WO2007014434A1 - Biomarqueur pour maladie cardiovasculaire - Google Patents

Biomarqueur pour maladie cardiovasculaire Download PDF

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Publication number
WO2007014434A1
WO2007014434A1 PCT/AU2006/001108 AU2006001108W WO2007014434A1 WO 2007014434 A1 WO2007014434 A1 WO 2007014434A1 AU 2006001108 W AU2006001108 W AU 2006001108W WO 2007014434 A1 WO2007014434 A1 WO 2007014434A1
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Prior art keywords
levels
subject
cardiovascular disease
predisposition
amount
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PCT/AU2006/001108
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English (en)
Inventor
Carolyn L. Geczy
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Newsouth Innovations Pty Limited
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Publication of WO2007014434A1 publication Critical patent/WO2007014434A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • SA stable angina
  • the S100 Ca 2+ -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).
  • FC foam cell
  • 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).
  • 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.
  • a method for determining the level of S100A12 in a subject comprising the steps of:
  • 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.
  • 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.
  • a method for diagnosing cardiovascular disease or a predisposition thereto in a subject comprising the steps of:
  • 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,
  • 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.
  • elevated levels of both S100A12 and S100A8 compared to controls may be indicative of myocardial infarction or a predisposition thereto.
  • 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:
  • 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.
  • elevated levels of both S100A12 and S100A8 compared to controls may be indicative of myocardial infarction or a predisposition thereto.
  • a method for diagnosing in a subject the over-expression of S100A12 comprising the steps of:
  • 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 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.
  • 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.
  • a diagnostic kit for use in determining the level of S100A12 in a subject 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,
  • 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.
  • a diagnostic kit for use in diagnosing in a subject the over-expression of S100A12 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.
  • a diagnostic kit for use in diagnosing cardiovascular disease in a subject 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.
  • the subject may be a human or any other animal.
  • the subject is selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine.
  • S100A12 refers to the member of the S100 protein family known as S100A12, calgranulin C, CGRP, CAAF1, CO-Ag or EN-RAGE,
  • Cardiovascular disease 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.
  • angina as used herein includes stable angina or unstable angina.
  • expression 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.
  • 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.
  • equivalent 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.
  • Figure 1 shows S100A12 expression in an atherosclerotic lesion. Typical patterns of expression of S100A12 (A 1 B, C) and RAGE (C) are shown in atherosclerotic arterial walls.
  • 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.
  • FIG. 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- i 5 S100A12 antibody. Arrows indicate the direction of migration of S100A12 standards (100ng) and molecular weight markers.
  • FIG. 3 shows levels of IL6 released from peripheral blood mononuclear cells (PBMCs) after stimulation with 5 ⁇ M S100A8, S100A9, S100A8/A9 and S100A12. Values are means from 2 blood donors +/- SEM.
  • FIG. 20 shows levels of IL6 released from peripheral blood mononuclear cells (PBMCs) after stimulation with 5 ⁇ M S100A12 at 6, 24 and 36 hours post-exposure. Values are means from 4 blood donors +/- SEM.
  • PBMCs peripheral blood mononuclear cells
  • FIG. compares separate levels of S100A12 in serum from patients with stable angina (SA), unstable angina (UA) and 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).
  • SA stable angina
  • U unstable angina
  • FIG. 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).
  • SA stable angina
  • U 3o unstable angina
  • CRP C reactive protein
  • 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),
  • 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.
  • the present inventor has developed an ELISA test to detect serum levels of S100A12,
  • 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.
  • 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).
  • ABTS 2,2'-azino-bis-(3- ethylbenzthiazoline-6-sulfonic acid
  • TMB tetramethylbenzidine
  • 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.
  • PBMCs peripheral blood mononuclear cells
  • 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.
  • S100A12 includes the product of the S100A12 gene and variant forms thereof.
  • 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.
  • the fragment is capable of being recognized by one or more agents such that the amount of the fragment can be determined.
  • a fragment may typically comprise an epitope recognized by an anti-S100A12 antibody.
  • the level of S100A12 may be measured in a variety of body fluids or tissues.
  • 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.
  • 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.
  • 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..
  • 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").
  • 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.
  • 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.
  • 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
  • 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.
  • 25 about 250 ng/mL or greater than about 300 ng/mL can also be considered as elevated.
  • 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.
  • 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.
  • the S100 proteins, and optionally additional biomarkers may be detected, and levels measured, by a variety of suitable methods.
  • 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.
  • 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:
  • 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.
  • CK-MB 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.
  • 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,
  • Troponins 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
  • 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.
  • antibodies raised against S100A12 are 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.
  • 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).
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • 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.
  • 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.
  • S100A12 polyclonal antibodies to S100A12, or fragments or analogues thereof.
  • 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.
  • 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).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • 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.
  • 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
  • BCG Bacille Calmette-Guerin
  • Corynebadehum parvum bacille Calmette-Guerin
  • 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.
  • 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),
  • 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.
  • the control sample may be a sample from an individual with normal levels of S100A12 and/or known not to have cardiovascular disease.
  • 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.
  • kits for carrying out a method of the invention contain all the necessary s reagents to carry out the method.
  • 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.
  • 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.
  • a signal generator such as a streptavidin peroxidase
  • 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.
  • ABTS 2,2'- azino-bis-(3-ethylbenzthiazo!ine-6-sulfonic acid
  • TMB tetramethylbenzidine
  • 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.
  • the anti-S100A12 antibody may comprise a marker, for example, biotin, o suitable for binding with a signal generator such as a streptavidin peroxidase.
  • kits of the invention may comprise reagents including for example, antibodies that recognize and bind at least one other biomarker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 ⁇ g 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.
  • nitrocellulose 0.1 ⁇ m was dissolved in 4 ml dimethyl sulphoxide (DMSO).
  • DMSO dimethyl sulphoxide
  • 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).
  • nitrocellulose particle suspension (NPS) was stored at -20 0 C prior to use.
  • IgG immunoglobulin G
  • 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 ⁇ g/ml and then immediately coated to 96-well Nunc Maxisorp microplates (Apogent, Denmark), (50 ⁇ l/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%
  • Anti- ⁇ -smooth- muscle-actin (Dako) and anti-RAGE (Chemicon) were used at 1:400 dilution.
  • Anti-S100A12 was used at 14 ⁇ g/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.
  • S100A9- and S100A12-positive monocytes reported in the ascending aortae of ApoE ⁇ ' ⁇ mice 5 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.
  • PBMC Peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • PBS Ca 2+ -/Mg 2+ -free Dulbecco's phosphate-buffered saline
  • RPMI 1640 Ca 2+ -/Mg 2+ -free Dulbecco's phosphate-buffered saline
  • BCS bovine calf serum
  • PBMCs Peripheral blood mononuclear cells
  • 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.
  • IL6 release from PBMCs was found to peak at 24 hours post exposure to S100A12, as shown in Figure 4.
  • IL-1 ⁇ IL-1 ⁇ release from peripheral blood mononuclear cells stimulated with S100A12 or bacterial endotoxin (LPS) (mean ⁇ SD pg/ml)
  • Table 2 TNF release from peripheral blood mononuclear cells stimulated with S100A12 or bacterial endotoxin (LPS) (mean ⁇ SD pg/ml)
  • 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 (p ⁇ .0001, non-parametric Mann Whitney test), as shown in Figure 5.
  • serum from 16 patients with unstable angina (UA) contained significantly elevated levels of S100A12
  • 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,
  • 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.
  • TF tissue factor
  • 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 serum-free RPMI were incubated at 37 0 C in 250 ⁇ l 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 ⁇ l RPMI and plates frozen at -8O 0 C.
  • 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/10 6 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.
  • 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

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Abstract

La présente invention concerne des procédés destinés à déterminer le niveau de S100A12 chez un sujet, le procédé comprenant les étapes consistant à obtenir un échantillon biologique provenant d’un sujet, et à analyser l’échantillon biologique afin de déterminer la quantité de S100A12 présente dans celui-ci. La quantité de S100A12 présente dans l’échantillon indique une maladie cardiovasculaire ou une prédisposition à celle-ci.
PCT/AU2006/001108 2005-08-04 2006-08-04 Biomarqueur pour maladie cardiovasculaire WO2007014434A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008107201A1 (fr) * 2007-03-08 2008-09-12 Roche Diagnostics Gmbh Utilisation de slim-1 dans l'évaluation d'une insuffisance cardiaque
EP2019318A1 (fr) * 2007-07-27 2009-01-28 Erasmus University Medical Center Rotterdam Marqueurs de protéines pour événements cardio-vasculaires
CN112114152A (zh) * 2020-09-09 2020-12-22 北京市心肺血管疾病研究所 血清s100a8/a9复合体水平在cabg术预后判断中的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KOSAKI A. ET AL.: "S100A12: A potential new marker for accelerated atherosclerosis in end stage renal disease patients with diabetes mellitus", DIABETES, vol. 54, no. SUPPL. 1, June 2005 (2005-06-01), pages A189, XP008077160 *
MASUDA ET AL.: "Plasma calcitonin gene-related peptide levels in patients with various hypertensive diseases", JOURNAL OF HYPERTENSION, vol. 10, 1992, pages 1499 - 1504, XP008077161 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008107201A1 (fr) * 2007-03-08 2008-09-12 Roche Diagnostics Gmbh Utilisation de slim-1 dans l'évaluation d'une insuffisance cardiaque
US9267954B2 (en) 2007-03-08 2016-02-23 The Governing Council Of The University Of Toronto Use of SLIM-1 in the assessment of heart failure
EP2019318A1 (fr) * 2007-07-27 2009-01-28 Erasmus University Medical Center Rotterdam Marqueurs de protéines pour événements cardio-vasculaires
WO2009017405A2 (fr) * 2007-07-27 2009-02-05 Erasmus University Medical Center Rotterdam Marqueurs protéiques pour des évènements cardiovasculaires
WO2009017405A3 (fr) * 2007-07-27 2009-07-23 Univ Erasmus Medical Ct Marqueurs protéiques pour des évènements cardiovasculaires
CN101889205A (zh) * 2007-07-27 2010-11-17 卡瓦迪斯有限责任公司 用于心血管事件的蛋白质标志物
CN112114152A (zh) * 2020-09-09 2020-12-22 北京市心肺血管疾病研究所 血清s100a8/a9复合体水平在cabg术预后判断中的应用

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