Novel biomarker of acute coronary syndromes

Abstract

The present invention is concerned with a novel approach to the measurement of circulating biomarkers of cardiac disease. In particular, the present invention provides assays, methods and test kits for the detection of immunoglobulin-bound circulating cardiac troponins, such as immunoglobulin-bound cardiac troponin T (bTnT-IgG), and to the utility of these biomarkers for diagnosing cardiac disease in a patient (e.g.) an acute myocardial infarction and / or for distinguishing between patients presenting with a type 1 myocardial infarction and a type 2 myocardial infarction. The present invention further provides a unique approach to enhance the clinical performance / accuracy of existing troponin assays in triaging patients with an acute myocardial infarction for immediate therapeutic interventions by measuring immunoglobulin-bound cardiac troponins, such as bTnT-IgG.

Description

[0001] NOVEL BIOMARKER OF ACUTE CORONARY SYNDROMES

[0002] TECHNICAL FIELD

[0003] The present invention is concerned with a novel approach to the measurement of circulating biomarkers of cardiac disease. In particular, the present invention provides assays, methods and test kits for the detection of unbound circulating cardiac troponins and to the utility of these biomarkers for identifying cardiac disease in a patient, for example, acute coronary syndromes such as acute myocardial infarction including the distinction between type 1 myocardial infarction and type 2 myocardial infarction.

[0004] BACKGROUND OF THE INVENTION

[0005] The following includes information that may be useful in understanding the present invention. It is not an admission that any of the information, publications or documents specifically or implicitly referenced herein is prior art, or essential, to the presently described or claimed inventions. All publications and patents mentioned herein are hereby incorporated herein by reference in their entirety.

[0006] Approximately 65,000 patients present annually to hospital with chest pain in New Zealand making it one of the most common causes for presentation [1], In these patients, the accurate and timely diagnosis of acute coronary syndromes (ACS, comprising myocardial infarction and unstable angina) is of major importance due to the high prevalence (~25% of ACS patients), mortality and morbidity (6-fold increased risk of major adverse event within 2 years) associated with these conditions [2, 7], The diagnosis of myocardial infarction is made on the basis of thorough clinical evaluation and measurement of circulating cardiac troponins (cTn). More recently, the introduction of highly sensitive (hs) troponin assays has facilitated faster assessment pathways for myocardial infarction diagnosis [3-5]. However, a limitation with existing troponin assays is the inability to take account of troponin-troponin binding interactions (e.g.) a binding complex comprising cardiac troponin T and cardiac troponin I (cTnl-cTnT). Further, the inability to distinguish between physiological levels of troponin and troponin produced by infarcted myocardial tissue is also a limitation associated with existing commercial troponin assays. Accordingly, improvements in the specificity of universally adopted clinical tests such as (e.g.) Roche's high sensitivity troponin assay (hsTnT) would yield improved patient triage and treatment outcomes for the management of acute myocardial infarction, as well as in the differentiation between type 1 and type 2 myocardial infarction.

[0007] In its recent clinical evaluation and assay development work, the Applicant has made surprising discoveries which address these and other unmet clinical needs as contemplated by the data and inventions described herein. SUMMARY OF THE INVENTION

[0008] The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Summary of the Invention. It is not intended to be all inclusive and the inventions described and claimed herein are not limited to or by the features or embodiments identified in this Summary of the Invention, which is included for purposes of illustration only and not restriction.

[0009] In an aspect of the present invention there is provided a test kit for determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) in a biological sample obtained from a patient, the test kit comprising:

[0010] (i) an anti-cTnT antibody which selectively binds to SEQ ID NO: 1; and

[0011] (ii) an anti-IgG antibody which selectively binds to the IgG which is bound to cTnT.

[0012] In another aspect of the present invention there is provided a method for determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from a patient, the method comprising:

[0013] (i) contacting the biological sample with a reaction mix comprising an anti-cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0014] (ii) washing the reaction mix from (i) to remove non-selectively bound analytes;

[0015] (Hi) contacting the reaction mix from (ii) with an anti-IgG detection antibody for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind the bTnT-IgG complex; and

[0016] (iv) determining the level of bTnT-IgG in the biological sample.

[0017] In a further aspect of the present invention there is provided a method for diagnosing an acute coronary syndrome in a patient, the method comprising:

[0018] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0019] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0020] (b) washing the reaction mix from (a) to remove non-selectively bound analytes; (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0021] (d) determining the level of bTnT-IgG in the biological sample;

[0022] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample measured relative to the reference standard from a control population is indicative that the patient has an acute coronary syndrome.

[0023] In yet another aspect of the present invention there is provided a method for improving the diagnostic performance of a cardiac troponin I (cTnl) assay for the diagnosis of an acute myocardial infarction in a patient, the method comprising:

[0024] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0025] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0026] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0027] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0028] (d) determining the level of bTnT-IgG in the biological sample;

[0029] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the reference standard from a control population it is combined with the cTnl assay to improve performance of the cTnl assay for diagnosis of an acute myocardial infarction in the patient compared to a diagnosis which is achieved in the absence of a bTnT-IgG measurement.

[0030] In yet another aspect of the present invention there is provided a method for improving the diagnostic performance of a cardiac troponin I (cTnl) assay for the diagnosis of an acute myocardial infarction in a patient, wherein the concentration of cTnl in the patient is >19 ng / L, the method comprising: (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0031] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0032] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0033] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0034] (d) determining the level of bTnT-IgG in the biological sample;

[0035] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the reference standard from a control population it is combined with the cTnl assay to improve performance of the cTnl assay for the diagnosis of an acute myocardial infarction in the patient compared to a diagnosis which is achieved in the absence of a bTnT-IgG measurement.

[0036] In yet another aspect of the present invention there is provided a method for improving the diagnostic performance of a cardiac troponin T (cTnT) assay for the diagnosis of an acute myocardial infarction in a patient, wherein the concentration of cTnT in the patient is >14 ng / L, the method comprising:

[0037] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0038] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0039] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0040] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0041] (d) determining the level of bTnT-IgG in the biological sample; (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the reference standard from a control population it is combined with the cTnT assay to improve performance of the cTnT assay for the diagnosis of an acute myocardial infarction in the patient compared to a diagnosis which is achieved in the absence of a bTnT-IgG measurement.

[0042] In yet another aspect of the present invention there is provided a method for distinguishing between type 1 and type 2 myocardial infarction in a patient, the method comprising:

[0043] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0044] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0045] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0046] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0047] (d) determining the level of bTnT-IgG in the biological sample;

[0048] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample measured relative to the reference standard from a control population distinguishes a patient who has type 1 myocardial infarction from a patient who has type 2 myocardial infarction.

[0049] In yet another aspect of the present invention there is provided a method for diagnosing type 1 myocardial infarction in a patient, the method comprising:

[0050] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0051] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex; (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0052] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0053] (d) determining the level of bTnT-IgG in the biological sample;

[0054] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample measured relative to the reference standard from a control population is indicative that the patient has Type 1 myocardial infarction.

[0055] In yet another aspect of the present invention there is provided a method for diagnosing type 1 myocardial infarction in a patient having a cardiac troponin T (cTnT) concentration which is > 14 ng / L, the method comprising:

[0056] (i) determining the level of a bound protein complex comprising cTnT and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0057] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0058] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0059] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0060] (d) determining the level of bTnT-IgG in the biological sample;

[0061] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample measured relative to the reference standard from a control population is indicative that the patient has Type 1 myocardial infarction.

[0062] In yet another aspect of the present invention there is provided a method for improving the performance of a cardiac troponin T (cTnT) assay for the diagnosis of an acute myocardial infarction in a patient, wherein the sensitivity of the cTnT assay is fixed at >98% and wherein the concentration of cTnT in the patient is >14 ng / L, the method comprising: (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0063] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0064] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0065] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0066] (d) determining the level of bTnT-IgG in the biological sample;

[0067] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the reference standard from the control population it is combined with the cTnT assay to improve performance of the cTnT assay for diagnosis of an acute myocardial infarction in the patient compared to a diagnosis which is achieved in the absence of a bTnT-IgG measurement.

[0068] In yet another aspect of the present invention there is provided a method for improving the performance of a cardiac troponin T (cTnT) assay for the diagnosis of type 1 myocardial infarction in a patient, wherein the specificity of the cTnT assay is fixed at >95% and wherein the concentration of cTnT in the patient is >52 ng / L, the method comprising:

[0069] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0070] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0071] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0072] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0073] (d) determining the level of bTnT-IgG in the biological sample; (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the reference standard from the control population it is combined with the cTnT assay to improve performance of the cTnT assay for diagnosis of type 1 myocardial infarction in the patient compared to a diagnosis which is achieved in the absence of a bTnT-IgG measurement.

[0074] In yet another aspect of the present invention there is provided a method for improving the performance of a cardiac troponin I (cTnl) assay for the diagnosis of type 1 myocardial infarction in a patient, wherein the sensitivity of the cTnl assay is fixed at >98% and wherein the concentration of cTnT in the patient is >64 ng / L, the method comprising:

[0075] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0076] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0077] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0078] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0079] (d) determining the level of bTnT-IgG in the biological sample;

[0080] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the reference standard from the control population it is combined with the cTnl assay to improve performance of the cTnl assay for diagnosis of type 1 myocardial infarction in the patient compared to a diagnosis which is achieved in the absence of a bTnT-IgG measurement.

[0081] In yet another aspect of the present invention there is provided a method for improving the diagnostic performance of a cardiac troponin I (cTnl) assay to rule out acute myocardial infarction in a patient, where the concentration of cTnl in the patient is < 19 ng / L, the method comprising:

[0082] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises: (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0083] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0084] I contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0085] (d) determining the level of bTnT-IgG in the biological sample;

[0086] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the reference standard from a control population it is combined with the cTnl assay to improve performance of the cTnl assay to rule out diagnosis of an acute myocardial infarction in the patient compared to a diagnosis which is achieved in the absence of a bTnT-IgG measurement.

[0087] In yet another aspect of the present invention there is provided a method for improving the diagnostic performance of a cardiac troponin T (cTnT) assay to rule out acute myocardial infarction in a patient, where the concentration of cTnT in the patient sample is < 14 ng / L, the method comprising:

[0088] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0089] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0090] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0091] I contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0092] (d) determining the level of bTnT-IgG in the biological sample;

[0093] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the reference standard from a control population it is combined with cTnT assay to improve the performance of the cTnT assay to rule out a diagnosis of an acute myocardial infarction in the patient compared to a diagnosis which is achieved in the absence of bTnT-IgG.

[0094] In yet another aspect of the present invention there is provided a method for predicting an acute myocardial infarction in a patient having a concentration of high sensitivity troponin I (hsTnl) which exceeds at least 30 ng / L, wherein the patient has a history of cardiovascular disease and abnormal cholesterol levels, the method comprising:

[0095] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0096] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0097] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0098] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0099] (d) determining the level of bTnT-IgG in the biological sample;

[0100] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample higher compared to the reference standard from a control population predicts the patient is predisposed to an acute myocardial infarction.

[0101] In yet another aspect of the present invention there is provided a method for predicting an acute myocardial infarction in a male or female patient having a level of high sensitivity troponin T (hsTnT) which exceeds at least 50 ng / L, wherein the patient has an abnormal electrocardiogram and is at least 60 years of age, the method comprising:

[0102] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0103] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex; (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0104] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0105] (d) determining the level of bTnT-IgG in the biological sample;

[0106] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample higher compared to the reference standard from a control population predicts the patient is predisposed to an acute myocardial infarction.

[0107] In a further aspect of the present invention there is provided a method for diagnosing unstable angina pectoris in a patient, the method comprising:

[0108] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0109] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0110] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0111] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0112] (d) determining the level of bTnT-IgG in the biological sample;

[0113] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample compared to the reference standard from a control population is indicative that the patient has unstable angina pectoris.

[0114] In yet a further aspect of the present invention there is provided a method for predicting a future acute coronary syndrome event in a patient, the method comprising:

[0115] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0116] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0117] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0118] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0119] (d) determining the level of bTnT-IgG in the biological sample;

[0120] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample compared to the reference standard from a control population is predictive of a future acute coronary syndrome event in the absence of a therapeutic intervention.

[0121] In yet a further aspect of the present invention there is provided a method for predicting stroke in a patient, the method comprising:

[0122] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0123] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnl capture antibody to selectively bind to the bTnT-IgG complex;

[0124] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0125] (c) contacting the reaction mix from (b) with an anti-IgG antibody, for a time and under conditions sufficient for the anti-IgG antibody to selectively bind to the bTnT-IgG complex; and

[0126] (d) determining the level of bTnT-IgG in the biological sample;

[0127] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample compared to the reference standard from a control population is predictive that the patient could suffer from a future stroke in the absence of a therapeutic intervention.

[0128] In a further aspect of the present invention there is provided a peptide complex comprising cardiac troponin T (cTnT) and a human immunoglobulin G (IgG) which is bound to a binding agent which selectively binds to cTnT, and preferably to SEQ ID NO: 1. In another further aspect of the present invention there is provided a peptide complex comprising cardiac troponin T (cTnT) and a human immunoglobulin G (IgG) which is bound to a monoclonal antibody or antigen-binding fragment thereof which selectively binds to cTnT, and preferably to SEQ ID NO: 1.

[0129] In yet another aspect of the present invention there is provided a peptide complex comprising cardiac troponin T (cTnT) and a human immunoglobulin G (IgG) which is bound to an aptamer which selectively binds to cTnT, and preferably to SEQ ID NO: 1.

[0130] In yet a further aspect of the present invention there is provided a binding agent comprising a peptide framework comprising one or more complementarity determining regions derived from an antibody which selectively binds to cardiac troponin I (cTnl) or cardiac troponin T (cTnT).

[0131] In yet a further aspect of the present invention there is provided a binding agent comprising a peptide framework comprising three complementarity determining regions derived from an antibody which selectively binds to cardiac troponin I (cTnl) or cardiac troponin T (cTnT).

[0132] In yet another aspect of the present invention there is provided an aptamer or aptamer ligand binding domain which selectively binds to cardiac troponin I (cTnl) or cardiac troponin T (cTnT).

[0133] In yet a further aspect of the present invention there is provided a binding agent which selectively binds to cardiac troponin I (cTnl) or cardiac troponin T (cTnT).

[0134] In yet another aspect of the present invention there is provided an antibody or antigenbinding fragment which selectively binds to cardiac troponin I (cTnl) or cardiac troponin T (cTnT).

[0135] In yet a further aspect of the present invention there is provided a monoclonal antibody, a polyclonal antibody, a chimeric antibody or a humanized antibody which selectively binds to cardiac troponin I (cTnl) or cardiac troponin T (cTnT), or an antigen-binding fragment of a monoclonal, polyclonal, chimeric or humanized antibody which selectively binds to cardiac troponin I (cTnl) or cardiac troponin T (cTnT).

[0136] In a further aspect of the present invention there is provided a method for diagnosing an acute coronary syndrome in a patient, the method comprising:

[0137] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0138] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex; (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0139] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0140] (d) determining the level of bTnT-IgG in the biological sample;

[0141] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein a higher level of bTnT-IgG in the biological sample compared to the reference standard from the control population is indicative that the patient has an acute coronary syndrome; and

[0142] (iii) where the level of bTnT-IgG in the biological sample is higher than the reference standard from the control population, triaging and / or treating the human for the acute coronary syndrome.

[0143] In another aspect of the present invention there is provided a method for determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from a patient, the method comprising:

[0144] (i) contacting the biological sample with a reaction mix comprising an anti-cTnT capture binding member which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture binding member to selectively bind to the bTnT-IgG complex;

[0145] (ii) washing the reaction mix from (i) to remove non-selectively bound analytes;

[0146] (iii) contacting the reaction mix from (ii) with an anti-IgG detection binding member, for a time and under conditions sufficient for the anti-IgG detection binding member to selectively bind to the bTnT-IgG complex; and

[0147] (iv) determining the level of bTnT-IgG in the biological sample.

[0148] In another aspect of the present invention there is provided a method for determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from a patient, the method comprising:

[0149] (i) contacting the biological sample with a reaction mix comprising an anti-cTnT capture aptamer which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture aptamer to selectively bind to the bTnT-IgG complex;

[0150] (ii) washing the reaction mix from (i) to remove non-selectively bound analytes; (iii) contacting the reaction mix from (ii) with an anti-IgG detection aptamer, for a time and under conditions sufficient for the anti-IgG detection aptamer to selectively bind to the bTnT-IgG complex; and

[0151] (iv) determining the level of bTnT-IgG in the biological sample.

[0152] BRIEF DESCRIPTION OF THE FIGURES

[0153] Figure 1 shows a schematic illustrating the principles of an bTnT-IgG assay according to the present invention. Importantly, the assay depicted in this schematic will not bind a troponin complex comprising cardiac troponin I and cardiac troponin T (i.e. cTnl-cTnT). Only cardiac troponin T that is not complexed with cardiac troponin I will bind to an anti-TnT antibody because the anti-TnT antibody binds to exactly the same region / epitope of TnT to which Tnl binds. The amount of "free" or "unbound" TnT is then measured using an anti-IgG detection antibody which targets the IgG bound to TnT.

[0154] Figure 2 shows Receiver Operating Curve (ROC) performance for (i) bTnT-IgG (AUC = 0.548; light blue line), (ii) hsTnl (AUC = 0.925; turquoise line) and (iii) hsTnl + bTnT-IgG (AUC = 0.926; crimson line); (iv) hsTnT (AUC = 0.902; green line) and hsTnT + bTnT-IgG (AUC = 0.902; purple line) for patients with an adjudicated diagnosis of myocardial infarction (n = 179) from within the combined SPACE and FAST-TRAC cohorts (n = 1,665). In these measurements all concentrations of bTnT-IgG were used in the ROC analyses; the median of bTnT-IgG was 0.47991 U / mL.

[0155] Figure 3 shows Receiver Operating Curve (ROC) performance for (i) history of cardiovascular disease + abnormal cholesterol levels + hsTnl + bTnT-IgG (< 0.47991 U / mL) (AUC = 0.698; 90% sensitivity + 24% specificity) and (ii) history of cardiovascular disease + abnormal cholesterol levels + hsTnl + bTnT-IgG (> 0.47991 U / mL) (AUC = 0.736; 90% sensitivity + 52% specificity) to predict inducible cardiac ischemia (n = 83 / 599 total patients tested).

[0156] Figure 4 shows Receiver Operating Curve (ROC) performance for (i) age + abnormal ECG + hsTnT (> 52 ng / L) (AUC = 0.794 ± 0.036; light blue line), and (ii) age + abnormal ECG + hsTnT (> 52 ng / L) + bTnT-IgG (AUC = 0.820 ± 0.033; crimson blue line) to predict patients with acute myocardial infarction when hsTnT is >52 ng / L at presentation (n=X) from within the combined SPACE and FAST-TRAC cohorts (n = 1,665). In these measurements concentrations of bTnT-IgG were restricted to > 0.406 U / mL were used.

[0157] Figure 5 shows Receiver Operating Curve (ROC) performance for (i) age + abnormal ECG + sex + hsTnl (AUC = 0.806 ± 0.028; light blue line), and (ii) age + abnormal ECG + sex + hsTnl + bTnT-IgG (AUC = 0.822 ± 0.026; crimson blue line) to predict patients with acute myocardial infarction when hsTnl is >34 ng / L at presentation (n=X) from within the combined SPACE and FAST-TRAC cohorts (n = 1,665). In these measurements concentrations of bTnT-IgG were restricted to > 0.406 U / mL were used.

[0158] Figure 6 shows the human cardiac troponin T amino acid sequence. The residues presented in bold text show the region of the protein to which the anti-cTnT capture antibody binds (i.e.) amino acid residues 106-183 as set forth in SEQ ID NO: 1.

[0159] Figure 7 shows Receiver Operating Curve (ROC) performance for (i) bTnT-IgG measured at index presentation (i.e. T=0 h; AUC = 0.574 ± 0.027 [grey line]) and (ii) bTnT- IgG measured two hours from index presentation (i.e. T=2 h; AUC = 0.581 ± 0.027 [black line]) in patients with an adjudicated diagnosis of Type 1 myocardial infarction with a hsTnT concentration of > 14 ng / L at index presentation. A total of 456 samples were obtained from a combined patient cohort (SPACE, APACE and FAST-TRAC) with 209 / 456 having an adjudicated diagnosis of Type 1 myocardial infarction.

[0160] Figure 8 shows Receiver Operating Curve (ROC) performance for (i) bTnT-IgG measured at index presentation (i.e. T=0 h; AUC = 0.483 ± 0.050 [grey line]) and (ii) bTnT- IgG measured two hours from index presentation (i.e. T=2 h; AUC = 0.505 ± 0.050 [black line]) in patients with an adjudicated diagnosis of Type 2 myocardial infarction with a hsTnT concentration of > 14 ng / L at index presentation. A total of 456 samples were obtained from a combined patient cohort (SPACE, APACE and FAST-TRAC) with 40 / 456 having an adjudicated diagnosis of Type 2 myocardial infarction.

[0161] Figure 9 shows Receiver Operating Curve (ROC) performance for (i) bTnT-IgG measured at index presentation (i.e. T=0 h; AUC = 0.616 [dotted grey line]), (ii) hsTnT measured at index presentation (i.e. T=0 h; AUC = 0.768 [light grey striped line]), (iii) bTnT- IgG + hsTnT measured at index presentation (i.e. T=0 h; AUC = 0.787 [dotted black line]), (iv) hsTnT measured two hours from index presentation (i.e. T=2 h; AUC = 0.844 [light grey solid line]) and (v) bTnT-IgG + hsTnT measured two hours from index presentation (i.e. T=2 h; AUC = 0.855 [solid black line]) in patients with an adjudicated diagnosis of Type 1 myocardial infarction. A total of 898 samples were obtained from a combined patient cohort (SPACE, APACE and FAST-TRAC) with 287 / 898 having an adjudicated diagnosis of Type 1 myocardial infarction. For all measurements, the sensitivity of the hsTnT assay was fixed at 98.6% and the concentration of hsTnT was > 14 ng / L at index presentation.

[0162] Figure 10 shows Receiver Operating Curve (ROC) performance for (i) bTnT-IgG measured at index presentation (i.e. T=0 h; AUC = 0.615 [light grey striped line]), (ii) hsTnT measured at index presentation (i.e. T=0 h; AUC = 0.770 [grey striped line]), (iii) hsTnT measured at index presentation + abnormal electrocardiogram + sex (i.e. T=0 h; AUC = 0.788 [dotted black line]), (iv) hsTnT measured two hours from index presentation (i.e. T=2 h; AUC = 0.846 [solid grey line]) and (v) hsTnT measured two hours from index presentation + abnormal electrocardiogram + sex (i.e. T=2 h; AUC = 0.851 [solid black line]) in patients with an adjudicated diagnosis of Type 1 myocardial infarction. A total of 898 samples were obtained from a combined patient cohort (SPACE, APACE and FAST-TRAC) with 287 / 898 having an adjudicated diagnosis of Type 1 myocardial infarction. For all measurements, the sensitivity of the hsTnT assay was fixed at 98.6% and the concentration of hsTnT was > 14 ng / L at index presentation.

[0163] Figure 11 shows Receiver Operating Curve (ROC) performance for (i) bTnT-IgG measured at index presentation (i.e. T=0 h; AUC = 0.626 [striped dark grey line]), (ii) hsTnT measured at index presentation (i.e. T=0 h; AUC = 0.697 [striped mid grey line]), (iii) bTnT- IgG + hsTnT measured at index presentation (i.e. T=0 h; AUC = 0.733 [dotted black line]), (iv) hsTnT measured two hours from index presentation (i.e. T=2 h; AUC = 0.800 [solid grey line]), and (v) bTnT-IgG + hsTnT measured two hours from index presentation (i.e. T=2 h; AUC = 0.815 [solid black line]) in patients with an adjudicated diagnosis of Type 1 myocardial infarction with a hsTnT concentration of > 52 ng / L at index presentation (n = 156).

[0164] Figure 12 shows Receiver Operating Curve (ROC) performance for (i) bTnT-IgG measured at index presentation (i.e. T=0 h; AUC = 0.647 [solid light grey line]), (ii) hsTnl measured at index presentation (i.e. T=0 h; AUC = 0.658 [striped mid grey line]), (iii) bTnT- IgG + hsTnl measured at index presentation (i.e. T=0 h; AUC = 0.701 [dotted dark grey line]), (iv) hsTnl measured two hours from index presentation (i.e. T=2 h; AUC = 0.761 [solid mid grey line]), and (v) bTnT-IgG + hsTnl measured two hours from index presentation (i.e. T=2 h; AUC = 0.788 [solid black line]) in patients with an adjudicated diagnosis of Type 1 myocardial infarction with a hsTnl concentration of > 64 ng / L at index presentation (n = 174).

[0165] Figure 13 shows Receiver Operating Curve (ROC) performance for (i) hsTnl + abnormal electrocardiogram + sex measured at index presentation (i.e. T=0 h; AUC = 0.800 [solid mid grey line]), (iv) bTnT-IgG + hsTnl + abnormal electrocardiogram + sex measured two hours from index presentation (i.e. T=2 h; AUC = 0.819 [solid black line]) in patients with an adjudicated diagnosis of Type 1 myocardial infarction with a hsTnl concentration of > 64 ng / at index presentation (n = 174).

[0166] DETAILED DESCRIPTION

[0167] General Definitions

[0168] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art to which the inventions belong (for example, in immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0169] Unless otherwise indicated, the recombinant protein and immunological techniques utilized in the present invention are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley 8i Sons (including all updates until present).

[0170] The term "and / or", e.g., "X and / or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

[0171] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0172] It is intended that reference to a range of numbers disclosed herein (for example 1 to 10) also incorporates reference to all related numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0173] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0174] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0175] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0176] Any example or embodiment described herein shall be taken to apply mutatis mutandis to any other example or embodiment unless specifically stated otherwise.

[0177] Selected Definitions

[0178] The term "ACS" as used herein means acute coronary syndrome. Examples of acute coronary syndromes include, but are not limited to, unstable angina or unstable angina pectoris; cardiac ischemia and myocardial ischemia; Type 1 and Type 2 (acute) myocardial infarction including ST-elevation myocardial infarction (STEMI) and non-ST myocardial infarction (NSTEMI); acute cardiac injury; acute cardiac damage resulting from acute drug toxicity, acute cardiomyopathies and cardiac transplant rejection.

[0179] The term "angina" as used herein means any form of chest pain whether that chest pain was experienced historically (e.g. "history of angina") or in an acute setting.

[0180] For any avoidance of doubt the term "history of angina" is taken to mean a patient who has had any history of cardiovascular disease or has a history of chest pain complaints. The terms "history of angina", "history of cardiovascular disease" and "history of chest pain" as used herein are therefore synonymous.

[0181] The term "antibody" refers to an immunoglobulin molecule capable of selectively binding to a target, such as a cardiac troponin (e.g. cTnl), by virtue of an antigen binding site contained within at least one variable region. This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primatized antibodies, de-immunized antibodies, half antibodies, bispecific antibodies) and single domain antibodies such as domain antibodies and heavy chain only antibodies (e.g., camelid antibodies or cartilaginous fish immunoglobulin new antigen receptors (IgNARs)). An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallisable (Fc). Preferred forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (~50-70 kDa) covalently linked and two light chains (~23 kDa each). A light chain generally comprises a variable region and a constant domain and in mammals is either a K light chain or a A light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types a, 5, E, y, or p. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL wherein each are ~110 amino acids in length) and one or more constant domains at the C- terminus. The constant domain of the light chain (CL which is ~110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CH which is -330-440 amino acids in length). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region can be identified between the CHI and Cm constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (preferably human) antibody. The term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also variants, fusion proteins comprising an antibody portion with an antigen binding site, humanised antibodies, human antibodies, chimeric antibodies, primatised antibodies, de-immunised antibodies or veneered antibodies.

[0182] The term "antigen-binding fragment" or "antigen-binding antibody fragment" shall be taken to mean any fragment of an antibody that retains the ability to bind to its target antigen. This term includes a Fab fragment, a Fab' fragment, a F(ab') fragment, a single chain antibody (SCA or SCAB) amongst others. A "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain. A "Fab1fragment" of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab1fragments are obtained per antibody molecule treated in this manner. A "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A "Fv fragment" is a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains. A "single chain antibody" (SCA) is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.

[0183] The term "chimeric antibody" refers to antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species (e.g., murine, such as mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g., primate, such as human) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl Acad. Sci USA 81:6851-6855).

[0184] The term "humanized antibody" shall be understood to refer to a chimeric molecule, generally prepared using recombinant techniques, having an epitope binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and / or sequence of a human immunoglobulin. The antigen-binding site preferably comprises the complementarity determining regions (CDRs) from the non-human antibody grafted onto appropriate framework regions in the variable domains of human antibodies and the remaining regions from a human antibody. Epitope binding sites may be wild type or modified by one or more amino acid substitutions. It is known that the variable regions of both heavy and light chains contain three complementaritydetermining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs. When non-human antibodies are prepared with respect to a particular epitope, the variable regions can be "reshaped" or "humanized" by grafting CDRs derived from non-human antibody on the FRs present in the human antibody to be modified.

[0185] The term "binding agent" as used herein is intended to refer to any molecule that binds a target antigen and includes isoforms thereof, and the term binding agent includes small molecules, antibodies from any species whether polyclonal or monoclonal, antigen-binding fragments such as Fab and Fab2, humanized antibodies, chimeric antibodies, or antibodies modified in other ways including substitution of amino acids, and / or fusion with other peptides or proteins (e.g. PEG). It also includes receptors or binding proteins from any species or modified forms of them.

[0186] As used herein, the term "antigenic variant" refers to polypeptide sequences different from the specifically identified sequences, wherein one or more amino acid residues are deleted, substituted, or added. Substitutions, additions or deletions of 1, 2, 3 or 4 amino acids are specifically contemplated. Variants may be naturally-occurring allelic antigenic variants, or non-naturally occurring antigenic variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, antigenic variants of the polypeptides useful in the invention have biological activities including hormone function or antigenic-binding properties that are the same or similar to those of the parent polypeptides. The term "antigenic variant" with reference to (poly)peptides encompasses all forms of polypeptides as defined herein. The term "antigenic variant" encompasses naturally occurring, as well as recombinant and synthetic produced polypeptides. The term "AUC" means Area Under the Curve which yields information about the strength of a correlation determined by the Receiver Operating Curve analysis. Typical ROC values where the AUC is greater than or equal to 0.70 yields a statistically significant correlation.

[0187] The term "biological sample" as used herein includes biological fluids selected from blood including venous blood and arterial blood, plasma, serum, intertistial fluid, or any other body fluid. The term "biological sample" also includes heart tissue sample. The term "biological sample" and "body fluid sample" as used herein refers to a biological sample or a sample of bodily fluid obtained for the purpose of, for example, diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient. In certain embodiments, such a sample may be obtained for diagnosing a cardiac disorder, for performing risk stratification of a cardiac disorder, for making a prognosis of a disease course in a patient with a cardiac disorder, for identifying a patient with elevated risk of a cardiac disorder, or combinations thereof. In addition, a person skilled in the art would realise that certain body fluid samples would be more readily analysed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

[0188] The term "comparing" has used herein has an ordinary meaning attached to it and is intended to mean a side-by-side comparison between the measured level of a particular biomarker from (e.g.) a test sample and the measured level of the same biomarker from a control sample, such as that obtained from an individual or a population of individuals. In other examples, the level of a biomarker measured from a test sample is compared to a test sample taken from an identical patient source at an earlier time point(s).

[0189] The terms "control population" and "suitable control population" according to the present invention refers to the mean circulating levels of a biomarker from sex- and age- matched subjects for which their cardiac disease or disorder status is known. The control population is used to provide a suitable reference interval by which a measured (e.g.) protein or isoform level is compared.

[0190] The term "Dx" as used herein means diagnosis or diagnostic.

[0191] The term "effective amount" as used herein refers to the amount of a therapy that is sufficient to result in the prevention of the development, recurrence, or onset of a disease or condition and one or more symptoms thereof, to enhance or improve the prophylactic effect(s) of another therapy, reduce the severity, the duration of disease, ameliorate one or more symptoms of the disease or condition, prevent the advancement of the disease or condition, cause regression of the disease or condition, and / or enhance or improve the therapeutic effect(s) of another therapy.

[0192] The term "ELISA" as used herein means enzyme-linked immunosorbent assay. The term "epitope" includes any antigenic (e.g., a protein) determinant capable of specific binding to an antibody. Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope typically includes, for example, at least 3, 5 or 8-10 amino acids. The amino acids may be contiguous, or non-contiguous amino acids juxtaposed by tertiary folding. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0193] As used in this specification, the term "fragment" or "functional derivative" in relation to a polypeptide is a subsequence of a polypeptide that may be detected using a binding agent. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant or derivative thereof.

[0194] The term "hsTnT" as used herein means high sensitivity Troponin T, and includes high sensitivity cardiac Troponin T (i.e. hscTnT).

[0195] The term "Hx" as used herein means history, for example "Hx CVD" means history of cardiovascular disease.

[0196] The terms "HxHF" or "HFHx" as used herein mean a patient who has a history of heart failure.

[0197] The terms "HxMI" or "MIHx" as used herein mean a patient who has a history of myocardial infarction.

[0198] An "increase" or "decrease" in the level of a particular biomarker (e.g. bTnT-IgG) compared with a control, or a "change" or "deviation" from a control (level) in one example is statistically significant. An increased level, decreased level, deviation from, or change from a control level or mean or historical control level can be considered to exist if the level differs from the control level by about 5% or more, by about 10% or more, by about 20% or more, or by about 50% or more compared to the control level. Statistically significance may alternatively be calculated as P<0.05. Increased levels, decreased levels, deviation, and changes can also be determined by recourse to assay reference limits or reference intervals. These can be calculated from intuitive assessment or non-parametric methods. Overall, these methods may calculate the 0.025, and 0.975 fractiles as 0.025* (n+1) and 0.975 (n+1). Such methods are well known in the art. Presence of a marker absent in a control may be seen as a higher level, deviation or change. Absence of a marker present in a control may be seen as a lower level, deviation or change.

[0199] The term "index presentation" as used herein means the point at which a patient presents to (e.g.) an emergency department, a clinic, a hospital, a surgery, a doctor's practice, a doctor or any other relevant medical forum, and information about the cardiac status of the patient is measured, including the patient's cardiac troponin levels levels. For any avoidance of doubt, the term "index presentation" also includes determining the levels of cardiac troponin(s) in a patient or subject who has a new or recurring complaint of chest pain.

[0200] The term "isolated" as applied to the polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular or other naturally-occurring biological environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques. The polypeptide sequences may be prepared by at least one purification step.

[0201] The term "level" as used herein is intended to refer to the amount per weight or weight per weight of an analyte of interest, (e.g.) a troponin such as cTnT. It is also intended to encompass "concentration" expressed as amount per volume or weight per volume. The term "circulating level" is intended to refer to the amount per weight or weight per weight or concentration of, for example, cardiac troponins present in the circulating fluid such as plasma, serum or whole blood.

[0202] As used herein, the terms "manage", "managing", and "management" in the context of the administration of a therapy to a subject refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent) or a combination of therapies, while not resulting in a cure of the disease or condition. In certain examples, a subject is administered one or more therapies (e.g., one or more prophylactic or therapeutic agents) to "manage" the disease or condition so as to prevent the progression or worsening of the disease or condition.

[0203] The term "MACE" as used herein means major acute cardiac event.

[0204] The terms "marker" or "biomarker" in the context of an analyte means any antigen, molecule or other chemical or biological entity that is specifically found in circulation or associated with a particular tissue (e.g. heart muscle) that it is desired to be identified in or on a particular tissue affected by a disease or disorder, for example unstable angina. In specific examples, the marker is a circulating cardiac troponin (e.g.) bTnT-IgG.

[0205] The terms "MI" and "AMI" as used herein mean (acute) myocardial infarction, a type of acute coronary syndrome.

[0206] The term "NCCP" as used herein means non-cardiac chest pain.

[0207] The term "NSTEMI" as used herein means non-ST elevation myocardial infarction, a type of myocardial infarction typically characterised by a depressed ST wave or T-wave inversion, no progression to Q wave and partial blockage of the coronary artery.

[0208] The term "NT-proBNP" as used herein means N-Terminal pro B-Type Natriuretic Peptide.

[0209] The terms "peptide" and "polypeptide" or "selectively binds" may be used interchangeably throughout this specification, and encompass amino acid chains of any length, including full length sequences in which amino acid residues are linked by covalent peptide bonds. Polypeptides useful in the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term "polypeptide" may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof. Polypeptides herein may have chain lengths of at least 40 amino acids, at least 50 amino acids, or at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least

[0210] 180, at least 190, at least 200, at least 210, at least 211, at least 212, at least 213, at least

[0211] 214, at least 215, at least 216, at least 217, at least 218, at least 219, at least 220, at least

[0212] 221, at least 222, at least 223, at least 224, at least 225, at least 226, at least 227, at least

[0213] 228, at least 229, at least 230, at least 231, at least 232, at least 233, at least 234, at least

[0214] 235, at least 236, at least 237, at least 238, at least 239, at least 240, at least 241, at least

[0215] 242, at least 243, at least 244, at least 245, at least 246, at least 247, at least 248, at least

[0216] 249, at least 250, at least 251, at least 252, at least 253, at least 254, at least 255, at least

[0217] 256, at least 257, at least 258, at least 259, at least 260, at least 261, at least 262, at least

[0218] 263, at least 264 amino acids. Reference to other polypeptides of the invention or other polypeptides described herein should be similarly understood.

[0219] The term "purified" as used herein does not require absolute purity. Purified refers in various embodiments, for example, to at least about 80%, 85%, 90%, 95%, 98%, or 99% homogeneity of a polypeptide, for example, in a sample. The term should be similarly understood in relation to other molecules and constructs described herein.

[0220] The term "Px" as used herein means prediction or prognostic.

[0221] Specifically, the term "reference interval" or "reference standard" as used herein is intended to refer to a figure within a statistical band of a representative concentration or alternatively a figure with an upper or lower concentration. The reference interval or reference standard will typically be obtained from subjects that do not have any pre-existing conditions that could result in changes to the level of circulating cardiac troponins.

[0222] The term "ROC" means Receiver Operating Curve and a ROC plot depicts the overlap between two distributions by plotting the sensitivity versus 1-specificity for a complete range of decision thresholds.

[0223] The term "subject" or "patient" may be used interchangeably in this specification and it intended to refer to a human or non-human primate. In one example, the subject or patient is a human.

[0224] The terms "specifically binds" or "selectively binds" may be used interchangeably throughout this specification, and shall be taken to mean that the binding agent reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity to a particular substance than it does with alternative substances. For example, a binding agent that specifically binds to bTnT-IgG, as well as isoforms thereof, or an epitope or immunogenic fragment thereof with greater affinity, avidity, more readily, and / or with greater duration than it binds to unrelated protein and / or epitopes or immunogenic fragments thereof. It is also understood by reading this definition that, for example, a binding agent that specifically binds to a first target (e.g. bTnT-IgG) may or may not specifically bind to a second target. As such, "specific binding" does not necessarily require exclusive binding or non- detectable binding of another molecule. Generally, but not necessarily, reference to binding means specific binding.

[0225] In addition to computer / database methods known in the art, polypeptide antigenic variants may be identified by physical methods known in the art, for example, by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) by recombinant DNA techniques also described by Sambrook et al. or by identifying polypeptides from natural sources with the aid of such antibodies.

[0226] The term "STEMI" as used herein means ST elevation myocardial infarction, a type of acute myocardial infarction typically characterised by elevated ST wave, progression to Q wave and full blockage of the coronary artery.

[0227] As used herein, the term "therapeutic agent" refers to any molecule, compound, and / or substance that is used for the purpose of treating and / or managing a disease or disorder, such as unstable angina. Examples of therapeutic agents include, but are not limited to, proteins, immunoglobulins (e.g., multi-specific Igs, single chain Igs, Ig fragments, polyclonal antibodies and their fragments, monoclonal antibodies and their fragments), peptides (e.g., peptide receptors, selectins), binding proteins, biologies, proliferation-based therapy agents, hormonal agents, radioimmunotherapies, targeted agents, epigenetic therapies, differentiation therapies, biological agents, and small molecule drugs.

[0228] As used herein, the terms "therapies" and "therapy" can refer to any method(s), composition(s), and / or agent(s) that can be used in the prevention, treatment and / or management of a disease or condition or one or more symptoms thereof.

[0229] The term "T1MI" as used herein means type 1 myocardial infarction.

[0230] The term "T2MI" as used herein means type 2 myocardial infarction.

[0231] The term "TID" as used herein means transient ischaemia dilation, which may be confirmed, for example, using spectral imaging or ultrasound.

[0232] The term "TnT" means Troponin T, preferably derived from a cardiac source (i.e. and "cTnT").

[0233] The term "bTnT-IgG" refers to a bound complex of cardiac troponin T (cTnT) and an immunoglobulin G (IgG) including, without limitation, IgGl, IgG2, IgG3 and IgG4. As used herein, the terms "treat", "treatment" and "treating" in the context of the administration of a therapy to a subject refer to the reduction, inhibition, elimination or amelioration of the progression and / or duration of (e.g.) an acute coronary syndrome, the reduction, inhibition, elimination or amelioration of the severity of (e.g.) acute coronary syndrome, and / or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies.

[0234] The term "UA" and "UAP" as used herein means unstable angina or unstable angina pectoris, a type of acute coronary syndrome.

[0235] The term "UDCP" as used herein means undifferentiated chest pain.

[0236] Term "variant" as used herein refers to polypeptide sequences different from the specifically identified sequences, wherein 1 to 6 or more or amino acid residues are deleted, substituted, or added. Substitutions, additions or deletions of one, two, three, four, five or six amino acids are contemplated. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides useful in the invention have biological activities including signal peptide activity or antigenic-binding properties that are the same or similar to those of the parent polypeptides. The term "variant" with reference to polypeptides encompasses all forms of polypeptides as defined herein.

[0237] Variant polypeptide sequences exhibit at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a sequence of the present invention. With regard to polypeptides, identity is found over a comparison window of at least 233 to 291 amino acid positions.

[0238] Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences, including those which could not reasonably be expected to have occurred by random chance.

[0239] Polypeptide sequence identity and similarity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.18 [April 2008]]) in bl2seq, which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

[0240] The similarity of polypeptide sequences may be examined using the following UNIX command line parameters: bl2seq— i peptideseql-j peptideseq2-F F-p blastp. The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. Variant polypeptide sequences commonly exhibit an E value of less than 1 x IO-5, less than 1 x 10s, less than 1 x IO-9, less than 1 x 1012, less than 1 x 1015, less than 1 x IO-18or less than 1 x IO-21when compared with any one of the specifically identified sequences. Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polypeptide sequences using global sequence alignment programs. EMBOSS-needle (available at http: / www. ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. Use of BLASTP is preferred for use in the determination of polypeptide variants according to the present invention.

[0241] DETAILED DESCRIPTION

[0242] The troponin market is estimated to be worth in excess of US$1B.

[0243] Medical cardiac troponin I (cTnl) measurement is provided by multiple suppliers.

[0244] Medical cardiac troponin T (cTnT) measurement is provided by one main supplier, Roche Diagnostics. Cardiac troponin I (cTnl) measurements are provided by more than twenty suppliers. Accordingly, cTnT measurements benefit from standardization from one supplier whereas cTnl measurement does not.

[0245] Despite these observations, auto-antibodies produced by the body can interfere with the binding sites that both cTnT and cTnl tests utilise, especially in patients who present with symptoms of myocardial infarction and a history of cardiovascular disease. This can lead to false low values, potentially missing a heart attack diagnosis. Conversely, false high values can be found as auto-antibody bound troponin has reduced clearance from the body. The prevalence of this is ~10% in healthy individuals and up to 30% of those with cardiovascular disease, but prior identification of individuals is difficult meaning the entire population would need to be tested in an acute setting.

[0246] An issue not contemplated by existing commercial troponin assays is the interference caused by troponin-troponin interactions (i.e.) cTnT directly binding to cTnl and influencing measured levels. In other words, the combination of cTnl-cTnT interactions and autoantibody levels to influence diagnostic troponin assay performance has not been addressed.

[0247] The Applicant has therefore conceived, designed and developed a clinically meaningful test (two-hour turn-around) that measures auto-antibody bound cTnT, free from cTnl interactions.

[0248] Specifically, and with reference to the assay concept illustrated in Figure 1, the Applicant was able to measure free / non-troponin bound cTnT-Ab complex (i.e. not bound to cTnl) in a sandwich ELISA which utilises an anti-cTnT antibody as a capture antibody and an anti-IgG antibody as a detection antibody. This simplistic but effective approach enables the measurement of a bound complex comprising cTnT and IgG, referred to herein as "bTnT-IgG". Importantly, bTnT-IgG and may be used to as an adjunct biomarker to reflect the amount of cTnT bound to cTnl (i.e.) reduced levels of bTnT-IgG measured relative to a reference standard from a control population would reflect increased amounts of cTnT that is bound to cTnl in a Tnl-TnT complex. In other words, lower test values in the bTnT-IgG assay reflect a greater proportion of cTnT that is bound up in Tnl-TnT complexes, which is more likely to be present during an acute cardiac event.

[0249] Accordingly, in an aspect of the present invention there is provided a method for determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from a patient, the method comprising: (a) contacting the biological sample with a reaction mix comprising an anti-cTnl capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0250] (b) washing the reaction mix from (i) to remove non-selectively bound analytes;

[0251] (c) contacting the reaction mix from (ii) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0252] (d) determining the level of bTnT-IgG in the biological sample.

[0253] In an example according to this and other aspects of the present invention, the anti- cTnT capture antibody selectively binds to SEQ ID NO: 1.

[0254] In another example according to this and all other aspects of the present invention, the anti-cTnT capture antibody selectively binds to a region defined by amino acid residues 106- 183 of human cTnT as set forth in SEQ ID NO: 1.

[0255] In another example according to this and all other aspects of the present invention, the anti-cTnT capture antibody is a monoclonal anti-cTnT antibody.

[0256] In a further example according to this and all other aspects of the present invention, the anti-IgG detection antibody is a human anti-IgG antibody.

[0257] In yet a further example according to this and all other aspects of the present invention, the anti-cTnT monoclonal antibody is immobilized on a solid substrate. Specific examples of solid substrates are provided in more detail below.

[0258] In yet another example according to this and all other aspects of the present invention, the anti-IgG antibody, including anti-human IgG antibody, comprises a detectable label. In a related example, the detectable label is an enzymatic detection label.

[0259] In yet another example according to this and all other aspects of the present invention, the biological sample is selected from plasma, serum, whole blood, arterial blood, venous blood, saliva, bone marrow tissue, heart tissue, vascular tissue and interstitial fluid sample.

[0260] In a related example according to this and all other aspects of the present invention, the biological sample is a circulating sample selected from plasma, serum, whole blood, arterial blood and venous blood.

[0261] In a further related example according to this and all other aspects of the present invention, the biological sample is plasma.

[0262] In yet a further example according to this and all other aspects of the present invention, step (a) is performed at room temperature for about one hour.

[0263] A person skilled in the art would recognise the term "about 1 hour" to mean a period of time which is about an hour and includes, without limitation, about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65 minutes, although by way of illustration (e.g.) 50 and 70 minutes would still meet this definition.

[0264] In yet a further example according to this and all other aspects of the present invention, step (b) comprises a first wash and a second wash, wherein the first and second washes are spaced apart by about 30 minutes.

[0265] A person skilled in the art would recognise the term "about 30 minutes" to mean a period of time which is about half an hour and includes, without limitation, about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 minutes, although by way of illustration (e.g.) 20 and 40 minutes would still meet this definition.

[0266] In another example according to this and all other aspects of the present invention, the level of bTnT-IgG in the biological sample is between about 0.01 and 100 ug / mL, and includes without limitation about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 ug / mL.

[0267] In another example according to this and all other aspects of the present invention, the method further comprises comparing the measured level of bTnT-IgG level against a reference standard from a control population.

[0268] In a related example, the reference standard is from a control population of sex and age-matched subjects who have not been identified as having an acute coronary syndrome or a history of an acute coronary syndrome.

[0269] The hypothesis that higher levels of bTnT-IgG (i.e. relevant to a reference standard from a control population) are more likely to reflect an acute cardiac event was subsequently validated using clinical samples derived from the 'SPACE' patient cohort (Christchurch School of Medicine, New Zealand; n = 257) and the 'FAST-TRAC' patient cohort (reference; n= 1489) to form a combined patient cohort of n = 1746 (refer to Example 1 for further details). The data is presented in Table 1 in Example 3 and shows median interquartile ranges of bTnT-IgG were lower in patients with an adjudicated diagnosis of a cardiovascular disease, acute myocardial infarction, abnormal cholesterol levels as compared patients with no history of cardiovascular disease, no previous myocardial infarction or normal cholesterol levels.

[0270] Importantly, the data presented in Table 2 also in Example 3 reflects a negative relationship between bTnT-IgG and NT-proBNP according to Spearman's rank correlation coefficient analysis.

[0271] Accordingly, in a further aspect of the present invention there is provided a method for diagnosing an acute coronary syndrome in a patient, the method comprising:

[0272] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises: (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0273] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0274] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0275] (d) determining the level of bTnT-IgG in the biological sample;

[0276] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, a higher level of bTnT-IgG in the biological sample measured relative to the reference standard from a control population is indicative that the patient has an acute coronary syndrome.

[0277] In an example according to this and all other aspects of the present invention, the acute coronary syndrome is selected from unstable angina or unstable angina pectoris; cardiac ischemia and myocardial ischemia; Type 1 and Type 2 (acute) myocardial infarction including ST-elevation myocardial infarction and non-ST myocardial infarction; acute cardiac injury; acute cardiac damage resulting from acute drug toxicity, acute cardiomyopathies and cardiac transplant rejection.

[0278] In a related example, the acute coronary syndrome is acute myocardial infarction or unstable angina pectoris.

[0279] The clinical significance of the bTnT-IgG assay developed by the Applicant is further realised by the data presented in Example 4 and Figure 2 for the diagnosis of acute myocardial infarction. Specifically, using Receiver Operating Curve analysis, the performance of hsTnl was enhanced by its combination with bTnT-IgG where the area under the curve increased according to the analyses performed. A person skilled in the art would recognise that this increase represents a statistically significant improvement for the diagnosis of an acute myocardial infarction.

[0280] The novel biomarker disclosed herein may advantageously be used to improve the diagnostic performance of existing cardiac troponin assays, for example, cTnT (including hsTnT) assays as well as cTnl (including hsTnl) assays. According to the data provided herein, the improved performance is selected from improved assay specificity, improved assay sensitivity, improved positive predictive value (PPV) for the diagnosis of (e.g.) an acute myocardial infarction and improved negative predictive value (NPV) for the diagnosis of (e.g.) an acute myocardial infarction. Accordingly, in another aspect of the present invention there is provided a method for enhancing the diagnostic performance of a cardiac troponin I (cTnl) assay for the diagnosis of an acute myocardial infarction in a patient, the method comprising:

[0281] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0282] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0283] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0284] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0285] (d) determining the level of bTnT-IgG in the biological sample;

[0286] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the level of bTnT-IgG of a reference standard from a control population it is combined with the cTnl assay to improve the diagnostic accuracy of the cTnl assay which is achieved in the absence of a bTnT-IgG measurement.

[0287] In another example according to this and other aspects of the present invention, the concentration of cTnl in the patient sample is greater than or equal to 14 ng / L, which includes without limitation a cTnl concentration that is greater than or equal to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 ng / L.

[0288] In another aspect of the present invention there is provided a method for enhancing the diagnostic performance of a cardiac troponin T (cTnT) assay for the diagnosis of an acute myocardial infarction in a patient, the method comprising:

[0289] (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0290] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0291] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0292] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0293] (d) determining the level of bTnT-IgG in the biological sample;

[0294] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein, when the level of bTnT-IgG in the biological sample is higher compared to the level of bTnT-IgG of a reference standard from a control population it is combined with the cTnT assay to improve the diagnostic accuracy of the cTnT assay which is achieved in the absence of a bTnT-IgG measurement.

[0295] In another example according to this and other aspects of the present invention, the concentration of cTnT in the patient sample is greater than or equal to 14 ng / L, which includes without limitation a cTnl concentration that is greater than or equal to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 ng / L.

[0296] In another example according to this and all other aspects of the present invention, the level of bTnT-IgG in the patient sample is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,

[0297] 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,

[0298] 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,

[0299] 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or at least 10 times lower than the level of bTnT-IgG in the reference standard from a control population.

[0300] In another example according to this and all other aspects of the present invention, the level of bTnT-IgG in the patient sample is at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

[0301] 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,

[0302] 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,

[0303] 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

[0304] 94, 95, 96, 97, 98, 99 or at least 100 times lower than the level of bTnT-IgG in the reference standard from a control population.

[0305] In another example according to this and all other aspects of the present invention, the reference standard is from a control population of sex and age-matched subjects who have not been identified as having acute myocardial infarction or a history of acute myocardial infarction.

[0306] In other examples according to any of the methods described herein, the concentration or level of bTnT-IgG is obtained from a biological sample taken from a patient at index presentation. This includes, without limitation, at a time when a patient (e.g.) presents to an emergency department, a clinic, a hospital, a surgery, a doctor's practice, a doctor or any other relevant medical forum, and information about the cardiac status of the patient is measured, including the patient's bTnT-IgG levels. Alternatively index presentation means a time when a patient identifies or presents with a complaint of chest pain during the hospital admission period, during a follow-up consultation or during participation in a clinical trial. The term "0" in the tables and figures which follow (e.g. "hsTnTO") is intended to mean the levels of highly sensitive troponin T an index presentation, for example.

[0307] In another example according to these and all other aspects of the present invention, multiple samples are taken from a patient while in the acute clinical setting. By way of illustration only, a first sample and second sample may be obtained from a patient and the time point between obtaining the first sample and second samples may be between about half an hour to about 10 hours. This includes, without limitation, about 0.5h, about lh, about 1.5h, about 2h, about 2.5h, about 3h, about 3.5h, about 4h, about 4.5h, about 5h, about 5.5h, about 6h, about 6.5h, about 7h, about 7.5h, about 8h, about 8.5h, about 9h, about 9.5h or about lOh.

[0308] In further examples according to the methods described herein, bTnT-IgG levels may be measured together with one or more other cardiac risk factors including, without limitation, a history of cardiovascular disease (e.g. HxMI or HxUAP), an increased heart rate relevant to a reference standard, an abnormal electrocardiogram, a decreased level of high-density lipoprotein relevant to a reference standard, a diagnosis of ischaemia, optionally by imaging, or dyslipidemia or a history of dyslipidemia, age threshold and sex bias, in isolation or in any combination.

[0309] A person skilled in the art would appreciate the terms "measured together with" or "measured together with one or more risk factors" in the context of (e.g.) the clinical risk factors identified above is intended to mean that (i) the patient has presented with one or more of those risk factors (ii) or it is determined that one or more of those risk factors exist when interrogating bTnT-IgG levels.

[0310] Advantageously, the diagnosis of an acute coronary syndrome made in accordance with methods described herein may be useful to inform a therapeutic regime to control, reverse, mitigate or treat ACS in the patient.

[0311] As such, in a further aspect of the present invention there is provided a method for treating an acute coronary syndrome in a patient, the method comprising: (i) determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:

[0312] (a) contacting the biological sample with a reaction mix comprising an anti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;

[0313] (b) washing the reaction mix from (a) to remove non-selectively bound analytes;

[0314] (c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG detection antibody to selectively bind to the bTnT-IgG complex; and

[0315] (d) determining the level of bTnT-IgG in the biological sample;

[0316] (ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control population wherein a higher level of bTnT-IgG in the biological sample compared to the reference standard from the control population is indicative that the patient has an acute coronary syndrome; and

[0317] (iii) where the level of bTnT-IgG in the biological sample is higher than the reference standard from the control population, triaging and / or treating the human for the acute coronary syndrome.

[0318] In an example according to this aspect of the present invention, bTnT-IgG is considered alongside other risk factors including circulating cardiac troponins such as cTnT and / or cTnl.

[0319] The therapeutic regimes administered in accordance with the methods of the present invention include, by way of illustration and example only, those outlined by the Mayo Clinic in its medication guidelines: https: / / www.mayoclinic.org / diseases-conditions / myocardial-ischemia / diagnosis- treatment / drc-20375422.

[0320] By way of illustration only, the therapeutic regimes administered in accordance with the present invention include, without limitation, administration of one or more drugs selected from the group consisting of aspirin, nitrates, beta-blockers, calcium channel blockers, cholesterol lowering medications, angiotensin-converting enzyme inhibitors, ranolazine, and any combination thereof.

[0321] Antibodies and Antigen Binding Fragments

[0322] The present invention contemplates various antibodies and antigen-binding fragments thereof which selectively bind to cardiac troponins or antigenic variants of cardiac troponins including isoforms thereof. The present invention also contemplates various antibodies and antigen-binding fragments thereof which selectively bind to an immunoglobulin (IgG) bound to cardiac troponin I or cardiac troponin T.

[0323] Accordingly, in yet another aspect of the present invention there is provided an antibody or antigen-binding fragment which selectively binds to cardiac troponin I or cardiac troponin T. In yet another aspect the present invention alternatively provides an antibody or antigenbinding fragment which selectively binds to an immunoglobulin (IgG) bound to cardiac troponin I or cardiac troponin T.

[0324] In yet a further aspect of the present invention there is provided a monoclonal antibody, a polyclonal antibody, a chimeric antibody or a humanized antibody which selectively binds to cardiac troponin I or cardiac troponin T, or an antigen-binding fragment of a monoclonal, polyclonal, chimeric or humanized antibody which selectively binds to cardiac troponin I or cardiac troponin T. In yet a further aspect the present invention alternatively provides a monoclonal antibody, a polyclonal antibody, a chimeric antibody or a humanized antibody which selectively binds to an immunoglobulin (IgG) bound to cardiac troponin I or cardiac troponin T.

[0325] In yet another aspect of the present invention there is provided a monoclonal antibody or antigen-binding fragment thereof which selectively binds to cardiac troponin I or cardiac troponin T. In yet another aspect the present invention alternatively provides a monoclonal antibody which selectively binds to an immunoglobulin (IgG) bound to cardiac troponin I or cardiac troponin T.

[0326] In yet a further aspect of the present invention there is provided an antibody or antigenbinding fragment which selectively binds to cardiac troponin I or cardiac troponin T, which antibody or antigen-binding fragment comprises a detectable label. In yet a further aspect of the present invention alternatively provides an antibody or antigen-binding fragment which selectively binds to an immunoglobulin (IgG) bound to cardiac troponin I or cardiac troponin T.

[0327] In yet another aspect of the present invention there is provided an antibody or antigenbinding fragment which selectively binds to cardiac troponin I or cardiac troponin T, which antibody or antigen-binding fragment is immobilized on a solid substrate. In yet another aspect the present invention alternatively provides an antibody or antigen-binding fragment which selectively binds to an immunoglobulin (IgG) bound to cardiac troponin I or cardiac troponin T, which antibody or antigen-binding fragment is immobilized on a solid substrate.

[0328] In yet a further aspect of the present invention there is provided a binding agent comprising a peptide framework comprising one or more complementarity determining regions derived from an antibody which selectively binds to cardiac troponin I or cardiac troponin T. In yet a further aspect the present invention alternatively provides a binding agent comprising a peptide framework comprising one or more complementarity determining regions derived from an antibody which selectively binds to an immunoglobulin (IgG) bound to cardiac troponin I or cardiac troponin T.

[0329] In yet a further aspect of the present invention there is provided a binding agent comprising a peptide framework comprising at least three complementarity determining regions derived from an antibody which selectively binds to cardiac troponin I or cardiac troponin T. In yet a further aspect the present invention alternatively provides a binding agent comprising a peptide framework comprising at least three complementarity determining regions derived from an antibody which selectively binds to an immunoglobulin (IgG) bound to cardiac troponin I or cardiac troponin T.

[0330] As noted above, antibody or antibodies as used herein refers to a peptide or polypeptide derived from, modelled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. As foreshadowed in the definition section of this specification, the term antibody includes antigen binding fragments such as, for example, fragments, subsequences, complementarity determining regions (CDRs) that retain capacity to bind to an antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody."

[0331] Also included is antiserum obtained by immunizing an animal such as a mouse, rat or rabbit with an antigen, such as for example, cardiac troponin I or cardiac troponin T. In brief, methods of preparing polyclonal antibodies are known to a person skilled in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and / or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include cardiac troponin I or cardiac troponin T, antigenic variants thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, bovine serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. Monoclonal antibodies may be prepared using hybridoma methods well known in the art. The hybridoma cells may be cultured in a suitable culture medium, alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. Preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the American Type Culture Collection, Virginia, USA. Immunoassays may be used to screen for immortalized cell lines that secrete the antibody of interest. Sequences of cardiac troponin I or cardiac troponin T or antigenic variants thereof may be used in screening.

[0332] Well known means for establishing binding specificity of monoclonal antibodies produced by the hybridoma cells include immunoprecipitation, radiolinked immunoassay (RIA), enzyme-linked immunoabsorbent assay (ELISA) and Western blot. For example, as noted above, the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis. Samples from immunised animals may similarly be screened for the presence of polyclonal antibodies.

[0333] Monoclonal antibodies can also be obtained from recombinant host cells. DNA encoding the antibody can be obtained from a hybridoma cell line. The DNA is then placed into an expression vector, transfected into host cells (e.g., COS cells, CHO cells, E. coli cells) and the antibody produced in the host cells. The antibody may then be isolated and / or purified using standard techniques.

[0334] The monoclonal antibodies or fragments may also be produced by recombinant DNA means. DNA modifications such as substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences are also possible. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. Production of chimeric, bivalent antibodies and multivalent antibodies are also contemplated herein.

[0335] Other known art techniques for monoclonal antibody production such as from phage libraries, may also be used.

[0336] The monoclonal antibodies secreted by the cells may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, reverse phase HPLC, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0337] Bispecific antibodies may also be useful. These antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. Antibodies with greater than two specificities for example trispecific antibodies are also contemplated herein.

[0338] Antibodies used in the immunoassays described herein selectively bind to IGFBP-3. The term "selectively binds" is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody "selectively binds" if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s). In certain examples, the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In other examples, antibodies bind with affinities of at least about 10-sM, or 10-7M, or at least about 10-8M, or 10-9M, or IO-10M, or 10-11M or 10-12M.

[0339] Affinity is calculated as Kd = kOff / kOn (korr is the dissociation rate constant, Konis the association rate constant and Kd is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labelled ligand at various concentrations (c). The data are graphed using the Scatchard equation: r / c=K(n-r): where r=moles of bound ligand / mole of receptor at equilibrium; c=free ligand concentration at equilibrium; K=equilibrium association constant; and n = number of ligand binding sites per receptor molecule. By graphical analysis, r / c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art.

[0340] Numerous publications discuss the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome that encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target binds to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means.

[0341] The antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labelled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a colour reaction will appear where antibody to the immobilized polypeptide(s) is present.

[0342] The antibodies so identified may then be further analysed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interact with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.

[0343] Aptamers

[0344] The present invention also contemplates aptamers that selectively bind to cardiac troponin I or cardiac troponin T or antigenic variants of cardiac troponin I or cardiac troponin T including isoforms thereof.

[0345] In yet another aspect of the present invention there is provided an aptamer or aptamer ligand binding domain which selectively binds to cardiac troponin I or cardiac troponin T, or to an immunoglobulin associated with cardiac troponin I or cardiac troponin T. In yet another aspect the present invention alternatively provides an aptamer or aptamer ligand binding domain which binds to an immunoglobulin (IgG) bound to a cardiac troponin I or cardiac troponin T.

[0346] Nucleic acid aptamers are nucleic acid molecules that have been engineered through repeated rounds of in vitro selection, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers offer molecular binding and recognition equivalent to antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in vitro, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.

[0347] According to an example of the present invention, the aptamer is a monomer (one unit). According to another example of the invention, the aptamer is a multimeric aptamer. The multimeric aptamer may comprise a plurality of aptamer units (mers). Each of the plurality of units of the aptamer may be identical. In such a case the multimeric aptamer is a homomultimer having a single specificity but enhanced avidity (multivalent aptamer).

[0348] Alternatively, the multimeric aptamer may comprise two or more aptameric monomers, wherein at least two mers of the multimeric aptamer are non-identical in structure, nucleic acid sequence or both. Such a multimeric aptamer is referred to herein as a heteromultimer. The heteromultimer may be directed to a single binding site i.e., monospecific (such as to avoid steric hindrance). The heteromultimer may be directed to a plurality of binding sites i.e., multispecific. The heteromultimer may be directed to a plurality of binding sites on different analytes. Further description of a multimeric aptamer is provided below.

[0349] A plurality of multimeric aptamers may be conjugated to form a conjugate of multimeric aptamers. The multimeric aptamer may comprise, two (dimer), three (trimer), four (tetramer), five (pentamer), six (hexamer), and even more units.

[0350] Aptamers of the invention can be synthesized and screened by any suitable methods in the art.

[0351] For example, aptamers can be screened and identified from a random aptamer library by SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind to an antigen of interest can be suitably screened and selected by a modified selection method herein referred to as cell-SELEX or cellular-SELEX.

[0352] A random aptamer library can be created that contains monomeric, dimeric, trimeric, tetrameric or other higher multimeric aptamers. A random aptamer library (either ssDNA or RNA) can be modified by including oligonucleotide linkers to link individual aptamer monomers to form multimeric aptamer fusion molecules. In other examples, a random oligonucleotide library is synthesized with randomized 45 nt sequences flanked by defined 20 nt sequences both upstream and downstream of the random sequence, i.e., known as 5'-arm and 3'-arm, which are used for the amplification of selected aptamers. A linking oligonucleotide (i.e., linker) is designed to contain sequences complementary to both 5'-arm and 3'-arm regions of random aptamers to form dimeric aptamers. For trimeric or tetrameric aptamers, a small trimeric or tetrameric (i.e., a Holiday junction-like) DNA nanostructure is engineered to include sequences complementary to the 3'-arm region of the random aptamers, therefore creating multimeric aptamer fusion through hybridization. In addition, 3-5 or 5-10 dT rich nucleotides can be engineered into the linker polynucleotides as a single stranded region between the aptamer-binding motifs, which offers flexibility and freedom of multiple aptamers to coordinate and synergize multivalent interactions with cellular ligands or receptors. Alternatively, multimeric aptamers can also be formed by mixing biotinylated aptamers with streptavidin.

[0353] A modified cellular SELEX procedure can be employed to select target-binding aptamers. Multimeric aptamers may be multivalent but be of single binding specificity (i.e., homomultimeric aptamers). Alternatively, the multimeric aptamer may be multivalent and multi- specific (i.e., heteromultimeric aptamers).

[0354] Thus, each monomer of the homomultimeric aptamer binds the target protein (e.g., cardiac troponins as well as antigenic variants thereof) in an identical manner. Thus according to an example of the invention, all monomeric components of the homomultimeric aptamer are identical.

[0355] Conversely, a heteromultimeric aptamer comprises a plurality of monomeric aptamers at least two of which bind different sites on a single target protein or bind at least two different target proteins.

[0356] Selection of RNA-aptamers is well-established using protocols described in the scientific literature.

[0357] In certain examples, a suitable nucleotide length for an aptamer ranges from about 15 to about 100 nucleotide (nt), and in various other examples, 12-30, 14-30, 15-30 nt, 30-100 nt, 30-60 nt, 25-70 nt, 25-60 nt, 40-60 nt, or 40-70 nt in length.

[0358] Multimerization can be done at the library level as follows.

[0359] In certain examples, a linker polynucleotide has a length between about 5 nucleotides (nt) and about 100 nt; in various examples, 10-30 nt, 20-30 nt, 25-35 nt, 30-50 nt, 40-50 nt, 50-60 nt, 55-65 nt, 50-80 nt, or 80-100 nt. It is within the ability of one of skill in the art to adjust the length of the linker polynucleotide to accommodate each monomeric aptamer in the multimeric structure.

[0360] In certain examples, the multimeric aptamers can be identified and screened from a random multimeric aptamer library as described herein. In other examples, the monomeric aptamers are linked to each other by one or a plurality of linker polynucleotides to form multimeric aptamers. Monomeric aptamers can be linked to form multimeric aptamers by any suitable means and in any configurations.

[0361] It will be appreciated that the monomeric structures of the invention can be further multimerized by post SELEX procedures.

[0362] Multimers can be linearly linked by continuous linear synthesis of DNA without spacers or with nucleic acid spacers. Aptamer synthesis usually relies on standard solid phase phosphoramitide chemistry.

[0363] Thus, dimers, trimers and tetramers or higher oligomeric structures (e.g., pentamers, hexamers, heptamers, octamers etc.) can be linked by a polymeric spacer.

[0364] In certain examples, the aptamers are further modified to protect the aptamers from nuclease and other enzymatic activities. The aptamer sequence can be modified by any suitable methods known in the art. For example, phosphorothioate can be incorporated into the backbone, and 5'-modified pyrimidine can be included in 5' end of ssDNA for DNA aptamer. For RNA aptamers, modified nucleotides such as substitutions of the 2'-OH groups of the ribose backbone, e.g., with 2'-deoxy-NTP or - fluoro-NTP, can be incorporated into the RNA molecule using T7 RNA polymerase mutants. The resistance of these modified aptamers to nuclease can be tested by incubating them with either purified nucleases or nuclease from mouse serum, and the integrity of aptamers can be analyzed by gel electrophoresis. The monomeric or multimeric aptamer of the invention can be further attached or conjugated to a detectable or therapeutic moiety (i.e., a pharmaceutical moiety).

[0365] Thus, as noted above, a diagnostic or therapeutic moiety can be attached to an aptamer embodied herein to provide additional biological activity, such as for diagnosing, preventing, or treating a condition or disease. In one example a diagnostic moiety such as a detectable moiety e.g., label (e.g., His tag, flag tag), fluorescent, radioactive, biotin / avidin etc., can be bound to the aptamer, and imaging, immunohistochemistry, or other invasive or non-invasive methods used to identify the location(s) and extend of binding of the conjugate to locations within the body. For therapeutic uses, a cytotoxic agent such as a chemotherapeutic agent, radioactive moiety, toxin, antibody, nucleic acid silencing agents e.g., small interferring RNA (siRNA) or other molecule with therapeutic activity when delivered to cells expressing a molecule to which the aptamer is targeted, may be used to enhance the therapeutic activity of the aptamer or provide a biological activity where the aptamer is providing the targeting activity. Moreover, other conjugates to the aptamers described herein are contemplated, such as but not limited to scaffolds, sugars, proteins, antibodies, polymers, and nanoparticles, each of which have art-recognized therapeutic or diagnostic utilities and can be targeted to particular sites in vivo using an aptamer embodied herein.

[0366] Detection of Binding Agents Including Peptide Binding Assays

[0367] The present invention includes use of a detection system involving the binding bTnT- IgG to capture / detection antibodies so as to measure the amount of bTnT-IgG present in a biological sample under interrogation. A similar solution is to detect the amount of unbound binding agent in a sample to get an indication of unbound or bound bTnT-IgG. It is intended that such alternative methods fall within the scope of the present invention as functional alternatives to directly detecting the amount of bound binding agent. Persons skilled in the art will appreciate that the concentration of bTnT-IgG in a sample can be readily calculated from the amount of bTnT-IgG in a sample when the sample volume is known.

[0368] In the assays, methods and kits according to the present invention, the measuring steps comprise detecting binding between bTnT-IgG and a binding agent that binds, selectively or specifically, to bTnT-IgG, and has low cross-reactivity with other markers of biological events.

[0369] In certain examples, the binding agent is an antibody or an antigen-binding fragment thereof. The antibody may be a monoclonal, polyclonal, chimeric or humanized antibody or antigen-binding fragment thereof. As such, in one example the assay, as well as methods involving assays, of the present invention is an immunoassay.

[0370] The antibodies of the present invention are particularly useful in immunoassays for determining the presence and / or amount of bTnT-IgG in a sample. Due to variable binding affinities of different antibodies, the person skilled in the art will appreciate that a standard binding curve of measured values versus amount of bTnT-IgG in a sample should be established for a particular antibody to enable the amount of bTnT-IgG in a sample to be determined. Such a curve is used to determine the true amount of bTnT-IgG in a sample.

[0371] Sample materials include biological fluids but are not limited thereto. In terms of the present invention, biological fluids are typically selected from whole blood, plasma or serum.

[0372] Immunoassays specific for bTnT-IgG usually will require the production or sourcing of antibodies that specifically bind to bTnT-IgG. The antibodies can be used to construct immunoassays with broad specificity, as in competitive binding assays below, or used in conjunction with other antibodies described below in sandwich type assays to produce assays specific to bTnT-IgG. The person skilled in the art will appreciate that non-competitive assays are also possible. The latter antibodies for sandwich immunoassays include those specific for amino acid sequences including the specific epitope sequences defined within SEQ ID NO: 1.

[0373] In another example, indicators may also be used. Indicators may be employed in ELISA and RIA assay formats.

[0374] Polyclonal and monoclonal antibodies can be used in competitive binding or sandwich type assays. In one example of this method a liquid sample is contacted with the antibody and simultaneously or sequentially contacted with a labelled bTnT-IgG or modified peptide containing the epitope recognised by the antibody. The label can be a radioactive component such as125I,131I,3H,14C or a non-radioactive component that can be measured by time resolved fluorescence, fluorescence, fluorescence polarisation, luminescence, chemiluminescence or colorimetric methods. These compounds include europium or other actinide elements, acrinidium esters, fluorescein, or radioactive material such as those above, that can be directly measured by radioactive counting, measuring luminescent or fluorescent light output, light absorbance etc. The label can also be any component that can be indirectly measured such as biotin, digoxin, or enzymes such as horseradish peroxidase, alkaline phosphatase. These labels can be indirectly measured in a multitude of ways. Horseradish peroxidase for example can be incubated with substrates such as o-Phenylenediamine Dihyhdrochloride (OPD) and peroxide to generate a coloured product whose absorbance can be measured, or with luminol and peroxide to give chemiluminescent light which can be measured in a luminometer. Biotin or digoxin can be reacted with binding agents that bind strongly to them; e.g. avidin will bind strongly to biotin. These binding agents can in turn be covalently bound or linked to measurable labels such as horseradish peroxidase or other directly or indirectly measured labels as above. These labels and those above may be attached to the peptide or protein: during synthesis, by direct reaction with the label, or through the use of commonly available crosslinking agents such as MCS and carbodiimide, or by addition of chelating agents.

[0375] Following contact with the antibody, usually for 18 to 25 hours at 4° C, or 1 to 240 minutes at 30° C to 40° C, the labelled peptide bound to the binding agent (antibody) is separated from the unbound labelled peptide. In solution phase assays, the separation may be accomplished by addition of an anti-gamma globulin antibody (second-antibody) coupled to solid phase particles such as cellulose, or magnetic material. The second-antibody is raised in a different species to that used for the primary antibody and binds the primary antibody. All primary antibodies are therefore bound to the solid phase via the second antibody. This complex is removed from solution by centrifugation or magnetic attraction and the bound labelled peptide measured using the label bound to it. Other options for separating bound from free label include formation of immune complexes, which precipitate from solution, precipitation of the antibodies by polyethyleneglycol or binding free labelled peptide to charcoal and removal from solution by centrifugation of filtration. The label in the separated bound or free phase is measured by an appropriate method such as those presented above.

[0376] Competitive binding assays can also be configured as solid phase assays that are easier to perform and are therefore preferable to those above. This type of assay uses a solid support including plates with wells (commonly known as ELISA or immunoassay plates), solid beads or the surfaces of tubes. The primary antibody is either adsorbed or covalently bound to the surface of the plate, bead or tube, or is bound indirectly through a second anti gamma globulin or anti Fc region antibody adsorbed or covalently bound to the plate. Sample and labelled peptide (as above) are added to the plate either together or sequentially and incubated under conditions allowing competition for antibody binding between bTnT-IgG in the sample and the labelled peptide. Unbound labelled peptide can subsequently be aspirated off and the plate rinsed leaving the antibody bound labelled peptide attached to the plate. The labelled peptide can then be measured using techniques described above.

[0377] Sandwich type assays are more preferred for reasons of specificity, speed and greater measuring range. In this type of assay an excess of the primary antibody to bTnT-IgG is attached to the well of an ELISA plate, bead or tube via adsorption, covalent coupling, or an anti Fc or gamma globulin antibody, as described above for solid phase competition binding assays. Sample fluid or extract is contacted with the antibody attached to the solid phase. Because the antibody is in excess this binding reaction is usually rapid. A second antibody to a bTnT-IgG peptide complex is also incubated with the sample either simultaneously or sequentially with the primary antibody. This second antibody is chosen to bind to a site on bTnT-IgG that is different from the binding site of the primary antibody. These two antibody reactions result in a sandwich with the bTnT-IgG from the sample sandwiched between the two antibodies. The second antibody is usually labelled with a readily measurable compound as detailed above for competitive binding assays. Alternatively, a labelled third antibody which binds specifically to the second antibody may be contacted with the sample. After washing the unbound material the bound labelled antibody can be measured by methods outlined for competitive binding assays. After washing away the unbound labelled antibody, the bound label can be quantified as outlined for competitive binding assays.

[0378] A dipstick type assay may also be used. These assays are well known in the art. They may for example, employ small particles such as gold or coloured latex particles with specific antibodies attached. The liquid sample to be measured may be added to one end of a membrane or paper strip preloaded with the particles and allowed to migrate along the strip. Binding of the antigen in the sample to the particles modifies the ability of the particles to bind to trapping sites, which contain binding agents for the particles such as antigens or antibodies, further along the strip. Accumulation of the coloured particles at these sites results in colour development are dependent on the concentration of competing antigen in the sample. Other dipstick methods may employ antibodies covalently bound to paper or membrane strips to trap antigen in the sample. Subsequent reactions employing second antibodies coupled to enzymes such as horse radish peroxidase and incubation with substrates to produce colour, fluorescent or chemiluminescent light output will enable quantitation of antigen in the sample. Receiver Operating Characteristic (ROC) Analysis

[0379] The clinical performance of a laboratory test depends on its diagnostic / prognostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Prognostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.

[0380] In each case, a receiver operating characteristic (ROC) plot depicts the overlap between the two distributions by plotting the sensitivity versus 1-specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as (number of true-positive test results) / (number of true-positive+number of false-negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup. On the x-axis is the false-positive fraction, or 1-specificity [defined as (number of false-positive results) / (number of true- negative+number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false-positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of disease in the sample. Each point on the ROC plot represents a sensitivity / -specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two distributions of results) has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes. If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than" to "less than" or vice versa. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test.

[0381] One convenient objective to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number. The most common global measure is the area under the ROC plot. By convention, this area is always ^ 0.5 (if it is not, one can reverse the decision rule to make it so). Values range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distributional difference between the two groups of test values). The area does not depend only on a particular portion of the plot such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative, descriptive expression of how close the ROC plot is to the perfect one (area = 1.0). Test Kits & Articles of Manufacture

[0382] Typically, test kits or articles of manufacture will be formatted for assays known in the art (e.g.) ELISA assays.

[0383] Binding agents that selectively bind cardiac troponins as well as immunoglobulins bound to cardiac troponins (e.g. IgG), and which include antigenic variants, thereof are desirably included in the test kits or articles of manufacture.

[0384] Accordingly, in an aspect of the present invention there is provided a test kit or article of manufacture comprising:

[0385] (i) an anti-cTnT binding agent which selectively binds to SEQ ID NO: 1; and

[0386] (ii) an anti-human IgG binding agent which selectively binds to the IgG bound to cTnT.

[0387] In another aspect of the present invention there is provided a test kit or article of manufacture for:

[0388] (a) diagnosing an acute coronary syndrome in a patient including an acute myocardial infarction or unstable angina pectoris;

[0389] (b) enhancing the performance of cardiac troponin, including highly sensitive troponin I (hsTnl) and highly sensitive troponin T (hsTnT) assays, for diagnosing myocardial infarction;

[0390] (c) distinguishing between type 1 myocardial infarction and type 2 myocardial infarction;

[0391] (d) diagnosing type 1 myocardial infarction;

[0392] (e) predicting a new acute coronary syndrome event;

[0393] (f) predicting future stroke; and

[0394] (g) predicting mortality the test kit or article of manufacture comprising:

[0395] (i) an anti-cTnT binding agent which selectively binds to SEQ ID NO: 1;

[0396] (ii) an anti-human IgG binding agent which selectively binds to the IgG bound to cTnT; and

[0397] (iii) optionally instructions for diagnosing or predicting the conditions recited in any one of (a) to (g).

[0398] In an example according to this and all other aspects of the present invention, the binding agent is selected from an antibody or antigen-binding fragment and an aptamer.

[0399] In another example, the antibody is monoclonal antibody, for example, and may be prepared in any mammal as described above, and includes antigen binding fragments and antibodies prepared using native and fusion peptides, for example.

[0400] Accordingly, in yet another aspect of the present invention there is provided a test kit or article of manufacture for: (a) diagnosing an acute coronary syndrome in a patient including an acute myocardial infarction or unstable angina pectoris;

[0401] (b) enhancing the performance of cardiac troponin, including highly sesntivie troponin I (hsTnl) and highly sensitive troponin T (hsTnT), for diagnosing myocardial infarction;

[0402] (c) distinguishing between type 1 myocardial infarction and type 2 myocardial infarction;

[0403] (d) diagnosing type 1 myocardial infarction;

[0404] (e) predicting a new acute coronary syndrome event;

[0405] (f) predicting future stroke; and

[0406] (g) predicting mortality the test kit or article of manufacture comprising:

[0407] (i) an anti-cTnT monoclonal antibody which selectively binds to SEQ ID NO: 1;

[0408] (ii) an anti-human IgG monoclonal antibody which selectively binds to the IgG bound to cTnT; and

[0409] (iii) optionally instructions for diagnosing or predicting the conditions recited in any one of (a) to (g).

[0410] The test kits or articles of manufacture may be comprised of one or more containers and may also include collection equipment, for example, bottles, bags (such as intravenous fluids bags), vials, syringes, and test tubes. At least one container will be included and will hold a product which is effective for use in the assays and methods described herein. The product is typically a peptide binding agent, particularly an antibody or antigen-binding fragment according to the invention described herein, or a composition comprising any of these. In one example, an instruction or label on or associated with the container indicates that the composition is used for diagnosing or monitoring the acute coronary syndrome or for predicting a future acute coronary syndrome event, stroke or mortality. Other components may include diluents and buffers.

[0411] The test kits or articles of manufacture may also include detection or measurement means or measurement directions involving one or more additional markers or risk factors for a cardiac disease of interest (e.g.) heart rate, haemoglobin concentration, blood pressure, age, sex, weight, level of physical activity, family history of events including obesity, diabetes and cardiac events, and levels of NT-proBNP, BNP, BNPsp and BNPsp fragments including BNPsp(17-26). Again, this may include binding agent(s), aptamer(s), antibody(s) and antigen-binding fragment(s) thereof which selectively bind to other biomarker(s) of interest.

[0412] In certain examples of the test kits or articles of manufacture according to the present invention, the anti-cTnT monoclonal antibody which selectively binds to SEQ ID NO: 1 is immobilized on a solid substrate, surface or matrix, for example, a micro-titre plate, porous strip or chip to form at least one detection site for the bTnT-IgG complex.

[0413] Also included in the kits or articles of manufacture may be a device for sample analysis comprising a disposable testing cartridge with appropriate components (markers, antibodies and reagents) to carry out sample testing. The device will conveniently include a testing zone and test result window. Immunochromatographic cartridges are examples of such devices. See for example US 6,399,398; US 6,235,241 and US 5,504,013.

[0414] Alternatively, the device may be an electronic device which allows input, storage and evaluation of levels of the measured marker against control levels and other marker levels. US 2006 / 0234315 provides examples of such devices. Also useful in the invention are Ciphergen's Protein Chip® which can be used to process SELDI results using Ciphergen's Protein Chip® software package.

[0415] The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

[0416] EXAMPLES

[0417] EXAMPLE 1: ASSAY METHOD

[0418] A monoclonal antibody directed to amino acids X-Y of the cardiac troponin T protein was coated on an ELISA plate. The plate is washed and then blocked with a common blocking agent (e.g. bovine serum albumin, casein) resulting in a prepared test plate.

[0419] Test plates are then processed as follows.

[0420] 100 ul of plasma sample (diluted 1 :200 in PBS) is added to the plate wells and incubated at room temperature for 1 hour. Wells are washed with 300 ul of buffer (0.1% Tween?) aspirated dry. 100 ul of goat anti-human IgG antibody conjugated to HRP is added and incubated at room temperature for 30 minutes. Plates are then washed again, aspirated dry and 100 ul of TMB added to develop colour.

[0421] The assay is therefore a sandwich ELISA setup that produces a linear curve proportional to the amount of antibody bound TnT present. Crucial to the assay is the requirement that antibody bound TnT must be free of bound Tnl: the plate bound monoclonal antibody is directed to the region of TnT that binds to Tnl - bound Tnl will interfere with this reaction and thus TnT cannot bind if TnT and Tnl are complexed. Refer to Figure 1.

[0422] EXAMPLE 2: CLINICAL SAMPLES TESTED

[0423] Study Population and Design

[0424] Patients with chest pain suspicious of acute coronary syndromes (ACS) were prospectively enrolled into the Applicant's ongoing observational study known as Signal Peptides in Acute Coronary Events (SPACE, http: / / www.anzctr.org.au, ACTRN12609000057280). All patients were enrolled in accord with protocols approved by the Health and Disabilities Ethics Committee of the Ministry of Health, New Zealand. All participants gave informed consent before recruitment and all investigations conformed to the principles of the Declaration of Helsinki. Since March 2009, 1053 eligible patients aged 18 years or older with the primary complaint of acute chest pain clinically suspicious of ACS and <4 hours from onset were recruited and included in this study. More general / atypical symptoms (such as fatigue, nausea, vomiting, sweating and faintness) were not used as inclusion criteria. Patients with end stage renal disease on dialysis were excluded.

[0425] In addition, samples were also obtained from the FAST-TRAC patient cohort. Adjudicated Diagnosis

[0426] The adjudicated diagnosis of acute MI was made in accordance with published guidelines [8], by two independent cardiologists with access to all clinical data, but not BNP signal peptide or hsTnl assay results. In the case of disagreement, an independent third cardiologist adjudicated to resolve this. The biochemical component of the diagnosis of MI was based on a highly sensitive (hs) Tnl assay with 1 value > 99th URL within 12 hours of presentation. Atrial fibrillation (AF) during emergency department presentation was determined from the ECG, whereas the diagnosis of UAP was made on the basis of confirmatory provocative investigations (exercise tolerance testing (ETT) or dobutamine stress echocardiography testing (DSE)) or angiographic catheterisation findings. Other cardiac disorders were defined as non-ACS cardiac presentations comprising conduction disorders (sick sinus syndrome), arrhythmias (atrial fi bril lation / fl utter) and acute heart failure. Undifferentiated chest pain was defined as chest pain without definitive associated clinical findings or cardiac tests where doubt remains as to the aetiology. Non-cardiac chest pain was defined as present when a definite non-cardiac cause for symptoms was identified.

[0427] Follow-up and Prognostic End Points

[0428] Within X days post-discharge, patients were followed up by telephone or in writing. Reported clinical events were identified from the patients themselves (or their primary physician) corroborated by the records of the treating institution or by the centralised New Zealand Ministry of Health database registry entries on mortality and events. The postdischarge end points considered were death, new MI, new ACS, acute decompensated heart failure and stroke. Events were analysed by ROC analysis for three groups; all patients (n=X), MI patients (n=X) and non-MI patients (n=X).

[0429] Clinical Assessment and Sample Collection

[0430] For all patients, initial assessment included clinical history, physical examination, ECG recordings, standard blood tests, pulse oximetry and chest radiography. Patient management was at the discretion of the attending physicians. Only standard clinical core lab hsTnT (Roche) and other standard blood test results were available to treating staff.

[0431] After consent was given, serial blood samples for measurement of bTnT-IgG, hsTnl and NT-proBNP (EDTA tubes) and hsTnT and lipids (Heparin tubes) were taken at 0, 1, 2 and 12- 24 hours after presentation. Blood samples (10 mL) were drawn into EDTA tubes chilled on ice, centrifuged at 2500g for 10 minutes and the plasma frozen at -80°C prior to assays. Heparin samples were collected into 5ml tubes and immediately sent to the hospital core biochemistry unit for measurement of hsTnT and lipids. Statistical Analysis

[0432] Continuous variables are presented as median (interquartile range, (IQR)) and categorical variables as numbers and percentages. Bivariate associations between patient outcomes and continuous variables were analysed using non-para metric Mann-Whitney U test and categorical variables using the Pearson x2 test. Analysis of plasma analyte results employed Spearman rank order correlation testing and receiver operator characteristic curve (ROC) analysis and diagnostic performance (sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV)) were carried out using SPSS v28 (IBM). For ROC curve generation and biomarker panel comparisons, biomarker data were analysed as standardised variables (z-scores) or LoglO values, where appropriate.

[0433] Individual biomarkers (bTnT-IgG, NT-proBNP, hsTnl and hsTnT) were assessed by ROC analysis for the prediction of index MI and UAP.

[0434] ROC curve comparisons were made using the approach of Hanley and McNeill [9] in SPSS v28. In all analyses, a p-value <0.05 was considered significant.

[0435] EXAMPLE 3: QUANTIFICATION OF bTnT-IgG LEVELS IN SPACE AND FAST-TRAC COHORTS bTnT-IgG levels were measured in patients from the SPACE (n= 257) and FAST-TRAC (n = 1489) cohorts. The total number of patients was n = 257 + 1489 = 1746.

[0436] The median (interquartile range, 25th-75thpercentile) values of bTnT-IgG in individuals with or without a history of cardiovascular disease (CAD), myocardial infarction (MI), elevated cholesterol levels (Choi) and DM are reflected in Table 1, below.

[0437] Table 1: Median bTnT-IgG levels in patients with a specific history

[0438] Table 2: Spearman's rho analysis of different analytes in SPACE and FAST-TRAC patients

[0439] EXAMPLE 4: bTnT-IgG LEVELS ENHANCE DX PERFORMANCE OF hsTnl

[0440] The data presented in Fig. 2 and Table 3 below reflects diagnostic receiver operator characteristic (ROC) curves for the diagnosis of acute myocardial infarction (AMI) (n = 179) by hsTnT (green line), hsTnl (turquoise line), bTnT-IgG (blue line), hsTnl + bTnT-IgG (crimson line) and hsTnT + bTnT-IgG (purple line) in 1665 patients presenting to hospital ("index presentation") with the primary complaint of chest pain suspicious of an acute coronary syndrome. In these particular measurements all concentrations of bTnT-IgG were used (median value = 0.47991 U / mL), and the addition of bTnT-IgG to hsTnl did not significantly add to the performance of hsTnl where the respective AUCs were 0.925 (hsTnl) and 0.926 (hsTnl + bTnT-IgG).

[0441] Table 3: bTnT-IgG assisted performance of hsTnT / hsTnT for Dx of MI (all bTnT-IgG cone.)

[0442] The Applicants next examined the enhanced performance of hsTnT and hsTnl for the diagnosis of acute myocardial infarction in the presence of bTnT-IgG both below and above the median value of 0.47991 U / mL. The data are presented in Tables 4-9 below.

[0443] Table 4: bTnT-IgG assisted performance of hsTnT for Dx of MI

[0444] Table 5: bTnT-IgG assisted performance of hsTnl for Dx of MI

[0445] Table 6: bTnT-IgG assisted performance of hsTnT for Dx of MI ["rule in test"] when hsTnT >14 ng / L Table 7: bTnT-IgG assisted performance of hsTnl for Dx of MI ["rule in test"] when hsTnT > 19 ng / L

[0446] Table 8: bTnT-IgG assisted performance of hsTnl for Dx of MI ["rule out test"], when hsTnl < 14 ng / L

[0447] Table 9: bTnT-IgG assisted performance of hsTnT for Dx of MI ["rule out test"], when hsTnT < 14 ng / L

[0448] Accordingly, bTnT-IgG does not improve the ability to hsTnT to rule out MI when below hsTnT <14 ng / L.

[0449] EXAMPLE 5: bTnT-IgG TO PREDICT POSITIVE CARDIAC STRESS TEST OUTCOME bTnT-IgG has no prognostic utility for new ACS within one year, in all patients and in sub-groups of index MI and index non-MI. However, the data presented in Figure 3 and Tables 10-12 below shows bTnl-IgG does have predictive ability for individuals who will test positive on a provocative stress test for inducible cardiac ischemia (n=83 / 589 total tests).

[0450] Table 10: Regression analysis for prediction of inducible ischemia

[0451] According to the data presented in Tabel 11, bTnT-IgG was most predictive when levels where above the median (0.47991 U / mL). Table 11: Utility of bTnT-IgG to predict inducible ischemia

[0452] The data presented in Figure 3, when read in conjunction with the specificity and sensitivity data presented in Table 12 below demonstrates that when the sensitivity of bTnT- IgG was held at 90%, a marked difference in specificity was observed when levels of bTnT- IgG above median (specificity = 24%) as opposed to levels of bTnT-IgG below median (specificity = 52%) reflecting the clinical value in utilising bTnT-IgG above a specified concentration.

[0453] Table 12: Specificity and sensitivity of bTnT-IgG to predict inducible ischemia

[0454] EXAMPLE 6: PREDICTION OF ACUTE MI WHEN hsTnT AT PRESENTATION IS >52 ng / L

[0455] According to the data presented in Figure 4, when read in conjunction with Tables 13 and 14, below, the inclusion of bTnT-IgG in an acute myocardial infarction prediction model that includes Age, ECG results and hsTnT, improves the ROC value and therefore prediction of acute myocardial infarction when hsTnT is >52 ng / L at index presentation. Specifically, the area under the curve increased from 0.794 ± 0.036 to 0.820 ± 0.033.

[0456] Table 13: ROC analysis for bTnT-IgG in predicting acute myocardial infarction when hsTnT >52 ng / L in the presence of other risk factors including age, abnormal ECG and hsTnT

[0457] Table 14: Pairwise regression analysis EXAMPLE 7: PREDICTION OF ACUTE MI WHEN hsTnl AT PRESENTATION IS >34 ng / L

[0458] According to the data presented in Figure 5, when read in conjunction with Tables 15 and 16, below, the inclusion of bTnT-IgG in an acute myocardial infarction prediction model that includes Age, ECG results and hsTnl, improves the ROC value and therefore prediction of acute myocardial infarction when hsTnl is > 34 ng / L at index presentation. Specifically, the area under the curve increased from 0.806 ± 0.028 to 0.822 ± 0.026.

[0459] Table 15: ROC analysis for bTnT-IgG in predicting acute myocardial infarction when hsTnl > 34 ng / L in the presence of other risk factors including age and abnormal ECG

[0460] Table 16: Pairwise regression analysis

[0461] EXAMPLE 8: MONOMERIC IgG BOUND cTnT (bTnT-IgG) DISTINGUISHES BETWEEN T1MI & T2MI

[0462] The data presented in Figure 7, when read in conjunction with Tables 17-19 (i.e. data related to T1MI patients; n = 209 / 456), and compared to the data presented in Figure 8, when read in conjunction with Tables 20-22 (i.e. data related to T2MI patients; n = 40 / 456), reveals that bTnT-IgG was elevated at index presentation in T1MI patients (AUC = 0.574 ± 0.027; bTnT_406_0) compared to T2MI patients (AUC = 0.483 ± 0.050; bTnT_406_0) when hsTnT levels were > 14 ng / L.

[0463] The bTnT-IgG values measured at index presentation (i.e. bTnT_406_0) do not appear to differ in significance to the values measured after two hours from index presentation (i.e. bTnT_406_2) reflecting that a delta bTnT-IgG value (AbTnT-IgG) has little or no clinical significance. By way of illustration the area under the curve for the T=0 h and T=2 h time points for bTnT-IgG in T1MI patients was 0.574 ± 0.027 and 0.581 ± 0.027, respectively. Conversely, this means that bTnT-IgG values at index presentation (i.e. bTnT-IgG[T=oh]) are applicable across any time point measured. These data demonstrate a bone fide clinical utility for bTnT-IgG in distinguishing T1MI patients from T2MI patients which has the potential to significantly enhance triaging / treatment pathways for myocardial infarction patients presenting to (e.g.) an emergency department, clinic or other medical forum.

[0464] Table 17: Case Processing Summary - Type I Myocardial Infarction

[0465] Table 18: Type I Myocardial Infarction Values

[0466] Table 19: Receiver Operating Curve Analysis for Type 1 Myocardial Infarction

[0467] Table 20: Case Processing Values - Type II Myocardial Infarction Table 21: Type II Myocardial Infarction Values

[0468] Table 22: Receiver Operating Curve Analysis for Type II Myocardial Infarction

[0469] EXAMPLE 9: bTnT-IgG IMPROVES DIAGNOSTIC PERFORMANCE OF hsTnT

[0470] The data presented in Figure 9, when read in conjunction with Table 23 reveals that bTnT-IgG at index presentation improves the performance of a highly sensitive troponin T assay for the diagnosis of an acute myocardial infarction when the sensitivity of the assay was fixed at 98.6%. Specifically, the addition of bTnT-IgG to hsTnT at index presentation (i.e bTnT_0 + hsTnT_0) improved the negative predictive value (NPV) for the diagnosis of myocardial infarction from 87.0% to 94.1% whereas the positive predictive value improved from 32.6% to 34.1%. Similarly, when bTnT-IgG levels at index presentation (bTnT_0) were added to hsTnT measured 2 hours from index presentation (i.e. hsTnT_2) the NPV improved from 94.0% to 97.5% and PPV improved from 34.1% to 38.3%.

[0471] These data further reveal that the combined performance of bTnT-IgG and hsTnT at index presentation (i.e. bTnT_0 + hsTnT_0) in terms of NPV and PPV essentially matched the performance of hsTnT measured 2 hours from index presentation (i.e. hsTnT_2) whereas the combined performance of bTnT-IgG at index presentation and hsTnT measured 2 hours from index presentation (i.e. bTnT_0 + hsTnT_2) out-performed hsTnT measured two hours from index presentation. Table 23: Addition of bTnTO improves hsTnT NPV and PPV

[0472] Applicant further interrogated the effect of sex and an abnormal electrocardiogram on the performance of hsTnT for the diagnosis of an acute myocardial infarction, at index presentation and two hours from index presentation. These data are presented in Figure 10 when read in conjunction with Table 24 below and reveal that the addition of these variables failed to improve the diagnostic performance of hsTnT for the diagnosis of an acute myocardial infarction when compared to the addition of bTnT-IgG at index presentation. Specifically, the NPV performance of [hsTnT_0 + ECG + sex] was poorer compared to [hsTnT_0 + bTnT- IgG_0] (i.e. 92.2% versus 94.1%); similarly, the PPV performance of [hsTnT_0 + ECG + sex] was poorer compared to [hsTnT_0 + bTnT-IgG_0] (i.e. 33.0% versus 34.1%). Similarly, the NPV performance of [hsTnT_2 + ECG + sex] was poorer compared to [hsTnT_2 + bTnT- IgG_0] (i.e. 96.4% versus 97.5%); similarly, the PPV performance of [hsTnT_2 + ECG + sex] was poorer compared to [hsTnT_2 + bTnT-IgG_0] (i.e. 35.5% versus 38.3%).

[0473] Table 24: Addition of ECG + Sex doesn't improve hsTnT NPV and PPV as well as bTnT_0 EXAMPLE 10: bTnT-IgG ENHANCES DIAGNOSTIC PERFORMANCE OF hsTnT TO DIAGNOSE T1MI

[0474] The data presented in Figure 11, when read in conjunction with Table 25 reveals that bTnT-IgG could improve the performance of hsTnT for the diagnosis of type 1 myocardial infarction when the specificity of the assay is fixed (e.g. in the data presented below specificity of the assay was fixed at 95.2%) in those patients with a hsTnT concentration greater than or equal to 52 ng / L.

[0475] Receiver Operating Curve analysis reveals that the area under the curve improved from AUC = 0.697 to 0.733 with the addition of bTnT-IgG to hsTnT at index presentation (hsTnT_0 ± bTnT 0); similarly the area under the curve improved from AUC = 0.800 to 0.815 with the addition of bTnT-IgG at index presentation to hsTnT measured two hours from index presentation.

[0476] Table 25: Addition of bTnTO improves sensitivity curve parameters of hsTnT

[0477] EXAMPLE 11: bTnT-IgG ENHANCES DIAGNOSTIC PERFORMANCE OF hsTnl TO DIAGNOSE T1MI

[0478] The data presented in Figure 12, when read in conjunction with Table 26 reveals that bTnT-IgG could improve the performance of hsTnT for the diagnosis of type 1 myocardial infarction when the sensitivity of the assay is fixed (e.g. in the data presented below sensitivity of the assay was fixed at 99%) in those patients with a hsTnl concentration greater than or equal to 64 ng / L.

[0479] Receiver Operating Curve analysis reveals that the area under the curve improved from AUC = 0.697 to 0.733 with the addition of bTnT-IgG to hsTnT at index presentation (hsTnT_0 ± bTnT 0); similarly the area under the curve improved from AUC = 0.800 to 0.815 with the addition of bTnT-IgG at index presentation to hsTnT measured two hours from index presentation. Further, the NPV improved from 53.6% to 89.0% for hsTnl measured at index presentation (hsTnI_0) with the addition of bTnT_0; whereas the NPV improved from 82.2% to 92.4% for hsTnl measured two hours from index presentation (hsTnI_2) with the addition of bTnT_0.

[0480] Table 26: Addition of bTnTO improves curve parameters of hsTnl

[0481] Applicant further interrogated the effect of sex and an abnormal electrocardiogram on the performance of hsTnl for the diagnosis of type 1 myocardial infarction, when measured two hours from index presentation with an without bTnT-IgG at index presentation. These data are presented in Figure 13 when read in conjunction with Table 27 below and reveal that the addition bTnT-IgG at index presentation significantly improved the diagnostic performance [hsTnl + abnormal ECG + sex] where the NPV improved from 79.7% to 92.2%.

[0482] Table 27: Addition of bTnTO + addition of ECG + Sex to hsTnI2 Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

[0483] REFERENCES

[0484] 1. Munro AR, Jerram T, Morton T, Hamilton S, 2015. Use of an accelerated diagnostic pathway allows rapid and safe discharge of 70% of chest pain patients from the emergency department. NZ Med J 128:62-71.

[0485] 2. Ministry of Health, 2012. Mortality and demographic data 2009. Wellington: New Zealand.

[0486] 3. Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD; Joint ESC / ACCF / AHA / WHF Task Force for the Universal Definition of Myocardial Infarction, 2012. Third Universal Definition of Myocardial Infarction. Circulation 126:2020-2035.

[0487] 4. eichlin T, Hochholzer W, Bassetti S, Steuer S, Stelzig C, Hartwiger S, Biedert S, Schaub N, Buerge C, Potocki M, Noveanu M, Breidthardt T, Twerenbold R, Winkler K, Bingisser R, Mueller C, 2009. Early diagnosis of myocardial infarction with sensitive cardiac troponin assays. New England J. Med. 361 :858-867.

[0488] 5. Keller T, Zeller T, Peetz D, Tzikas S, Roth A, Czyz E, Bickel C, Baldus S, Warnholtz A, Frohlich M, Sinning CR, Lackner KJ, Munzel TF, Blankenberg S, 2009. Sensitive troponin I assay in early diagnosis of myocardial infarction. New England J. Med. 361 :868-877.

[0489] 8. Thygesen K, Alpert JS, Jaffe AS, et al. Fourth Universal Definition of Myocardial Infarction (2018). J Am Coll Cardiology 72(18) :2231-2264.

[0490] 9. Hanley JA, McNeil BJ. A method of comparing the areas under receiver operating characteristic curves derived from the same cases. Radiology (1983) 148(3) :839-43.

Claims

CLAIMS1. A method for determining the level of a bound protein complex comprising cardiac Troponin T (cTnT) and an immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from a patient, the method comprising:(i) contacting the biological sample with a reaction mix comprising an a nti- cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti-cTnT capture antibody to selectively bind to the bTnT-IgG complex;(ii) washing the reaction mix from (i) to remove non-selectively bound analytes;(Hi) contacting the reaction mix from (ii) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti-IgG capture antibody to selectively bind to the bTnT-IgG complex; and(iv) determining the level of bTnT-IgG in the biological sample.

2. A method for improving the diagnostic performance of a cardiac troponin assay for the diagnosis of an acute myocardial infarction in a patient, the method comprising:(i) determining the level of a bound protein complex comprising cardiac Troponin T(cTnT) and a human immunoglobulin G (IgG) (bTnT-IgG) in a biological sample obtained from the patient which comprises:(a) contacting the biological sample with a reaction mix comprising an anti-cTnT capture antibody which selectively binds to SEQ ID NO: 1, for a time and under conditions sufficient for the anti- cTnT capture antibody to selectively bind to the bTnT-IgG complex;(b) washing the reaction mix from (a) to remove non-selectively bound analytes;(c) contacting the reaction mix from (b) with an anti-IgG detection antibody, for a time and under conditions sufficient for the anti- IgG detection antibody to selectively bind to the bTnT-IgG complex; and(d) determining the level of bTnT-IgG in the biological sample;(ii) comparing the level of bTnT-IgG from (i) against a reference standard from a control populationwherein, when the level of bTnT-IgG in the biological sample is higher relative to the reference standard from a control population it is combined with the cardiac troponin assay to improve the performance of the cardiac troponin assay for the diagnosis of an acute myocardial infarction in the patient compared to a diagnosis which is achieved in the absence of a bTnT-IgG measurement.

3. The method according to claim 2, wherein the cardiac troponin assay is a cardiac troponin T (cTnT) assay or a cardiac troponin I (cTnl) assay.

4. The method according to claim 1 or claim 2, wherein when the cardiac troponin assay is a cTnT assay, the concentration of cTnT in the biological sample obtained from the patient is greater than or equal to 14 ng / L.

5. The method according to claim 4, wherein when the concentration of cTnT is greater than or equal to 52 ng / L the acute myocardial infarction is type 1 myocardial infarction.

6. The method according to claim 1 or claim 2, wherein when the cardiac troponin assay is a cTnl assay, the concentration of cTnl in the biological sample obtained from the patient is greater than or equal to 19 ng / L.

7. The method according to claim 6, wherein when the concentration of cTnl is greater than or equal to 64 ng / L the acute myocardial infarction is type 1 myocardial infarction.

8. The method according to any one of claims 1 to 5, wherein the improved diagnostic accuracy of the cardiac troponin assay is selected from improved assay specificity, improved assay sensitivity, positive predictive value for the diagnosis of an acute myocardial infarction, negative predictive value for the diagnosis of an acute myocardial infarction and improved accuracy for the diagnosis of an acute myocardial infarction.

9. The method according to any one of claims 1 to 8, wherein the anti-cTnT antibody selectively binds to SEQ ID NO: 1.

10. The method according to claim 1, wherein the antiOcTnT antibody selectively binds to a region defined by amino acid residues 106-183 as set forth in SEQ ID NO: 1.

11. The method according to any one of claims 1 to 10, wherein the anti-cTnT antibody is a monoclonal antibody.

12. The method according to any one of claims 1 to 11, wherein the anti-cTnT antibody is immobilized on a solid substrate.

13. The method according to any one of claims 1 to 12, wherein the anti-IgG antibody is a human monoclonal antibody.

14. The method according to any one of claims 1 to 13 wherein the anti-IgG antibody comprises a detectable label.

15. The method according to claim 14, wherein the detectable label is an enzymatic detection label.

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