 in a biological sample, wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease or a predisposition thereto.

The kit may further comprise at least one agent for measuring S100A8 or S100A9.

For the purposes of the above aspects and embodiments, the subject may be a human or any other animal. In particular embodiments the subject is selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine.

Definitions

In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

The term "S100A12" as used herein refers to the member of the S100 protein family known as S100A12, calgranulin C, CGRP, CAAF1, CO-Ag or EN-RAGE,

The term "cardiovascular disease" as used herein refers to any condition, disorder or disease state associated with, resulting from or causing a structural or functional abnormality of the heart, or of the blood vessels supplying the heart, that impairs its normal functioning. Cardiovascular disease may comprise coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina, deep vein thrombosis, stroke, congestive heart failure or arrhythmia.

The term "angina" as used herein includes stable angina or unstable angina. The term "expression" as used herein refers interchangeably to expression of a gene or gene product, including the encoded protein. Expression of a gene product may be determined, for example, by immunoassay using an antibody(ies) that bind with the polypeptide. Accordingly, in the context of the present invention, expression may refer to the expression of an S100A12 polypeptide. As used herein the term "polypeptide" means a polymer made up of amino acids linked together by peptide bonds. Additionally or alternatively, the term "polypeptide" may include proteolytic fragments derived from S100A12.

The term "equivalent" as used herein means substantially the same as but not necessarily identical to. For example, in comparing levels of expression between samples, "equivalent" means that expression is not substantially increased nor substantially reduced in one sample compared to another.

Brief Description of the Drawings

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings: Figure 1. shows S100A12 expression in an atherosclerotic lesion. Typical patterns of expression of S100A12 (A₁ B, C) and RAGE (C) are shown in atherosclerotic arterial walls. A: S100A12 was localized in foam cells (open arrows) and non-foam cells (solid arrows) in an area of superficial plaque. B: S100A12+ cells (arrows) around neovessels (asterisks), showing S100A12 5 weakly expressed by neointimal endothelial cells. C: distribution of RAGE+ (brown) and S100A12+ (rose) inflammatory cells in the deep layer of atherosclerotic intima. Arrows indicate RAGE+/S100A12+ cells. D: S100A12+ cells in a section of an atherosclerotic plaque were recognized by an anti-S100A12 IgG antibody. L indicates the lumen of the vessel. Macrophage-like cells and foam cells (large arrows) and endothelial cells (small arrows) are indicated. Weak io S100A12 expression is seen in some smooth muscle cells (arrows with asterisks). The strongly positive cells within the lumen of the vessels are blood neutrophils which constitutively express S100A12.

Figure 2. shows a Western blot of plaque extracts expressing S100A12. Extracts (S1, S4, S5 and S6) were separated with and without 10OmM dithiothreitol (DTT) and blotted with an anti- i₅ S100A12 antibody. Arrows indicate the direction of migration of S100A12 standards (100ng) and molecular weight markers.

Figure 3. shows levels of IL6 released from peripheral blood mononuclear cells (PBMCs) after stimulation with 5μM S100A8, S100A9, S100A8/A9 and S100A12. Values are means from 2 blood donors +/- SEM.

20 Figure 4. shows levels of IL6 released from peripheral blood mononuclear cells (PBMCs) after stimulation with 5μM S100A12 at 6, 24 and 36 hours post-exposure. Values are means from 4 blood donors +/- SEM.

Figure 5. compares separate levels of S100A12 in serum from patients with stable angina (SA), unstable angina (UA) and control patients (control).

2₅ Figure 6. shows levels of S100A12 in serum from patients with either stable angina (SA) or unstable angina (UA) compared with control patients (control).

Figure 7. shows levels of (a) S100A12, (b) S100A8 and (c) S100A9 in serum from patients with either stable angina (SA) or unstable angina (UA) compared with control patients (control).

Figure 8. compares levels of S100A12 in serum from patients with stable angina (SA) and 3o unstable angina (UA) and from controls with levels of C reactive protein (CRP).

Figure 9. summarizes the results of analyzing levels of S100A8 in subsets of coronary artery disease patients, namely: (1) controls, (2) those with stable angina (SA), (3) those with acute coronary syndrome (ACS) and (4) those with acute myocardial infarction (Ml). Figure 10. summarizes the results of analyzing levels of S100A12 in subsets of coronary artery disease patients, namely: (1) controls, (2) those with stable angina (SA), (3) those with acute coronary syndrome (ACS) and (4) those with acute myocardial infarction (Ml),

Best Mode of Performing the Invention

The present invention provides a biomarker that is shown herein to be useful in determining levels of disease states in subjects, The biomarker is S100A12. S100A12 levels are shown to be positively correlated with cardiovascular disease. As exemplified herein, the present inventor has developed an ELISA test to detect serum levels of S100A12, In one embodiment, the ELISA test comprises a sandwich ELISA with two polyclonal antibodies that each recognize and bind a different epitope on S100A12. A polyclonal anti-S100A12 capture antibody is immobilized onto a solid surface, such as the well of a microtitre plate or a bead, and a polyclonal anti-S100A12 detection antibody is conjugated to a marker such as biotin. The presence of S100A12 in a test sample results in the formation of a capture antibody-antigen- detection antibody complex. A signal generator, such as a streptavidin peroxidase, is then bound to the polyclonal detection antibody, followed by binding with a substrate such as 2,2'-azino-bis-(3- ethylbenzthiazoline-6-sulfonic acid) (ABTS) or tetramethylbenzidine (TMB).

The inventor has found significant expression of S100A12 in macrophages and foam cells (FC) in human atheroma, in both atherosclerotic arterial walls and in plaque extracts. S100A12 was also seen in some smooth muscle cells (SMC) and co-expression of S100A12 and RAGE was evident in some FC. Furthermore, S100A12 was found to be capable of inducing 1L6 release by peripheral blood mononuclear cells (PBMCs). IL6 induces the acute phase response and causes production of C-reactive protein (CRP) which is associated with poor prognosis in patients with cardiovascular disease. Increased IL6 levels are also associated with coronary events. Serum levels of A12 correlated positively with serum levels of CRP in 32 samples from normal individuals or those with cardiovascular disease. Moreover, application of the ELISA developed by the inventor revealed that S100A12 serum levels were significantly elevated in patients with angina. Accordingly, the present invention can be used to identify individuals with over- or under-production of S100A12 and to diagnose cardiovascular disease and/or a predisposition thereto. Accordingly, one aspect of the present invention relates to a method for determining the level of S100A12 in a subject, the method comprising the steps of obtaining a biological sample from the subject and analysing the biological sample to determine the amount of S100A12 present, wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease and/or a predisposition thereto. For the purposes of the present invention S100A12 includes the product of the S100A12 gene and variant forms thereof. For example, a variant of S100A12 may include one or more amino acid substitutions such that although the primary sequence of the polypeptide is altered, the activity of the polypeptide is fully or partially retained. The present invention also relates to peptide fragments of S100A12, for example proteolytic fragments. Typically, the fragment is capable of being recognized by one or more agents such that the amount of the fragment can be determined. For example, a fragment may typically comprise an epitope recognized by an anti-S100A12 antibody.

Further, those skilled in the art will appreciate that the level of S100A12 may be measured in a variety of body fluids or tissues. For example, the level of S100A12 may be measured in whole blood, blood serum, blood plasma, urine or other bodily fluids. Additionally or alternatively, the level of S100A12 may be measured in blood leukocytes.

Persons skilled in the art will further appreciate and understand that the level of S100A12 may be determined in conjunction with determining the level of at least one other biomarker in the subject, wherein the amount of the at least one other biomarker is indicative of cardiovascular disease or a predisposition thereto. By way of non-limiting example, the at least one other biomarker may be selected from the group comprising, but not limited to CRP, troponin I, creatine kinase, creatine kinase MB, a cardiac index, myoglobin or interleukin-6..

Moreover, persons skilled in the art will appreciate that the level of S100A12 in a test sample obtained from a subject may be compared with the level of S100A12 from one or more control samples. Typically a control sample may comprise a sample from a subject with normal levels of S100A12 and/or known not to have the cardiovascular disease or a predisposition thereto ("normal subjects"). In particular, samples from normal subjects may be tested in order to establish a normal range of S100A12 levels against which test samples may be compared. For example, levels of S100A12 less than about 400 ng/mL are generally considered to be normal. Further, S100A12 levels of less than about 100 ng/mL, less than about 200 ng/mL, less than about 225 ng/mL, less than about 250 ng/mL, less than about 275 ng/mL, less than about 300 ng/mL, less than 325 ng/mL, less than about 350 ng/mL, less than about 375 ng/mL or less than about 400 ng/mL can also be considered as within a normal range. Levels of S100A12 greater than about 400 ng/mL are generally considered to be elevated.

Further, S100A12 levels of greater than about 400 ng/mL, greater than about 425 ng/mL, greater than about 450 ng/mL, greater than about 475 ng/mL greater than about 500 ng/mL, greater than about 625 ng/mL, greater than about 650 ng/mL, greater than about 675 ng/mL, greater than about 700 ng/mL, greater than about 725 ng/mL, greater than about 750 ng/mL, greater than about 775 ng/mL, greater than about 800 ng/mL, greater than about 825 ng/mL, greater than about 850 ng/mL, greater than about 875 ng/mL, greater than about 900 ng/mL, greater than about 925 ng/mL, greater than about 950 ng/mL, greater than about 975 ng/mL, greater than about 1000 ng/mL, greater than about 1200 ng/mL, greater than about 1300 ng/mL, greater than about 1400 ng/mL, greater than about 1500 ng/mL, greater than about 1600 ng/mL, greater than about 1700

5 ng/mL, greater than about 1800 ng/mL, greater than about 1900 ng/mL, greater than about 2000 ng/mL, greater than about 2500 ng/mL or greater than about 3000 ng/mL can also be considered as elevated.

The present inventor has also surprisingly found that serum S100A12 levels are elevated and serum S100A8 and S100A9 levels are not elevated in angina patients. This finding appears in

I₀ sharp contrast with other inflammatory conditions, for example, rheumatoid arthritis, malaria and influenza, where serum levels of S100A12, S100A8 and S100A9 are all elevated.

Furthermore, the present inventor has found that serum levels of both S100A12 and S100A8 are elevated in myocardial infarction patients.

For example, levels of S100A8 less than about 30 ng/mL are generally considered to be is normal, Further, S100A8 levels of less than about 2 ng/mL, of less than about 5 ng/mL, of less than about 10 ng/mL, of less than about 12 ng/mL of less than about 15 ng/mL, of less than about 17 ng/mL, of less than about 20 ng/mL, of less than about 25 ng/mL, of less than about 27 ng/mL, can also be considered as within a normal range.

Levels of S100A8 greater than about 40 ng/mL are generally considered to be elevated.

2o Further, S100A8 levels of greater than about 40 ng/mL, greater than about 45 ng/mL, greater than about 50 ng/mL, greater than about 55 ng/mL, greater than about 60 ng/mL, greater than about 65 ng/mL, greater than about 70 ng/mL, greater than about 75 ng/mL, greater than about 80 ng/mL, greater than about 85 ng/mL, greater than about 90 ng/mL, greater than about 100 ng/mL, greater than about 125 ng/mL, greater than about 150 ng/mL, greater than about 200 ng/mL or greater than

25 about 250 ng/mL or greater than about 300 ng/mL can also be considered as elevated.

Accordingly, the present invention also contemplates measurement of S100A12 and one or more of S100A8 or S100A9 as indicative of inflammation specific to cardiovascular disease, wherein comparison of test samples with controls indicate elevated levels of S100A12 and equivalent levels of one or more of S100A8 or S100A9. This can provide a useful means by which o to distinguish between different inflammatory conditions, for example, between cardiovascular disease and other inflammatory conditions such as rheumatoid arthritis, malaria or influenza in which CRP may also be elevated.

For example, levels of S100A9 less than about 10 ng/mL are generally considered to be normal. Further, S100A9 levels of less than about 0.5 ng/mL, less than about 1 ng/mL, of less than

35 about 2 ng/mL, of less than about 3 ng/mL, less than about 4 ng/mL, less than about 5 ng/mL, less than about 6 ng/mL, less than about 7 ng/mL, less than about 8 ng/mL or less than about 9 ng/mL, can also be considered as within a normal range,

Levels of S100A9 greater than about 10 ng/mL are generally considered to be elevated.

Further, S100A9 levels of greater than about 11 ng/mL, greater than about 12 ng/mL, greater than about 13 ng/mL, greater than about 14 ng/mL, greater than about 15 ng/mL, greater than about 16 ng/mL, greater than about 17 ng/mL, greater than about 18 ng/mL, greater than about 19 ng/mL, greater than about 20 ng/mL, greater than about 21 ng/mL, greater than about 22 ng/mL, greater than about 23 ng/mL, or greater than about 24 ng/mL, greater can than about 25 ng/mL, greater than about 26 ng/mL, greater than about 27 ng/mL, greater than about 30 ng/mL or greater than about 50 ng/mL are be considered as elevated.

In embodiments of the invention, the S100 proteins, and optionally additional biomarkers, may be detected, and levels measured, by a variety of suitable methods. Typically, detection is via immunoassay such as an enzyme-linked immunoassay, a radioimmunoassay, or an immunoassay comprising a biosensor. Detection may also include nephelometric, immunonephelometric and/or turbidometric methods.

In relation to the use of additional biomarkers in concert with the biomarkers of the present invention, several laboratory tests are known in the art for the diagnosis of coronary artery disease, and in particular, for the diagnosis of acute myocardial infarction (Ml). However, none of these tests are completely sensitive or specific, particularly following the onset of symptoms, with the accuracy of such tests being dependent upon parameters including sampling time and correlation with patient symptoms, electrocardiograms, and angiographic studies.

Biomarkers that are known in the art as useful for the detection of acute Ml, and which are contemplated for use in concert with the biomarkers of the present invention, include:

1. Creatine Kinase - Total: The total CK is a simple and inexpensive test that is readily available using many laboratory instruments. However, an elevation in total CK is not specific for myocardial injury, because most CK is located in skeletal muscle, and elevations are possible from a variety of non-cardiac conditions.

2. Creatine Kinase - MB Fraction (specific for cardiac muscle): CK-MB is a good marker for acute Ml, because of specificity, and rises in serum within 2 to 8 hours of onset. Serial measurements every 2 to 4 hours for a period of 9 to 12 hours after the patient is first seen will provide a pattern to determine whether the CK-MB is rising, indicative of myocardial injury. The CK- MB is also useful for diagnosis of reinfarction or extensive of an Ml because it begins to fall after a day, dissipating in 1 to 3 days, so subsequent elevations are indicative of another event.

3. A "cardiac index" can provide a useful indicator for early Ml. This is calculated as a ratio of total CK to CK-MB, and is a sensitive indicator of myocardial injury when the CK-MB is elevated,

4. Troponins: Troponin I and T are structural components of cardiac muscle released into the bloodstream with myocardial injury, They are more specific for myocardial injury than CK-MB and help to exclude elevations of CK with skeletal muscle trauma. Troponins will begin to increase

5 following Ml within 3 to 12 hours, about the same time frame as CK-MB. However, the rate of rise for early infarction may not be as dramatic as for CK-MB, Troponins will remain elevated longer than CK-up to 5 to 9 days for troponin I and up to 2 weeks for troponin T.

5. Myoglobin: Myoglobin is a protein found in skeletal and cardiac muscle which binds oxygen, It is a sensitive indicator of muscle injury. The rise in myoglobin can help to determine the Q size of an infarction, A negative myoglobin can help to rule out Ml, It is elevated even before CK- MB, However, it is not specific for cardiac muscle, and can be elevated with any form of injury to skeletal muscle.

Particular embodiments of the invention provide for the use of one or more antibodies raised against S100A12, either free or in association with other molecules, for the detection of S100A12 s and the determination of S100A12 levels. The antibodies may be polyclonal or monoclonal and may be raised by the use of S100A12 or an antigenic fragment or portion thereof as an antigen, As exemplified herein, the antibodies may be polyclonal rabbit anti-S100A12 antibodies, although persons skilled in the art will readily understand and appreciate that alternative methods of generation may be used to produce antibodies, either monoclonal or polyclonal, suitable for 0 performance of the invention. Antibodies suitable for use in the methods of the present invention can be raised against S100A12 using techniques known to those in the art. Suitable antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanised, single chain, Fab fragments, and a Fab expression library.

Suitable antibodies may be prepared from discrete regions or fragments of the S100A12 s polypeptide. An antigenic S100A12 polypeptide contains at least about 5, and typically at least about 10, amino acids. Methods for the generation of suitable antibodies will be readily appreciated by those skilled in the art. For example, an anti-S100A12 monoclonal antibody, typically containing Fab portions, may be prepared using the hybridoma technology described in Antibodies - A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbour Laboratory, N.Y. (1988). In Q essence, in the preparation of monoclonal antibodies directed toward S100A12, a fragment or analogue thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include the hybridoma technique originally developed by Kohler et al., Nature, 256:495-497 (1975), as well as the trioma technique, the human B-cell hybridoma technique [Kozbor et al., Immunology Today, 4:72 (1983)], and the EBV- ₅ hybridoma technique to produce human monoclonal antibodies [Cole et a/., in Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., (1985)]. Immortal, antibody- producing cell lines can be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al, "Hybridoma Techniques" (1980); Hammerling et al, "Monoclonal Antibodies and T- cell Hybridomas" (1981); Kennett et al,, "Monoclonal Antibodies" (1980).

A monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques.

Similarly, there are various procedures known in the art which may be used for the production of polyclonal antibodies to S100A12, or fragments or analogues thereof. For the production of polyclonal antibody, various host animals can be immunized by injection with the S100A12 polypeptide, or a fragment or analogue thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. Further, the S100A12 polypeptide or fragment or analogue thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Also, various adjuvants may be used to increase the immunological response, including but not limited to Freund's (complete and incomplete), nitrocellulose, cellulose acetate, mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebadehum parvum.

Screening for the desired antibody can also be accomplished by a variety of techniques known in the art. Assays for immunospecific binding of antibodies may include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, Western and dot blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, biosensors and the like (see, for example, Ausubel et al,, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).

Antibody binding may be detected by virtue of a detectable label on the primary anti- Si 00A12 antibody. Alternatively, the anti-S100A12 antibody may be detected by virtue of its binding with a secondary antibody or reagent that is appropriately labeled to enable detection. A variety of methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example determinations of S100A12 levels can be accomplished by any one of a number of techniques known in the art including, for example enzyme-linked immunosorbent assays (ELISA); sandwich immunoassays, competitive immunoassays, immunoradiometric assays (IRMA), radioimmunoassays (RIA),

5 Immunoelectrophoresis assays, in situ immunoassays, immunodiffusion assays, immunofluorescence assays, Western blots, ligand-binding assays, biosensors and the like.

Methods of the invention for determining the significance of S100A12 levels may include the step of comparing the level of S100A12 in a sample obtained from the subject of interest, for example an individual suspected of cardiovascular disease, with the level of S100A12 from one or o more control samples. Typically the control sample may be a sample from an individual with normal levels of S100A12 and/or known not to have cardiovascular disease.

The present invention also provides kits for the determination of the level of S100A12, or the diagnosis of cardiovascular disease, wherein the kits facilitate the employment of methods of the invention. Typically, kits for carrying out a method of the invention contain all the necessary s reagents to carry out the method. For example, In one embodiment the kit may comprise a first container containing a capture antibody raised against S100A12, and a second container containing a detection antibody raised against S100A12. The anti-S100A12 capture antibody may be immobilized onto a solid surface, such as the well of a microtitre plate or a bead. The anti- Si 00A12 detection antibody may be conjugated to a marker such as biotin. o Typically, the kits described above will also comprise one or more other containers, containing for example, wash reagents, and/or other reagents capable of quantitatively detecting the presence of bound antibodies. For example, a signal generator such as a streptavidin peroxidase, may be provided for binding to the detection antibody, and a substrate such as 2,2'- azino-bis-(3-ethylbenzthiazo!ine-6-sulfonic acid) (ABTS) or tetramethylbenzidine (TMB) may be ₅ provided for binding to the signal generator.

Additionally or alternatively, kits of the invention may comprise a competitive ELISA, wherein S100A12 may be immobilized onto a solid surface. The immobilized S100A12 may then compete with endogenous S100A12 present in test sample for binding with an anti-S100A12 antibody. Additionally or alternatively, the anti-S100A12 antibody may comprise a marker, for example, biotin, o suitable for binding with a signal generator such as a streptavidin peroxidase.

Additionally or alternatively, kits of the invention may comprise reagents including for example, antibodies that recognize and bind at least one other biomarker. By way of non-limiting example, the at least one other biomarker may be selected from the group comprising, but not limited to, CRP, troponin I, creatine kinase MB or interleukin-6. In the context of the present invention, a compartmentalised kit includes any kit in which reagents are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of reagents from one compartment to another compartment whilst avoiding cross-contamination of the samples and reagents, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion. Such kits may also include a container which will accept the test sample, a container which contains the antibody(s) used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and like), and containers which contain the detection reagent. Typically, a kit of the present invention will also include instructions for using the kit components to conduct the appropriate methods.

Kits and methods of the invention may be used in conjunction with automated analysis equipment and systems, such as diagnostic systems enabling the analysis of multiple samples and/or multiple biomarkers, for example, the automated bead-based multiplexing BioRad BioPlex 2200 analyser. For example, an automated analyser may be used to determine the level of S100A12 in conjunction with determining the level of at least one other biomarker in a subject, wherein the amount of the at least one other biomarker is indicative of cardiovascular disease or a predisposition thereto. The at least one other biomarker may be selected from the group comprising CRP, troponin I, creatine kinase MB or interleukin-6. Methods and kits of the present invention are equally applicable to any animal, including humans, and also including but not limited to non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline and canine species. Accordingly, for application to different species, a single kit of the invention may be applicable, or alternatively different kits, for example containing reagents specific for each individual species, may be required. Methods and kits of the present invention find application in any circumstance in which it is desirable to determine S100A12 levels or to obtain an indication of cardiovascular disease.

The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention. Examples

Example 1. Development of S100A12 ELISA

1.1. Preparation of antibodies: Polyclonal antibodies (Abs) were raised in New Zealand white rabbits by standard techniques known to those skilled in the art. However, an additional adjuvant developed by the inventor (9, 10) was used when injecting rabbits, allowing for the production of high-titre Abs with high specificity. For the initial injection into rabbits, recombinant S100A12 (50 μg in phosphate buffered saline (PBS) (30 μl)) was mixed with 200 μl nitrocellulose particle suspension (NPS) for 2 hours at room temperature. The mixture was then made up to 0.5 ml with PBS and combined with 0.5 ml of complete Freund's adjuvant (CFA, Sigma) and emulsified with two glass syringes attached to a 3-way stopcock. Intradermal injections of 1 ml per rabbit were then made with a 25-gauge needle. Booster injections were then given two weeks after the initial injection and then every 4 weeks until a high titre was obtained. The emulsion for the boost comprised 100 μg of S100A12 in PBS mixed with an equal volume of incomplete Freund's adjuvant per rabbit. Rabbits were bled two weeks after the second booster injection, with collection of 5-10 ml from the marginal ear vein. Serum was separated and tested by ELISA and/or Western blot.

A 4cm² piece of nitrocellulose (0.1 μm) was dissolved in 4 ml dimethyl sulphoxide (DMSO). The nitrocellulose was precipitated by adding it dropwise to 40 ml carbonate buffer (0.015M Na2CO3, 0.03M NaHCCb, pH 9.6) with vortexing over 2 min. Large particles were allowed to settle by gravity for 1 min and the remaining particles in suspension were aspirated through a 21-gauge needle to generate smaller particles which were sedimented by centrifugation at 50Og for 10 min. The pellet was washed twice in carbonate buffer (50 ml per wash). A 0.5 ml packed volume of nitrocellulose particles was resuspended in 2 ml carbonate buffer and the suspension aspirated several times through a 25-gauge needle to further break up particles. The nitrocellulose particle suspension (NPS) was stored at -20 ⁰C prior to use. To determine cross-reactivities between S100A12 and other S100 protein family members, immunoglobulin G (IgG) was prepared from immune sera using standard Protein A-Sepharose protocols known to those skilled in the art. Standard SDS-PAGE and Western blotting techniques were then used to determine cross-reactivity of the IgG with S100B, S100A1, S100A8 and S100A9. In the event of cross-reactivity, particularly with the S100 protein family members with highest structural and amino acid homologies (for example, S100A8 and S100A9), the IgG was absorbed on columns of Sepharose 4B to which recombinant S100A8 and S100A9 were coupled using standard techniques. Removal of cross-reacting Abs was then confirmed again by Western blotting.

1.2 S100A12 ELISA: The rabbit anti-S100A12 IgG capture antibody was diluted in coating buffer (0.05 M carbonate, pH 9,6) at 5 μg/ml and then immediately coated to 96-well Nunc Maxisorp microplates (Apogent, Denmark), (50 μl/well). The coated plated were then sealed and incubated overnight at room temperature. Each well was then aspirated and washed three times with 0.05%

Tween 20 in PBS. Potential non-specific protein binding to plates was blocked by adding 100 μl of block buffer (1% bovine serum albumin (Sigma) in PBS) to each well. Plates were then covered and incubated at room temperature for a minimum of 1 hour, after which they were aspirated and washed as described above. 100 μl of each test sample or recombinant S100A12 standards was then diluted in PBS, For serum samples, the dilution started at 1:200 v/v. S100A12 standards were serially diluted from 250 ng/ml, giving serial dilutions of 250, 125, 62.5, 31.25, 15.6 and 7.8 ng/ml. Plates were then covered and incubated at room temperature for 2 hours, after which they were again aspirated and washed as above. 100 μl of detection antibody (biotinylated rabbit anti- Si 00A12 IgG prepared using standard protocols), diluted in PBS to 4 mg/ml, was then added to each well. Plates were then once more covered and incubated at room temperature for 2 hours, after which they were again aspirated and washed as above. 100 μl of working dilution of Streptavidin-horseradish peroxidase (Amersham Life Sciences; (1:2000 v/v) was then added to each well. Plates were again covered and incubated at room temperature for 2 hours, after which they were aspirated and washed as above. 100 μl of substrate solution (tetramethylbenzidine (PanBio, Australia)) was then added to each well. Plates were then incubate for 20 minutes at room temperature in the dark. 50 μl stop solution (2N H2SO4) was then added to each well. The optical density (OD) of each well was then immediately determined using an automated microplate reader set to A450 nm. Standard curves were then constructed using the optical density (OD) of the S100A12 standards, from which S100A12 levels in the serum test samples were determined.

Example 2. S100A12 in human atherosclerotic arterial walls

Carotid artery specimens containing atherosclerotic lesions and areas of apparently normal arterial wall, collected according to the Declaration of Helsinki, were obtained from 16 patients (aged 32-68 years) undergoing endarterectomy at St Vincent's Hospital, Sydney. Tissue processing, single and double immunostaining were undertaken using anti-CD3 (T-cell marker, Sigma), anti-CD68 (monocyte/macrophage marker), anti-von-Willebrand-factor (endothelial cell (EC) marker) and fascin (dendritic cell (DC) marker, all from Dako) at 1 :50 dilution. Anti-α-smooth- muscle-actin (Dako) and anti-RAGE (Chemicon) were used at 1:400 dilution. Anti-S100A12 was used at 14 μg/ml. Secondary antibodies used were biotinylated equine anti-mouse, caprine anti- rabbit and rabbit anti-caprine (Vector Laboratories) IgGs. Counter-staining was undertaken with Mayer's hematoxylin.

RAGE was absent in non-atherosclerotic areas but was variably and weakly expressed in atherosclerotic areas in 16 specimens. It co-localized on S100A12^"1" macrophage-like cells in 3 of 6 specimens (with no documented clinical differences between patients), as shown in Figures 1A, 1B and 1C.

S100A12⁺ cells were located along the arterial lumen in early and advanced lesions and S100A12 was weakly expressed by some microvascular EC. Normal areas of arteries did not contain the S100 proteins tested, as shown in Figure 1 D. Example 3. S100A12 in human plaque extracts

SDS-PAGE (10% gels) and electroblotting was performed on human plaque extracts using ovalbumin (7% w/v) as the blocking agent, with Tween 20 (0.05%) in TBS for washes. Immunoreactivity with anti-S100A12 (14 μg) IgG in 10 ml 2% ovalbumin in TBS for 2 h was visualized by chemiluminescence, Extracts were seen to contain varying levels of the S100A12 monomer (~10 kDa), as shown in Figure 2. Varying amount of other S100A12-containing complexes of undefined nature were also obvious in 6 of 8 samples, indicating some inter- specimen variability (Figure 2). S100A9- and S100A12-positive monocytes reported in the ascending aortae of ApoE^~'^~ mice⁵ were suggested to regulate monocyte transmigration and endothelial activation but because there is no S100A12 in the murine or rat genomes (6) the observation of A12 in mice is due to nonspecific cross-reactivity of the antibody used. High expression of A8, A9 and A12 was found in human plaque but not normal intima. S100A12 was strongly expressed by foam cells and its presence in SMC strongly supports the notion that it may mediate RAGE signaling, leading to proliferation and neointimal thickening.

Example 4. S100A12 induces IL6

Peripheral blood mononuclear cells (PBMC) from citrate-anticoagulated blood of normal donors was used. Erythrocytes were sedimented in 4.5% dextran T500 (Phamacia, Uppsala,

Sweden) at a ratio of 1 :5 (v/v) at 37°C for 20 minutes. Buffy coat cells were layered onto Ficoll- paque™ Plus (Amersham Biosciences, Uppsala Sweden) and centrifuged at 700 g for 30 minutes at 25°C. PBMCs were collected from the interface, washed with Ca²⁺-/Mg²⁺-free Dulbecco's phosphate-buffered saline (PBS, GIBCO, Invitrogen Corporation, Grand Island NY), and resuspended in RPMI 1640 supplemented with 0.5% bovine calf serum (BCS) at 2x10"⁶M Cell suspensions (0.5 ml/well) in 24-well cell culture plates were incubated for the desired times with

S100A12 (final concentration, 5μM) and supematants collected for IL-6 assay by ELISA. Peripheral blood mononuclear cells (PBMCs) from four normal donors were studied.

S100A12 was found to induce significantly higher amounts of IL6 release from PBMCs than S100A8, S100A9 or S100A8/A9 in combination, as shown in Figure 3.

When examined in more detail over time, IL6 release from PBMCs was found to peak at 24 hours post exposure to S100A12, as shown in Figure 4.

In addition, other cytokines involved in atherogenesis and inflammation, namely IL-1β and tumour necrosis factor (TNF) by activating mast cells and blood mononuclear cells, as shown in Table 1 and Table 2. Like mononuclear cells, mast cells also play a role in atherogenesis. Table 1: IL-1β release from peripheral blood mononuclear cells stimulated with S100A12 or bacterial endotoxin (LPS) (mean ± SD pg/ml)

Media A12 LPS

2 hr 1.90 ± 0.41 12.65 ± 2.37 * 136.32 ± 59# 6 hr 9.16 ± 3.85 50.39 ± 23.9 ** 1257.39 ± 246.9##

P < 0.05 compared with A12 at 2 hr

_*, P < 0.001 compared with media at 2 hr _**. P < 0.02 compared with media at 6hr

#:^' P < 0.005 compared with media at 2 hr ##: P < 0.0001 compared with media at 6 hr

Table 2: TNF release from peripheral blood mononuclear cells stimulated with S100A12 or bacterial endotoxin (LPS) (mean ± SD pg/ml)

Media A12 LPS

2 hr 5.63 ± 6.25 14.27 ± 9.04 580.5 ± 177 #

6 hr 8.4 ± 2.5 92.3 ± 68.4* 2191.25 ± 877.39 ##

_*. P < 0.05 compared with media at 6 hr

#^': P < 0.001 compared with media at 2 hr

##: P < 0.005 compared with media at 6 hr

Example 5. S100A12 is elevated in the serum of patients with angina

Serum samples from 24 patients with stable angina (SA) were tested by ELISA for levels of S100A12 and found to contain significantly higher levels of S100A12 than serum from age-matched controls (pθ.0001, non-parametric Mann Whitney test), as shown in Figure 5. Similarly, serum from 16 patients with unstable angina (UA) contained significantly elevated levels of S100A12

(p=0.0007). Differences in S100A12 levels were not significant between SA and UA patients

(P=0.8576). When SA and UA levels were combined, the significance was P<0.0001 compared to control levels as shown in Figure 6. These results are summarized in Table 3.

Table 3: Serum S100A12 levels (mean+ASEM)

Group No. Patients Serum S100A12

Control 24 289+/-27

SA 24 997+/-211

UA 16 834+/-142

SA+UA 40 931+/-138

Serum samples from these patients were also tested by ELISA for levels of S100A8 and S100A9. As shown in Figure 7, whilst combined figures for SA and UA samples showed that the significance was P<0.0001 for elevated S100A12 levels compared to control levels, serum levels for S100A8 and S100A9 were, in contrast, not significantly different between combined angina patients and controls,

Example 6. Relationship between C-reactive protein and S100A12

Serum CRP levels of subjects were measured by nephelometry using a high sensitivity CRP reagent from Beckman Coulter (Brea, CA)₁ as shown in Figure 8. This assay was undertaken using standard methods, for example as shown at http://www,cdc.gov/nchs/data/nhanes/nhanes_01_02/l11_b_met_c_reactive_protein.pdf (12). The correlation of S100A12 levels with CRP levels was somewhat weaker than the correlation with tissue factor (TF) levels (see Example 7), suggesting that S100A12 levels are a better predictor of cardiovascular events than CRP. In any event, serum S100A12 levels were positively correlated with serum CRP levels using either Pearson (parametric; r=0,42, p<0.02) or Spearman (non-parametric; r=0.45, p<0.01) tests in all subjects. Data shown in Figure 8 contains values from both normal and angina patients to give a broad CRP range (n=32).

Example 7. Relationship between Procoagυlant Activity and S100A12

Procoagulant activity was measured by reference to Tissue Factor (TF), TF initiates the extrinsic coagulation cascade and accounts for most thrombotic events in acute coronary syndromes (11). PBMC (5χ10⁵) in serum-free RPMI were incubated at 37⁰C in 250 μl in 96-well plates (Nunc International, Roskilde, Denmark) in 5% CO2 in air for 4 hours then supernatants removed by centrifugation and cells resuspended in 250 μl RPMI and plates frozen at -8O⁰C. Following 2 cycles of freeze (-8O⁰C) and rapid thawing (37⁰C)₁ procoagulant activity was measured using a 1 -stage plasma recalcification test as described, with a coagulometer (Diagnostica Stago, France). Human brain extract (HBE; Sigma) was used to construct a standard curve for recalcification assays. Activity calculated from the standard curve was expressed as mU TF/10⁶ PBMC. Procoagulant activity is due to TF on monocytes and since monocyte differential counts vary among subjects, procoagulant activities were normalized to a relative monocyte count of 1% by dividing the original TF value by the percentage of monocytes in cells from each donor. Patients with symptomatic coronary artery disease had significantly higher basal monocyte

TF activity than age- and gender-matched controls, confirming other earlier studies (REF) and supporting the notion that monocytes from patients with angina may be activated. Basal TF activity on PBMC from UA patients was significantly higher than controls (p=0.01), whereas the difference between SA and controls was less (p=0,03), as shown in Table 4. Table 4: TF activity

Control SA UA SA+UA

171(115-242) 394(196-652)$ 433(330-1006)§ 431(210~955)§ t p=0.03; § p=0.01

Serum S100A12 levels in controls were significantly less than in patients with angina and correlated positively with basal TF activity on PBMC (Pearson r=0,46, p=0.03) in 22 controls and 22 angina patients.

These results indicate that the measurement of S100A12 in serum provides a novel means of detecting cardiovascular disease or a predisposition thereto.

10 Example 8. S100A12 Analysis in CAD Patients

A further series of serum samples from patients with coronary artery disease (CAD) was analysed for S100A8 and S100A12 levels by ELISA according to the protocol disclosed in Example 1. The patients were all males and consisted of the following groups: (1) Controls (no obvious disease); (2) Stable angina (SA); (3) Acute coronary syndrome (ACS: includes unstable angina and

I₅ patients with Troponin T levels >0.03 ng/mL); and (4) Acute myocardial infarction (heart attack). (Ml).

As shown in Table 5 and Figure 9, A8 levels in sera were not significantly raised in patients with stable or unstable angina/ACS. In contrast, and as shown in Table 6 and Figure 10, S100A12 levels were significantly raised in patients with SA and ACS.

20

In patients with Mi, both S100A8 and S100A12 were raised, as seen in Table 5 and Figure 9, and Table 6 and Figure 10, respectively. However S100A12 levels in patients with ACS and Ml together were significantly higher than those of S100A8, and all patients with CAD had significantly raised S100A12 levels, but not S100A8 levels.

Accordingly, these data show that a combination of S100A8 and S100A12 measurements are useful in monitoring cardiovascular disease (wherein raised levels of S100A12 compared to S100A8 are indicative of a disease state) and in diagnosis of acute Ml (wherein levels of both S100A12 and S100A8 are raised), in association with established markers including but not limited to total creatine kinase (total CK), creatine kinase - MB Fraction (specific for cardiac muscle), troponins and myoglobin.

References

1. Arroyo-Espliguero R, Avanzas P, Gosin-Sales J, Aldama G, Pizzi C, Kaski JC, C-reactive protein elevation and disease activity in patients with coronary artery disease, Eur Heart J. 2004 Mar;25(5):401-8.

2. Auer J, Berent R, Lassnig E, Eber B., C-reactive protein and coronary artery disease. Jpn ffearfJ. 2002 Nov;43(6):607-19.

3. Yen ET, Willerson JT. Coming of age of C-reactive protein: using inflammation markers in cardiology. Circulation. 2003; 107:370-1. 4. Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low- density lipoprotein cholesterol levels in the prediction of first cardiovascular events. New England Journal of Medicine. 2002;347:1557-1565.

5. Lau W, Devery JM, Geczy CL. A chemotactic S100 peptide enhances scavenger receptor and Mac-1 expression and cholesteryl ester accumulation in murine peritoneal macrophages in vivo. J Clin Invest 1995;95: 1957-65

6. T. Ravasi, K. Hsu, J. Goyette, K, Schroder, Z. Yang, F. Rahimi, LP. Miranda, P.F. Alewood, D.A. Hume and CL Geczy. Probing the S100 protein family through genomic and functional analysis. Genomics, 2004: 84:10-22

7. Yang Z, Tao T, Raftery MJ, et a/. Proinflammatory properties of the human S100 protein S100A12. J Leukoc Biol. 2001 ;69:986-94.

8. Hofmann MA, Drury S, Fu C, et at. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Ce//. 1999;97:889-901

9. Hoad R.B. and Geczy CL (1991). Characterisation of monoclonal antibodies to human factor X/Xa. Initial observations with a quantitative ELISA procedure. J. Immunol. Methods 136: 269-278.

10. lismaa SE, Hu S, Kocher M, et a/. Recombinant and cellular expression of the murine chemotactic protein, CP-10. DNA CeIIBbI. 1994; 13: 183-92

11. Moons AH, Levi M, Peters RJ. Tissue factor and coronary artery disease. Cardiovascular Research. 2002;53:313-25. 12. Hutchinson, K. C-reactive protein in serum by nephelometry. NHANES 2001-2002.

Claims

1. A method for determining the level of S100A12 in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and (b) analysing the biological sample to determine the amount of S100A12 present, wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease or a predisposition thereto.

2. The method according to claim 1 , further comprising assessment of clinical symptoms of the subject.

3. The method according to either claim 1 or claim 2, further comprising determining the level of at least one other biomarker in the subject, wherein the amount of the at least one other biomarker is indicative of cardiovascular disease or a predisposition thereto.

4. The method of claim 3, wherein the at least one other biomarker is selected from the group comprising CRP, troponin I₁ creatine kinase, creatine kinase MB, a cardiac index, myoglobin or interleukin-6.

5. The method according to any one of claims 1 to 4, wherein the biological sample comprises whole blood, blood serum, blood plasma, blood leukocytes, urine or other bodily fluids.

6. The method according to any one of claims 1 to 5, wherein the analysis of the amount of S100A12 is via immunoassay using at least an anti-S100A12 antibody.

7. The method according to any one of claims of 1 to 6, wherein the cardiovascular disease comprises coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina, deep vein thrombosis, stroke, congestive heart failure or arrhythmia.

8. The method according to claim 7, wherein the angina is stable angina or unstable angina.

9. The method according to any one of claims 1 to 8, further comprising comparing the amount of S100A12 in the sample obtained from the subject with an amount of S100A12 from one or more control samples.

10. The method according to claim 9, wherein the control sample comprises a sample from a subject with normal levels of S100A12 and/or known not to have the cardiovascular disease or a predisposition thereto.

11. A method for diagnosing cardiovascular disease or a predisposition thereto in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of S100A12 present, wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease or a predisposition thereto.

12, The method according to claim 11, further comprising assessment of clinical symptoms of the subject.

13. The method according to either claim 11 or claim 12, further comprising determining the level of at least one other biomarker in the subject, wherein the amount of the at least one other biomarker is indicative of cardiovascular disease or a predisposition thereto.

14. The method of claim 13, wherein the at least one other biomarker may be selected from the group comprising CRP, troponin I, creatine kinase, creatine kinase MB, a cardiac index, myoglobin or interleukin-6.

15. The method according to any one of claims 11 to 14, wherein the biological sample comprises whole blood, blood serum, blood plasma, blood leukocytes, urine or other bodily fluids.

16. The method according to any one of claims 11 to 15, wherein the analysis of the amount of S100A12 is via immunoassay using at least an anti-S100A12 antibody.

17. The method according to any one of claims of 11 to 16, wherein the cardiovascular disease comprises coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina, deep vein thrombosis, stroke, congestive heart failure or arrhythmia.

18. The method according to claim 17, wherein the angina is stable angina or unstable angina.

19. The method according to any one of claims 11 to 18, further comprising comparing the amount of S100A12 in the sample obtained from the subject with an amount of S100A12 from one or more control samples.

20. The method according to claim 19, wherein the control sample comprises a sample from a subject with normal levels of S100A12 and/or known not to have the cardiovascular disease or a predisposition thereto.

21. The method according to any one of claims 11 to 24, wherein elevated levels of S100A12 compared to controls are indicative of cardiovascular disease or a predisposition thereto.

22. The method according to any one of claims 11 to 21, further comprising determining levels of at least one of S100A8 or S100A9 and comparing said levels with determined levels of S100A12.

23. The method according to claim 22, wherein elevated levels of S100A12 compared to controls and equivalent levels of at least one of S100A8 or S100A9 compared to controls is indicative of angina or a predisposition thereto.

24. The method according to claim 22, wherein elevated levels of S100A12 and S100A8 compared to controls is indicative of myocardial infarction or a predisposition thereto.

25, A method for diagnosing cardiovascular disease or a predisposition thereto in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of S100A12 and at least one s of S100A8 or S100A9 present, wherein the amount of S100A12 and at least one of S100A8 or S100A9 present in the sample is indicative of cardiovascular disease or a predisposition thereto,

26. The method according to claim 25, wherein elevated levels of S100A12 compared to controls and equivalent levels of at least one of S100A8 or S100A9 compared to controls is o indicative of cardiovascular disease or a predisposition thereto.

27, A method for diagnosing in a subject the over-expression of S100A12, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of S100A12 present. s wherein over-expression of S100A12 in the sample is indicative of cardiovascular disease or a predisposition thereto.

28. The method according to claim 27, further comprising assessment of clinical symptoms of the subject.

29. The method according to either claim 27 or claim 28, further comprising determining 0 the level of at least one other biomarker in the subject, wherein the amount of the at least one other biomarker is indicative of cardiovascular disease or a predisposition thereto.

30, The method of claim 29, wherein the at least one other biomarker is selected from the group comprising CRP, troponin I, creatine kinase, creatine kinase MB, a cardiac index, myoglobin or interleukin-6. ₅

31. The method according to any one of claims 27 to 30, wherein the biological sample comprises whole blood, blood serum, blood plasma, blood leukocytes, urine or other bodily fluids.

32. The method according to any one of claims 27 to 31, wherein the analysis of the amount of S100A12 is via immunoassay using at least an anti-S100A12 antibody.

33. The method according to any one of claims of 27 to 32, wherein the cardiovascular 0 disease comprises coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina, deep vein thrombosis, stroke, congestive heart failure or arrhythmia.

34. The method according to claim 33, wherein the angina is stable angina or unstable angina.

35, The method according to any one of claims 27 to 34, further comprising comparing the amount of S100A12 in the sample obtained from the subject with an amount of S100A12 from one or more control samples,

36. The method according to claim 35, wherein the control sample comprises a sample from a subject with normal levels of S100A12 and/or known not to have the cardiovascular disease or a predisposition thereto,

37. The method according to any one of claims 1 to 36, wherein levels of S100A12 less than about 400 ng/mL are normal.

38. The method according to any one of claims 1 to 37, wherein levels of S100A12 greater than about 400 ng/mL are elevated.

39, The method according to any one of claims 1 to 37, wherein levels of S100A12 greater than about 1000 ng/mL are elevated.

40, The method according to any one of claims 1 to 37, wherein levels of S100A12 greater than about 1500 ng/mL are elevated.

41. The method according to any one of claims 1 to 37, wherein levels of S100A12 greater than about 2000 ng/mL are elevated.

42. The method according to any one of claims 22 to 26, wherein levels of S100A8 less than about 30 ng/mL are normal.

43. The method according to any one of claims 22 to 26, wherein levels of S100A8 greater than about 30 ng/mL are elevated.

44. The method according to any one of claims 22 to 26, wherein levels of S100A8 greater than about 75 ng/mL are elevated.

45. The method according to any one of claims 22 to 26, wherein levels of S100A8 greater than about 100 ng/mL are elevated.

46. The method according to any one of claims 22 to 26, wherein levels of S100A9 less than about 10 ng/mL are normal.

47. The method according to any one of claims 22 to 26, wherein levels of S100A9 greater than about 10 ng/mL are elevated.

48. The method according to any one of claims 22 to 26, wherein levels of S100A9 greater than about 15 ng/mL are elevated.

49. The method according to any one of claims 22 to 26, wherein levels of S100A9 greater than about 20 ng/mL are elevated.

50. A kit for use in determining the level of S100A12 in a subject, the kit comprising at least one agent for measuring S100A12 in a biological sample, wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease or a predisposition thereto.

51. The kit according to claim 50, wherein the agent comprises an antibody that recognizes and binds S100A12.

52. The kit according to claim 50 or claim 51, further comprising at least one agent for measuring S100A8 or S100A9.

53. A kit for use in diagnosing in a subject the over-expression of S100A12, the kit comprising at least one agent for measuring S100A12 in a biological sample, wherein the over- expression of S100A12 in the sample is indicative of cardiovascular disease or a predisposition thereto.

54. A kit for use in diagnosing cardiovascular disease in a subject, the kit comprising at least one agent for measuring S100A12 in a biological sample, wherein the amount of S100A12 present in the sample is indicative of cardiovascular disease or a predisposition thereto.