JP2010528306A - H-FABP as an early predictor of myocardial infarction - Google Patents

Abstract

The present invention relates to a method for diagnosing myocardial infarction in a subject suffering from acute coronary syndrome and having a myocardial troponin level that is detectable but has a level that is lower than what is considered an index of myocardial infarction. Furthermore, the present invention relates to a method for identifying a subject who can perform cardiac intervention. However, the subject suffers from acute coronary syndrome and has a myocardial troponin level that is detectable but lower than what is considered an indicator of myocardial infarction. The method according to the invention is based on measuring H-FABP and optionally myoglobin in the sample of the subject and comparing the amount of H-FABP and optionally myoglobin with a reference amount. Also encompassed by the present invention are kits or devices for performing the methods according to the present invention.
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Description

  The present invention relates to a method for diagnosing myocardial infarction in a subject suffering from acute coronary syndrome and having a myocardial troponin level that is detectable but has a level that is lower than what is considered an index of myocardial infarction. Furthermore, the present invention relates to a method for identifying a subject who can perform cardiac intervention. In this case, the subject is suffering from acute coronary syndrome and has a myocardial troponin level that is detectable but lower than what is considered an indicator of myocardial infarction. The method according to the invention comprises the steps of measuring heart-type fatty acid binding protein (H-FABP) and optionally myoglobin in the sample of the subject, and determining the amount of heart-type fatty acid binding protein (H-FABP) and optionally myoglobin. Based on comparing with at least one reference quantity. Also encompassed by the present invention are kits or devices for performing the methods according to the present invention.

  The goal of modern medicine is to provide a personal or individual treatment regimen. It is a treatment regimen that takes into account the needs or risks of each individual patient. A particularly significant risk is the presence of cardiovascular complications, in particular the presence of acute cardiovascular events. Cardiovascular complications are one of the leading causes of morbidity and mortality in the Western Hemisphere. In the individual treatment of a person suffering from cardiovascular complications, a reliable diagnosis has a significant impact on the success of the person's treatment. This is particularly important in patients who have symptoms of acute coronary syndrome (ACS).

  Acute coronary syndrome means a group of clinical symptoms caused by acute myocardial ischemia. Patients with acute coronary syndrome have a markedly increased risk of cardiac death and must be identified among patients with atraumatic chest symptoms (Morrow et al., National academy of clinical biochemistry guidelines: Clinical characteristics and utilization of biochemical markers in acute coronary syndrome, 2007, Circulation; 115; 356-375). Patients who present with symptoms of an acute cardiovascular event (eg, chest discomfort greater than 20 minutes) and undergo an emergency assessment are generally examined by electrocardiography. In addition, a blood sample is taken to determine the level of cardiac troponin. Myocardial troponin, such as troponin T, is a biomarker for myocardial infarction (MI). An electrocardiogram (ECG) provides important information for diagnosis. Specifically, when ECG shows an increase in the ST portion, it is diagnosed as ST-elevation myocardial infarction (STEMI). If the ECG does not show an ST segment elevation, it is diagnosed as non-ST elevation MI (NSTEMI) when cardiac troponin is detected in each patient sample. Patients who do not exhibit characteristic ECG and do not have detectable cardiac troponin are suspected to have unstable angina (UAP). Unstable angina and NSTEMI are thought to be closely related conditions that share similar clinical findings. However, they vary in severity. NSTEMI is distinguished from unstable angina by ischemia causing irreversible myocardial damage detectable by biomarkers of myocardial necrosis (Morrow et al., Supra). In any of the cases described, and thus in any of STEMI, NSTEMI, and UAP, the patient is treated according to the diagnosis.

  In the past 20 years, biomarkers such as cardiac troponin have become valuable tools for the diagnosis of heart related diseases. Additional markers for heart related diseases are, for example, NTproBNP or creatine kinase isoenzyme MB (CK-MB). Recently, myoglobin measurement has been shown to be a useful tool for the diagnosis of subjects with ACS symptoms (Brogan GX et al. Evaluation of a new rapid quantitative immunoassay for serum myoglobin versus CK-MB for ruling out acute myocardial infarction in the emergency department. 1994. Ann. Emerg. Med. 24 (4): 665-71). Measurement of myoglobin is advantageous because it can measure the increase in concentration immediately after the onset of symptoms (about 1 hour later), and studies have demonstrated that AMI detection sensitivity is high within the first few hours of ACS. However, the use of myoglobin for the diagnosis of myocardial infarction has some limitations. The concentration of myoglobin increases rapidly after onset of symptoms, but this concentration decreases rapidly after about 8-16 hours (James McCord J. at al. Ninety-Minute Exclusion of Acute Myocardial Infarction By Use of Quantitative Point -of-Care Testing of Myoglobin and Troponin I. 2001. Circulation 104: 1483; Morrow DA et al. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines: Clinical Characteristics and Utilization of Biochemical Markers in Acute Coronary Syndromes. 2007. Circulation, 115 : 356-375).

  More recently, heart-type fatty acid binding protein (H-FABP) has been suggested to be an early marker of myocardial infarction. Heart-type fatty acid binding protein (H-FABP) is a low molecular weight cytoplasmic protein and is present in large amounts in the myocardium. When the myocardium is damaged as in the case of myocardial infarction, low molecular weight cytoplasmic proteins such as H-FABP are released into the bloodstream, so that an increase in H-FABP level can be detected in the blood sample. (For example, Okamoto et al., Clin Chem Lab Med 38 (3): 231-8 (2000) Human heart-type cytoplasmic fatty acid-binding protein (H-FABP) for the diagnosis of acute myocardial infarction.Clinical evaluation of H -FABP in comparison with myoglobin and creatine kinase isoenzyme MB; O'Donoghue et al., Circulation, 114; 550-557 (2006) Prognostic Utility of Heart-Type Fatty Acid Binding Protein in patients with acute coronary syndrome; or Ruzgar et al ., Heart Vessels, 21; 209-314 (2006) The use of human heart-type fatty acid-binding protein as an early diagnostic marker of myocardial necrosis in patients with acute coronary syndrome, and its comparison with troponin-T and its creatine kinase-myocardial band).

  The finding that cardiac troponin, such as cardiac troponin T (TnT) or cardiac troponin I (TnI), is a marker for myocardial infarction has revolutionized the diagnosis and management of patients with symptoms of ACS. Specifically, myocardial troponin T is a very specific marker of myocardial damage, thus allowing discrimination between UAP and MI in patients with ACS symptoms. However, there are still some problems associated with using cardiac troponin as a diagnostic marker in patients with acute coronary syndrome. For example, cardiac troponin levels generally do not increase upon the onset of acute coronary event symptoms. In general, elevated troponin levels are detectable about 4-6 hours after the onset of ACS symptoms. Thus, within the first 0-6 hours of an acute cardiovascular event, the use of troponin as a diagnostic biomarker for myocardial infarction results in a relatively high rate of false negative results. Thus, myocardial infarction may not be recognized by the myocardial troponin assay, which can lead to inadequate or delayed treatment. Introducing a new generation myocardial troponin test that is more sensitive than previous generations of troponin tests and therefore can detect much lower myocardial troponin levels, so that detection of elevated myocardial troponin levels is more reliable and more It became possible to do early. Therefore, in the case of a myocardial event, it is possible to detect necrosis earlier. However, recent studies provide evidence that myocardial troponin can be reproducibly detected in patients with stable coronary heart disease who do not suffer from acute events when using more sensitive troponin tests (unpublished data ). Thus, if a person with stable coronary heart disease and still low but already elevated myocardial troponin levels showed symptoms of ACS, the detectable elevated myocardial troponin level is due to an acute event or has already Whether it is due to existing coronary heart disease is unknown. This increases the possibility of misdiagnosis, for example, the possibility of diagnosing MI instead of UAP, which can result in harmful, wrong, and / or delayed treatment.

Morrow et al., National academy of clinical biochemistry guidelines: Clinical characteristics and utilization of biochemical markers in acute coronary syndrome, 2007, Circulation; 115; 356-375 Brogan G.X et al. Evaluation of a new rapid quantitative immunoassay for serum myoglobin versus CK-MB for ruling out acute myocardial infarction in the emergency department. 1994. Ann. Emerg. Med. 24 (4): 665-71 James McCord J. at al. Ninety-Minute Exclusion of Acute Myocardial Infarction By Use of Quantitative Point-of-Care Testing of Myoglobin and Troponin I. 2001. Circulation 104: 1483 Morrow D. A. et al. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines: Clinical Characteristics and Utilization of Biochemical Markers in Acute Coronary Syndromes. 2007. Circulation, 115: 356-375 Okamoto et al., Clin Chem Lab Med 38 (3): 231-8 (2000) Human heart-type cytoplasmic fatty acid-binding protein (H-FABP) for the diagnosis of acute myocardial infarction.Clinical evaluation of H-FABP in comparison with myoglobin and creatine kinase isoenzyme MB O'Donoghue et al., Circulation, 114; 550-557 (2006) Prognostic Utility of Heart-Type Fatty Acid Binding Protein in patients with acute coronary syndrome Ruzgar et al., Heart Vessels, 21; 209-314 (2006) The use of human heart-type fatty acid-binding protein as an early diagnostic marker of myocardial necrosis in patients with acute coronary syndrome, and its comparison with troponin-T and its creatine kinase-myocardial band

  Thus, there is clearly a need for diagnostic and prognostic means and methods that allow a reliable and rapid diagnosis of MI in subjects who exhibit symptoms of acute coronary syndrome and have cardiac troponin levels near the detection limit. The means and methods allow for the diagnosis of the subject and the identification of subjects who can perform cardiac intervention, which is an appropriate treatment for the subject, and overcome the disadvantages of the current technology as described above. Will be avoided.

  Therefore, the technical problem underlying the present invention should be regarded as providing means and methods that meet the above-mentioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

Accordingly, the present invention relates to a method for diagnosing myocardial infarction in a subject suffering from acute coronary syndrome and having a myocardial troponin level lower than a level that is detectable but is considered an indicator of myocardial infarction. This method has the following steps:
(A) determining the amount of heart-type fatty acid binding protein (H-FABP) in the sample of the subject;
(B) comparing the amount of H-FABP determined in step (a) with at least one reference amount; and (c) based on the information obtained in steps (a) and (b) Including the step of diagnosing.

Receiver operating characteristic (ROC) curve for H-FABP. By calculating diagnostic sensitivity vs. (1-specificity) for a given diagnostic parameter (H-FABP) based on clinical outcome (non-myocardial infarction (MI) converter vs. myocardial infarction (MI) converter) ROC curve analysis was performed to determine the diagnostic accuracy. Incorporated into this ROC curve are data obtained from patients with stable coronary heart disease and patients with ACS (see Examples). The cut-off score for clinical outcome MI is 4950 pg / ml H-FABP. (ROC-AUC: area under the receiver operating characteristic curve, C.O. cutoff). Receiver operating characteristic (ROC) curve for myoglobin. ROC curve by calculating diagnostic sensitivity vs. (1-specificity) for a given diagnostic parameter (myoglobin) based on clinical outcome (non-myocardial infarction (MI) converter vs. myocardial infarction (MI) converter) Analysis was performed to determine diagnostic accuracy. Incorporated into this ROC curve are data obtained from patients with stable coronary heart disease and patients with ACS (see Examples). The cutoff score for clinical outcome MI is 61 ng / ml myoglobin. (ROC-AUC: area under the receiver operating characteristic curve, C.O. cutoff).

  In one embodiment of the above-described method according to the invention, additionally, an additional step (aa) determines the amount of myoglobin in the subject's sample and an additional step (bb) at least one reference amount of myoglobin. Compare with Thus, in step (c ′), the determined amount of myoglobin and H-FABP is compared with the amount of myoglobin and at least one reference amount of myoglobin, and the amount of H-FABP and at least one of H-FABP A myocardial infarction is diagnosed based on the comparison with the reference amount. Preferably, H-FABP is measured before measuring myoglobin. However, it is also conceivable to take measurements in any order, ie simultaneously or first in H-FABP and then in myoglobin order or first in myoglobin and then in H-FABP order.

  The method according to the present invention is preferably an in vitro method. In addition, it may include steps that are in addition to those explicitly stated above. For example, further steps may relate to sample pretreatment or evaluation of results obtained by the method. It is also possible to use the method according to the invention for diagnostic monitoring, confirmation and subclassification. The method can be performed manually or can be assisted by automation. Preferably, steps (a), (b) and / or (c) are carried out by automation, for example by a robot or sensor device suitable for measurement in step (a) or by a computer in step (b) Can be supported in whole or in part by the comparisons made.

  The term “diagnosing myocardial infarction” refers to a subject as defined in the present invention (thus a subject suffering from ACS and having a myocardial troponin level that is lower than a level that is detectable but considered an indicator of MI) ) Means assessing whether myocardial infarction has recently developed and, therefore, whether the underlying cause of ACS is myocardial infarction or unstable angina. The term “myocardial infarction” is known to those skilled in the art. The term refers to irreversible necrosis of the myocardium that occurs as a result of prolonged ischemia. As will be appreciated by those skilled in the art, the diagnosis is usually not intended to be correct for 100% of the subjects to be analyzed. However, this term requires that the diagnosis be valid for a statistically significant portion of the subject being examined. Those skilled in the art can use various well-known statistical evaluation means, for example, determination of confidence intervals, determination of p-values, Student's t test, Mann-Whitney test, etc. It is possible to determine whether a part is statistically significant. Detailed content can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the diagnosis would be correct for at least 60%, at least 70%, at least 80%, or at least 90% of subjects in a given cohort as a possibility envisaged by the present invention.

  As used herein, the term “subject” means an animal, preferably a mammal, more preferably a human.

  The subject preferably exhibits symptoms of ACS. The term “acute coronary syndrome” (ACS) is understood by those skilled in the art. The term refers to a group of clinical symptoms caused by acute myocardial ischemia. Ischemia itself results from the destruction of coronary atherosclerotic plaque. The symptoms of ACS are preferably chest pain over 20 minutes, shortness of breath, nausea, vomiting, and / or sweating. Furthermore, chest pain is often known to radiate radially to the left arm and left mandibular corner. In general, these clinical symptoms, particularly chest pain, occur suddenly and may appear at rest or after very little effort. Further, in the context of the present invention, the term “acute coronary syndrome” may also mean that there is a suspicion of ACS, presumed ACS, or a possibility of ACS. This is because these expressions are frequently used for patients who show symptoms consistent with ACS but whose diagnosis is not finally finalized (see Morrow, supra). ACS patients may develop unstable angina (UAP) or such individuals may have myocardial infarction (MI). The MI may be ST-elevated MI (STEMI) or non-ST-elevated MI (NSTEMI). MI is classified as belonging to coronary heart disease CHD, preceded by other events classified as belonging to CHD, such as unstable angina UAP. Chest pain alleviated by sublingual administration of nitroglycerin is a symptom of UAP. UAP results from partial occlusion of the coronary vessels that causes hypoxemia and myocardial ischemia. If the obstruction is too severe or total, irreversible myocardial necrosis (which is the pathological condition underlying myocardial infarction) results. In general, if the electrocardiogram (ECG) shows an increase in the ST portion, STEMI is diagnosed by an electrocardiogram. By determining myocardial troponin levels at least 6 hours after the onset of ACS symptoms, it is possible to distinguish between UAP and NSTEMI. If troponin levels are elevated (indicative of myocardial injury), it is estimated to be NSTEMI. MI may develop without clear symptoms. That is, the subject shows no discomfort and the MI is not preceded by stable angina or unstable angina. When MI occurs, left ventricular dysfunction (LVD) can subsequently occur.

  It is particularly envisaged that the subject suffers from coronary heart disease (often referred to as coronary artery disease) prior to ACS and is detectable at the time of the onset of ACS symptoms (and thus even before onset of symptoms). Assume that you already have a troponin level. Specifically, the subject shall have a cardiac troponin level that is detectable at the onset of ACS symptoms but lower than a level that is considered an indicator of MI. For the subject, the method according to the invention will be particularly advantageous. This is because, in the case of ACS, it is unclear whether an elevated level of cardiac troponin is an indicator of an acute event (ongoing necrosis) or is due to an already existing coronary heart disease.

  The term “cardiac troponin” refers to all troponin isoforms expressed in cardiac cells, preferably subendocardial cells. Such isoforms are well characterized in the art as described, for example, in Anderson 1995, Circulation Research, vol. 76, no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493. It is attached. Preferably, cardiac troponin means troponin T and / or troponin I, most preferably troponin T. Of course, the isoforms of troponin can be measured with the method according to the invention together, ie simultaneously or sequentially, or individually, ie without any other isoforms being quantified. The amino acid sequences of human troponin T and human troponin I are disclosed in Anderson, supra and Ferrieres 1998, Clinical Chemistry, 44: 487-493.

  The term “cardiac troponin” also encompasses the specific troponin variants described above, ie preferably troponin T (TnT) or troponin I (TnI) variants. Such variants have at least the same essential biological and immunological properties as the specific cardiac troponin. Specifically, if they are detectable by the same specific assay referred to herein, for example, an ELISA assay using a polyclonal or monoclonal antibody that specifically recognizes the cardiac troponin. , They share the same essential biological and immunological properties. Furthermore, it will be appreciated that variants referred to in accordance with the present invention have different amino acid sequences due to at least one amino acid substitution, deletion, and / or addition. In this case, the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97% with the amino acid sequence of the specific troponin, 98% or 99% identical. The variant can be an allelic variant, or any other species-specific homolog, paralog, or ortholog. In addition, variants referred to herein include specific cardiac troponins or fragments of variants of the types described above, which fragments are essential immunological properties and organisms referred to above. It must have scientific properties. Such a fragment can be, for example, a degradation product of troponin. Furthermore, different mutants may be mentioned due to post-translational modifications such as phosphorylation and myristylation.

  As used herein, the term “cardiac troponin level” means the concentration of cardiac troponin, preferably the concentration of TnT. Preferably, the term refers to the concentration of cardiac troponin in a subject's plasma sample or serum sample. The term “detectable cardiac troponin level” means any cardiac troponin level that is different from zero and detectable by means and methods known in the art, for example, by a commercially available cardiac troponin assay. Preferably, “detectable cardiac troponin level” means a concentration above the minimum detection limit of the assay used to determine troponin levels. Preferably, a detectable troponin level means any concentration of 0.001 ng / ml or more, 0.005 ng / ml or more, 0.0075 ng / ml or more, more preferably 0.01 ng / ml or more or 0.002 ng / ml or more. Most preferably, detectable myocardial troponin levels mean any concentration greater than or equal to 0.002 ng / ml. Of course, a detectable level of cardiac troponin (eg, greater than 0.002 ng / ml) is considered to be an increase in the level of cardiac troponin because such a level is not normally detected in healthy individuals. Furthermore, increased levels of cardiac troponin are indicative of necrosis.

  The term “troponin level regarded as an index of myocardial infarction” means a generally accepted troponin concentration that is an index of myocardial infarction. Preferably, a level considered as an index of myocardial infarction means a concentration that exceeds the 99th percentile concentration (cutoff score) of a suitable reference population. This level is based on a recommendation developed by the Joint Committee of The European Society of Cardiology and The American College of Cardiology to avoid false positive results (The Joint European Society of Cardiology / American College of Cardiology Committee. Myocardial infarction redefined--a consensus document of the joint European Society of Cardiology / American College of Cardiology Committee for the Redefinition of Myocardial Infarction. J Am Coll Cardiol 2000; 36: 959-969). The person skilled in the art knows how to select a suitable reference population and how to determine the 99th percentile concentration. Of course, this concentration may vary depending on the assay used to determine myocardial troponin concentration and depending on the reference population selected. Preferred myocardial troponin levels considered as indicators of MI in the context of the present invention are 0.05 ng / ml, 0.075 ng / ml, 0.099 ng / ml, 0.1 ng / ml, 0.2 ng / ml, and 0.3 ng / ml However, it is not limited to these. The most preferred myocardial troponin level considered as an indicator of MI in the context of the present invention is 0.1 ng / ml.

  In a preferred embodiment of the method according to the invention, the troponin level of a subject suffering from acute coronary syndrome and having a myocardial troponin level lower than that which is detectable but is considered an indicator of myocardial infarction, specifically Troponin T levels (as defined in this application) are in the range of 0.002 to 0.1 ng / ml (greater than 0.002 ng / ml but less than 0.1 ng / ml).

  Cardiac fatty acid binding protein, also referred to herein as H-FABP or cardiac fatty acid binding protein, is a small molecule cytosolic protein that functions as a major transporter of long chain fatty acids in cardiomyocytes. It is generally believed that H-FABP is present in the myocardium and is rapidly released into the bloodstream in response to myocardial injury. Some studies, such as Okamoto et al., Clin Chem Lab Med 38 (3): 231-8 (2000) Human heart-type cytoplasmic fatty acid-binding protein (H-FABP) for the diagnosis of acute myocardial infarction. Clinical evaluation of H-FABP in comparison with myoglobin and creatine kinase isoenzyme MB; O'Donoghue et al., Circulation, 114; 550-557 (2006) Prognostic Utility of Heart-Type Fatty Acid Binding Protein in patients with acute coronary syndrome; Or Ruzgar et al., Heart Vessels, 21; 209-314 (2006) The use of human heart-type fatty acid-binding protein as an early diagnostic marker of myocardial necrosis in patients with acute coronary syndrome, and its comparison with troponin T And its creatine kinase-myocardial band indicates that H-FABP is an early biochemical marker of myocardial infarction. H-FABP is well known in the art. In addition, assays for determining the amount of H-FABP are also well known.

  Myoglobin is a cytoplasmic heme protein consisting of a single polypeptide chain of 154 amino acids and is expressed almost exclusively in cardiomyocytes and aerobic skeletal muscle fibers. Like hemoglobin, myoglobin binds reversibly to oxygen and can therefore facilitate oxygen transport from erythrocytes to mitochondria during periods of increased metabolic activity, or function as an oxygen reservoir during hypoxia or anoxia . Ordway G. and Garry D. J., Myoglobin: an essential hemoprotein in striated muscle. 2004. Journal of Experimental Biology 207, 3441-3446 (2004). Myoglobin is well known in the art. In addition, assays for determining the amount of myoglobin are also well known.

  Myoglobin and H-FABP as used herein also encompass variants of myoglobin polypeptide and H-FABP polypeptide, respectively. Such variants have at least the same essential biological and immunological properties as the specific H-FABP polypeptide and the specific myoglobin polypeptide, respectively. Specifically, they use the same specific assay referred to herein, for example, using polyclonal or monoclonal antibodies that specifically recognize the H-FABP polypeptide and the myoglobin polypeptide, respectively. They share the same essential biological and immunological properties if detectable by the existing ELISA assays. Furthermore, it will be appreciated that variants referred to in accordance with the present invention have different amino acid sequences due to at least one amino acid substitution, deletion, and / or addition. In this case, the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90% with the amino sequence of the specific H-FABP polypeptide and specific myoglobin polypeptide, respectively. %, 92%, 95%, 97%, 98%, or 99% are identical. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity should be determined by comparing two sequences that are optimally aligned over the comparison window. In this case, fragments of the amino acid sequence within the comparison window may contain additions or deletions (eg, gaps or overhangs) compared to a reference sequence (which does not include additions or deletions) in optimal alignment. Determine the number of positions where the same amino acid residue is present in both sequences, determine the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to sequence Calculate the percent by finding the percent identity. The optimal alignment of the sequences for comparison is based on the homology of Needleman and Wunsch J. Mol. Biol. 48: 443 (1970) by the local homology algorithm of Smith and Waterman Add. APL. Math. 2: 482 (1981). By using the similarity search method of Pearson and Lipman Proc. Natl. Acad Sci. (USA) 85: 2444 (1988), these algorithms can be executed by computer (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably utilized to determine their optimal alignment and thus the degree of identity. Preferably, default values of 5.00 for gap weight and 0.30 for gap length weight are used. The variants referred to above can be allelic variants, or any other species-specific homolog, paralog, or ortholog. In addition, variants referred to herein include specific H-FABP polypeptides and specific myoglobin polypeptides or fragments of variants of the types described above, respectively, as referred to above. Must have essential immunological and biological properties. Such fragments can be, for example, degradation products of H-FABP polypeptide and myoglobin polypeptide, respectively. Furthermore, different mutants may be mentioned due to post-translational modifications such as phosphorylation and myristylation.

  The term “sample” refers to a sample of body fluid, a sample of isolated cells, or a sample derived from a tissue or organ. The body fluid sample can be obtained by well-known techniques and preferably includes a blood, plasma, serum or urine sample, more preferably a blood, plasma or serum sample. Tissue or organ samples can be obtained from any tissue or organ, for example, by biopsy. The separated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. Preferably, the cell sample, tissue sample, or organ sample is obtained from a cell, tissue, or organ that expresses or produces a peptide referred to herein. Preferably, the term “sample” means a plasma or serum sample, more preferably a serum sample.

  Samples are obtained at appropriate time points known to those skilled in the art. Preferably, the sample is obtained from a subject according to the invention immediately after onset of symptoms of acute coronary syndrome, preferably 2 hours or less (and thus within 2 hours), more preferably 4 hours or less, more preferably 6 hours or less. Is done. The method according to the present invention is particularly advantageous when a sample is obtained immediately after the onset of symptoms of ACS. In such cases, it is unclear whether the detectable elevated myocardial troponin level is due to an acute event or due to an already existing coronary heart disease.

  Determining the amount of H-FABP, preferably human H-FABP, or myoglobin, preferably human myoglobin, or any other peptide or polypeptide or protein referred to herein, prefers the amount or concentration. Means semi-quantitative or quantitative measurement. The terms polypeptide and protein are used interchangeably throughout this application. Measurement can be performed directly or indirectly. Direct measurement refers to measuring the amount or concentration of a peptide or polypeptide based on the signal obtained from the peptide itself or the polypeptide itself and the signal intensity that directly correlates with the number of molecules of the peptide present in the sample. means. Such a signal (sometimes referred to herein as an intensity signal) can be obtained, for example, by measuring the intensity value of a specific physical or chemical property of a peptide or polypeptide. Indirect measurements can be obtained from a secondary component (ie, a component that is not the peptide itself or the polypeptide itself) or a biological readout system, eg, a measurable cellular response, a ligand, a label, or an enzymatic reaction product. Measurement of the signal can be mentioned.

According to the present invention, determination of the amount of peptide or polypeptide can be achieved by all known means for determining the amount of peptide in a sample. Such means include immunoassay devices and methods that may utilize the labeled molecules in various sandwich assay formats, competitive assay formats, or other assay formats. The assay will generate a signal indicative of the presence or absence of the peptide or polypeptide. Furthermore, the signal intensity can preferably be directly or indirectly correlated (eg, inversely proportional) to the amount of polypeptide present in the sample. Further suitable methods include measuring physical or chemical properties specific for the peptide or polypeptide, such as its exact molecular mass or NMR spectrum. The method preferably includes a biosensor, an optical device coupled to an immunoassay, a biochip, an analytical device, such as a mass spectrometer, an NMR analyzer, or a chromatography device. In addition, methods include microplate ELISA-based methods, fully automated or robotized immunoassays (eg, can be performed using Elecsys ^™ analyzers), CBA (enzymatic cobalt binding assays, eg, Roche-Hitachi ^™ analyzers). And latex agglutination assays (for example, can be performed using a Roche-Hitachi ^™ analyzer).

  Preferably, the determination of the amount of peptide or polypeptide comprises: (a) contacting a cell whose intensity can elicit a cellular response that is indicative of the amount of peptide or polypeptide, with the peptide or polypeptide for an appropriate amount of time And (b) measuring a cellular response. In order to measure the cellular response, preferably a sample or treated sample is added to the cell culture to measure the internal or external cellular response. A cellular response can include measurable expression of a reporter gene or secretion of a substance such as a peptide, polypeptide, or small molecule. Expression or substance shall generate an intensity signal that correlates with the amount of peptide or polypeptide.

  Also preferably, determining the amount of peptide or polypeptide comprises measuring a specific intensity signal obtainable from the peptide or polypeptide in the sample. As described above, such signals can be observed signal intensities in peptide / polypeptide specific m / z variables observed in the mass spectrum or NMR spectra specific for the peptide or polypeptide.

Determination of the amount of peptide or polypeptide preferably comprises (a) contacting the peptide with a specific ligand, (b) (optionally) removing unbound ligand, and (c) the amount of bound ligand. Including the step of measuring. The bound ligand will generate an intensity signal. The bonds according to the present invention include both covalent and non-covalent bonds. A ligand according to the present invention can be any compound that binds to a peptide or polypeptide described herein, eg, a peptide, polypeptide, nucleic acid, or small molecule. Preferred ligands include antibodies, nucleic acids, peptides or polypeptides, such as receptors or binding partners for peptides or polypeptides and fragments thereof comprising a binding domain for peptides, and aptamers such as nucleic acid aptamers or peptide aptamers. . Methods for preparing such ligands are well known in the art. For example, suitable antibody or aptamer identification and production methods are also provided by the suppliers. Those skilled in the art are familiar with methods for developing derivatives of such ligands with higher affinity or specificity. For example, random mutations can be introduced into nucleic acids, peptides, or polypeptides. These derivatives can then be tested for binding by screening procedures known in the art, such as phage display. The antibodies referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, eg, Fv, Fab, and F (ab) ₂ fragments capable of binding to an antigen or hapten. The invention also encompasses single chain antibodies and humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody exhibiting the desired antigen specificity and the sequence of a human acceptor antibody are combined. The donor sequence will usually include at least the antigen-binding amino acid residues of the donor, but may also include other structurally and / or functionally compatible amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. Preferably, the ligand or agent specifically binds to the peptide or polypeptide. Specific binding according to the present invention is one in which the ligand or agent substantially binds ("cross-reacts with") other peptides, polypeptides, or substances present in the sample to be analyzed. It means not to be. Preferably, the specifically bound peptide or polypeptide binds with an affinity that is at least 3 times, more preferably at least 10 times, even more preferably at least 50 times the affinity of any other related peptide or polypeptide. It must be. Non-specific binding can be tolerated if it is still distinguishable and clearly measurable, for example, by size on a Western blot or by a relatively high abundance in the sample. Binding of the ligand can be measured by any method known in the art. Preferably, the method is semi-quantitative or quantitative. A preferred method is described below.

  First, ligand binding can be measured directly, for example, by NMR or surface plasmon resonance.

  Second, if the ligand also functions as a substrate for the enzymatic activity of the peptide or polypeptide of interest, it is possible to measure the enzymatic reaction product (eg, the amount of cleaved substrate on a Western blot). It is possible to measure the amount of protease by measuring by, for example). Alternatively, if the ligand can exhibit enzymatic properties per se, the “ligand / peptide or polypeptide” complex or ligand bound by the peptide or polypeptide, respectively, can be contacted with a suitable substrate to obtain strength. Detection can be performed by generating a signal. In measuring enzyme reaction products, preferably the amount of substrate is saturated. It is also possible to label the substrate with a detectable label before the reaction. Preferably, the sample is contacted with the substrate for an appropriate time. By appropriate time is meant the time required to produce a detectable (preferably measurable) amount of product. Instead of measuring the amount of product, it is also possible to measure the time required for a given (eg detectable) amount of product to appear.

Third, it is possible to detect and measure the ligand by binding the ligand covalently or non-covalently to the label. Labeling can be performed by direct or indirect methods. Direct labeling involves coupling the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves binding a secondary ligand (covalently or non-covalently) to a primary ligand. The secondary ligand must bind specifically to the primary ligand. The secondary ligand can be bound to a suitable label and / or can be the target (receptor) of a tertiary ligand that binds to the secondary ligand. Secondary, tertiary, or higher order ligands are often used to increase the signal. Suitable secondary and higher order ligands may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). It is also possible to “tag” a ligand or substrate with one or more tags, as is known in the art. In that case, such tags may be targets for higher order ligands. Suitable tags include biotin, digoxigenin, His tag, glutathione-S-transferase, FLAG, GFP, myc tag, influenza A virus hemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably present at the N-terminus and / or C-terminus. A suitable label is any label detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzyme activity labels, radioactive labels, magnetic labels (including “e.g. magnetic beads”, paramagnetic labels and superparamagnetic labels), And fluorescent labels. Examples of the enzyme activity label include horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and derivatives thereof. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3′-5,5′-tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro- 3-Indolyl-phosphate (available as a ready-made stock solution from Roche Diagnostics), CDP-Star ^™ (Amersham Biosciences), ECF ^™ (Amersham Biosciences). Using a suitable enzyme-substrate combination produces a colored reaction product (fluorescence or chemiluminescence) that can be measured by methods known in the art (eg, using a light-sensitive film or a suitable camera system). It is possible. For the determination of enzyme reactions, the criteria described above apply in the same way. Typical fluorescent labels include fluorescent proteins (eg, GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (eg, Alexa 568). Additional fluorescent labels are available from, for example, Molecular Probes (Oregon). Furthermore, the use of quantum dots as fluorescent labels is also conceivable. Typical radioactive labels include ³⁵ S, ¹²⁵ I, ³² P, ³³ P, and the like. The radioactive label can be detected by any known appropriate method, for example, a photosensitive film or a phosphor imager. Other suitable measurement methods according to the present invention include precipitation (specifically immunoprecipitation), electrochemiluminescence (electrolytic generation chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay). , Sandwich enzyme immunoassay, electrochemiluminescence sandwich immunoassay (ECLIA), dissociation enhanced lanthanide fluorescence immunoassay (DELFIA), scintillation proximity assay (SPA), turbidimetric method, ratiometric method, latex enhanced turbidimetric method or latex enhanced ratio Examples include sputum or solid phase immunity tests. Additional methods known in the art, such as gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and mass spectrometry, alone or labeled or as described above It can be used in combination with other detection methods.

  The amount of peptide or polypeptide is also preferably as follows: (a) contacting a solid support comprising a ligand for the peptide or polypeptide specified above with a sample comprising the peptide or polypeptide. (B) can be determined by measuring the amount of peptide or polypeptide bound to the support. The ligand (preferably selected from the group consisting of nucleic acids, peptides, polypeptides, antibodies, and aptamers) is preferably present on the solid support in solid phase form. Materials for producing solid supports are well known in the art, and in particular, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, nitro Cellulose strips, membranes, sheets, duracyte, reaction tray wells and walls, plastic tubes and the like. The ligand or agent can be bound to a wide variety of carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the present invention. Suitable methods for immobilizing / immobilizing the ligand are well known and include, but are not limited to, ionic interactions, hydrophobic interactions, covalent interactions and the like. It is also conceivable to use a “suspension array” as an array according to the present invention (Nolan 2002, Trends Biotechnol. 20 (1): 9-12). In such a suspension array, carriers such as microbeads and microspheres exist in a suspended state. The array is composed of various microbeads or microspheres that may be labeled carrying various ligands. Methods for producing such arrays, eg, arrays based on solid phase chemistry and photoactive protecting groups, are widely known (US Pat. No. 5,744,305).

  Preferably, the amount of H-FABP and the amount of myoglobin (when measuring myoglobin) are determined in a blood sample obtained from a subject as defined in the present invention. Preferably such measurement is performed by ELISA. Such measurements by ELISA can be performed, for example, by using the HBT ELISA Test Kit (HyCult Biotechnology, Uden, The Netherlands) for human heart-type fatty acid binding protein to determine the amount of H-FABP, respectively, and myoglobin This can be done by using the Tina-Quant® Myoglobin Test System (Roche Diagnostics) to determine the amount of urine.

  As used herein, the term “amount” refers to absolute amount (eg, absolute amount of H-FABP or myoglobin), relative amount or relative concentration (eg, relative amount or relative concentration of H-FABP or myoglobin), Furthermore, any value or parameter that correlates with them is included. Such values or parameters include intensity signal values derived from any specific physical or chemical property obtained from the peptide by direct measurement, eg, intensity values of mass spectra or NMR spectra. In addition, all values or parameters obtained by indirect measurements specified elsewhere herein, for example, expression levels determined from biological reading systems that respond to peptides, or specifically bound Intensity signals obtained from different ligands are included. Of course, any values that correlate with the quantities or parameters described above can also be obtained by standard mathematical operations.

  As used herein, the term “compare” refers to the amount of a suitable reference source specified elsewhere in the specification for the amount of peptide, polypeptide, or protein contained in a sample to be analyzed. Comparing with Of course, comparison as used herein means comparison of corresponding parameters or values. For example, the absolute amount is compared to the absolute reference amount, while the concentration is compared to the reference concentration, or the intensity signal obtained from the test sample is compared to the same type of intensity signal of the reference sample. The comparison referenced in step (b) of the method according to the invention can be performed manually or can be supported by a computer. In a computer assisted comparison, the determined amount of value can be compared by a computer program with a value corresponding to a suitable reference stored in the database. The computer program can further evaluate the result of the comparison. That is, it is possible to automatically provide a desired evaluation in a suitable output format. Based on a comparison between the amount determined in step (a) and a suitable reference amount, MI can be diagnosed in the subject. Of course, the amount of H-FABP determined in step (a) of the method according to the present invention is compared with the reference amount of H-FABP specified elsewhere in this application in step (b). And the amount of myoglobin is compared to the reference amount of myoglobin.

  Thus, as used herein, the term “reference amount” refers to (in a subject suffering from acute coronary syndrome and having a myocardial troponin level lower than a level that is detectable but is considered an indicator of myocardial infarction. Thus, in an subject as defined in the present invention, it means either an amount that allows the determination of the recent onset of MI or an amount that allows the exclusion of the recent onset of MI. A recent onset in this context preferably means that MI has developed within 6 hours, more preferably within 4 hours, most preferably within 2 hours prior to obtaining a sample from the subject. Preferably, the reference amount for determining the MI is preferably found to have suffered from MI within 6 hours, more preferably within 4 hours, most preferably within 2 hours prior to obtaining the sample. It can be derived from the subject defined in A reference amount for excluding a recent onset of MI can be derived from a subject as defined in the present invention known to have never suffered from MI. In addition, the reference amount to rule out recent onset of MI was derived from subjects with stable coronary heart disease who had low but detectable myocardial troponin levels (as specified above) and who did not suffer from MI. Is possible. The amount of H-FABP and myoglobin (when measuring myoglobin) in a subject as defined in the present invention greater than the reference amount for determining the onset of MI is an indicator of the recent onset of MI in the subject. (Thus, it shall be an indicator that the cause of ACS is MI). The amount of H-FABP and the amount of myoglobin (when measuring myoglobin) in a subject as defined in the present invention that is less than the reference amount to rule out the onset of MI is that the MI infarction has not recently developed, and therefore It shall be an indicator that the subject is preferably suffering from UAP (thus, an indicator that the cause of ACS is UAP). In the context of the present invention, it will be appreciated that the amount of H-FABP is the reference amount described above (the reference amount for determining the recent onset of MI and the reference amount for excluding the recent onset of MI). A subject as defined in the present invention in between will need to be diagnosed again. Preferably, this is also the case where the amounts of both myoglobin and H-FABP are determined and in rare cases where these amounts do not correspond, eg, one amount is greater (or less) than the respective reference amount but the other The amount will also occur in rare cases where the amount is not (or not less) than the respective reference amount. A new diagnosis is preferably made by determining the amount of the marker (s) and on a new (and therefore more recently acquired) sample of the subject. A new sample is preferably obtainable from the subject 1 hour, 2 hours and 3 hours after obtaining the first sample. In addition, it is possible to measure myoglobin in the new sample. After obtaining the sample, it is possible to determine the amount of H-FABP and the amount of myoglobin in the new sample. The result (s) thus obtained can then be compared with a reference amount (described elsewhere in this application). Preferably, the amount of cardiac troponin is also determined in the sample and used for diagnosis. Preferably, an amount of cardiac troponin T greater than 0.1 ng / ml at 6 hours after showing ACS symptoms is an indicator of MI.

  The person skilled in the art knows how to determine the reference amount. It will be appreciated that the reference amount can be selected based on the desired sensitivity or specificity of the diagnosis. Thus, one skilled in the art can select the reference amount based on the desired sensitivity and specificity. Means for determining a suitable reference amount are known to those of skill in the art and can be determined, for example, from a receiver operator curve (ROC) based on clinical trials.

  The reference amount of H-FABP to determine the recent onset of MI in a subject as defined in the present invention, which defines a threshold amount of H-FABP, is 4950 pg / ml, 5550 pg / ml, or 6000 pg / ml, or More preferably, it is 5700 pg / ml.

  An amount of H-FABP that is greater than the reference amount of H-FABP for determining the recent onset of MI is more preferably an indicator of the recent onset of MI (specifically NSTEMI).

  In addition to H-FABP, when determining the amount of myoglobin in a sample of a subject as defined in the present invention and comparing it to a reference amount, the MI of a subject as defined in the present invention is defined as a threshold amount of myoglobin. The reference amount of myoglobin for determining recent onset is 64 ng / ml or 69 ng / ml, more preferably 77 ng / ml.

  An amount of myoglobin greater than the reference amount of myoglobin for determining the recent onset of MI is more preferably the amount of H-FABP in the sample of the subject is also used to determine the recent onset of MI. If it is higher than the reference amount of H-FABP, it is an indicator of recent onset of MI (specifically NSTEMI).

  The reference amount of H-FABP, which defines the threshold amount of H-FABP and excludes recent onset of MI in subjects according to the present invention, is 2100 pg / ml or 2300 pg / ml, or more preferably 2500 pg / ml is there.

  An amount of H-FABP that is smaller than the reference amount of H-FABP for excluding recent onset is more preferably an indicator that no myocardial infarction has developed in the subject defined in the present invention. Preferably, as a result, the onset of MI can be ruled out, and for example, it can be presumed to be UAP.

  In addition to H-FABP, when determining the amount of myoglobin in a sample of a subject as defined in the present invention and comparing it to a reference amount, a recent onset of MI in a subject according to the present invention that defines a threshold amount of myoglobin The reference amount of myoglobin to exclude is 28 ng / ml or 61 ng / ml, or more preferably 55 ng / ml.

  An amount of myoglobin that is less than the reference amount of myoglobin to rule out the recent onset of MI, more preferably, the amount of H-FABP in the sample of the subject also excludes the recent onset of MI If it is less than the reference amount of H-FABP, it is an indicator that myocardial infarction has not developed in the subject defined in the present invention. Preferably, as a result, the onset of MI can be ruled out, and for example, it can be presumed to be UAP.

  The term “at least one reference amount” excludes one or more reference amounts (eg, two reference amounts), eg, a reference amount to establish a recent onset of MI and a recent onset of MI Refers to the reference amount.

  Advantageously, according to the research underlying the present invention, the subject defined in the present invention (thus lower than a level suffering from acute coronary syndrome and detectable but considered to be an indicator of MI) It has been found that determination of the amount of H-FABP in a subject with troponin levels and comparison of the determined amount with at least one reference amount is necessary for the diagnosis of MI in the subject. Since it has been found that H-FABP is required to assess the onset of MI when cardiac troponin T levels are low but immediately detectable after onset of ACS or putative ACS symptoms, More reliable than things. In the study underlying the present invention, H-FABP levels and TnT levels were determined in patients with ACS symptoms (within the first 2 hours after the onset of symptoms). TnT levels were determined by using a sensitive troponin T assay with a detection limit of 0.002 ng / ml. Control experiments were performed to determine the amount of TnT and H-FABP in patients with stable coronary disease (ie, patients without overt acute events). From these experiments, it was found that TnT can be detected even in subjects with stable coronary disease. In addition, receiver operating characteristic (ROC) curve analysis, including data from the above studies, showed that H-FABP is a powerful biochemical marker of myocardial infarction (Figure 1). Specifically, H-FABP levels in subjects as defined in the present invention greater than 5700 pg / ml are indicative of a recent onset of MI (confirmed), while quantities less than 2500 pg / ml Is an indicator that the disease has not recently developed (exclusion). In addition, when determining the amount of myoglobin in a subject's sample and comparing it to a reference amount of myoglobin, the sensitivity and specificity of the diagnosis based on the measurement of H-FABP in the subject's sample as defined in the present invention is , Will be further increased. Specifically, myoglobin levels in subjects as defined in the present invention greater than 77 ng / ml are indicative of a recent onset of MI (determined), while quantities less than 55 ng / ml are associated with a recent MI It is an indicator that it did not develop (exclusion) (Figure 2). Thanks to this aspect of the invention, more reliable diagnosis of patients with ACS or putative ACS with myocardial troponin levels that are low but detectable (lower than the level that is detectable but considered an indicator of MI) Is possible. The findings of the research underlying the present invention are (a) already having low but detectable myocardial troponin levels (and therefore elevated myocardial troponin levels) due to preexisting coronary heart disease and also exhibiting symptoms of ACS Subjects and (b) ACS subjects who have low but detectable myocardial troponin levels at the time of obtaining a sample to determine myocardial troponin levels (eg, because the sample was obtained too early) but recently developed MI It will be particularly advantageous for the diagnosis of In both cases (a) and (b), measurement of H-FABP may be a useful tool for diagnosis, specifically to distinguish between UAP and MI. After diagnosis, the subject can be treated accordingly. If H-FABP is not measured, the diagnosis may be misleading, resulting in the wrong treatment, harmful treatment, and / or delayed treatment of the designated subject. Furthermore, the sensitivity and specificity of the diagnosis can be further increased when measuring myoglobin.

  In a preferred embodiment of the method according to the invention, the method is used in a subject suffering from acute coronary syndrome and having a myocardial troponin level that is detectable but lower than a level considered as an indicator of myocardial infarction (MI). Enables discrimination between myocardial infarction (MI) and unstable angina (UAP).

Thus, the method according to the present invention, in one embodiment, in a subject suffering from acute coronary syndrome and having a myocardial troponin level that is detectable but lower than a level considered as an indicator of myocardial infarction (MI). It is a method for discriminating myocardial infarction from unstable angina. This method has the following steps:
(A) determining the amount of heart-type fatty acid binding protein (H-FABP) in the sample of the subject;
(B) comparing the amount of H-FABP determined in step (a) with at least one reference amount; and (c) based on the information obtained in steps (a) and (b) Discriminating from unstable angina.

  Specifically envisaged, the subject shall have a cardiac troponin level that is low but detectable at the onset of ACS symptoms (and thus lower than a level that is detectable but considered an indicator of MI) , Shall have that level before ACS). Preferably, the detectable level is due to coronary heart disease. As described above, in the case of ACS, it is difficult to determine whether the detectable level is due to an existing coronary heart disease or due to the ACS.

  Thus, in a preferred embodiment of the method according to the present invention, the method has a myocardial troponin level lower than a level that is suffering from acute coronary syndrome and is detectable but is considered an indicator of myocardial infarction (MI). And for the diagnosis of myocardial infarction in a subject who had a low but detectable myocardial troponin level at the onset of ACS symptoms.

Thus, the method according to the present invention, in one embodiment, has an acute coronary syndrome and has a myocardial troponin level that is lower than a level that is detectable but is considered an indicator of myocardial infarction (MI), And a method for diagnosing myocardial infarction in a subject who already had detectable myocardial troponin levels at the time of onset of ACS symptoms. This method has the following steps:
(A) determining the amount of heart-type fatty acid binding protein (H-FABP) in the sample of the subject;
(B) comparing the amount of H-FABP determined in step (a) with at least one reference amount; and (c) based on the information obtained in steps (a) and (b) Including the step of diagnosing.

  In accordance with the above, MI can be confirmed / excluded in subjects suffering from ACS using the present invention.

  Accordingly, in another preferred embodiment of the method according to the present invention, the method comprises a cardiac troponin suffering from acute coronary syndrome and detectable but below a level considered to be indicative of myocardial infarction (MI) For the exclusion of myocardial infarction in subjects who have a level and already had low but detectable myocardial troponin levels at the onset of ACS symptoms (and therefore just before the onset of symptoms). Preferably, MI is excluded if the level of H-FABP is lower than the reference amount for excluding MI. When measuring both H-FABP and myoglobin, MI is preferably excluded when both markers are less than their respective reference amounts to exclude MI.

Thus, the method according to the present invention, in one embodiment, has an acute coronary syndrome and has a myocardial troponin level that is lower than a level that is detectable but is considered an indicator of myocardial infarction (MI), And preferably a method for excluding myocardial infarction in a subject who already had a detectable but not detectable myocardial troponin level at the onset of ACS symptoms. This method has the following steps:
(A) determining the amount of heart-type fatty acid binding protein (H-FABP) in the sample of the subject;
(B) comparing the amount of H-FABP determined in step (a) with a reference amount for excluding myocardial infarction; and (c) a reference amount for determining the amount of H-FABP to exclude myocardial infarction. Excluding myocardial infarction if less.

  In another preferred embodiment of the method according to the invention, the method has a myocardial troponin level lower than a level that is suffering from acute coronary syndrome and is detectable but is considered an indicator of myocardial infarction (MI). And preferably for the determination of myocardial infarction in a subject who already had low but detectable myocardial troponin levels at the onset of ACS symptoms. Preferably, the MI is determined when the level of H-FABP is greater than the reference amount for determining the MI. When measuring both H-FABP and myoglobin, the MI is preferably determined when both markers are greater than their respective reference amounts for determining the MI.

  An explanation of the terms used in relation to the above method can be found elsewhere in this specification. Preferably they also include determining the amount of myoglobin and comparing the amount to a reference amount.

  Of course, according to the method according to the invention described hereinbefore and hereinafter, the amount of H-FABP and preferably further the amount of myoglobin or means for determining them is acute coronary syndrome And can be used to produce a diagnostic composition for diagnosing MI in a subject that is detectable but has a myocardial troponin level lower than that considered to be an indicator of myocardial infarction.

  The present invention further relates to a method for identifying a subject who can undergo cardiac intervention. In this case, the subject is suffering from acute coronary syndrome and has a myocardial troponin level that is detectable but lower than that considered as an indicator of myocardial infarction, the method comprising any of the methods described above Including steps (a) and (b) described in one and optionally performing steps (aa) and (bb), and (c) identifying a subject capable of undergoing cardiac intervention.

  Thanks to the method described above, it is possible to easily perform risk / success stratification before subjecting the patient to cardiac intervention. This risky, time consuming and / or costly therapy can be avoided if it turns out that the patient is unable to perform a cardiac intervention. Thus, besides ensuring that subjects do not suffer from the harmful and serious side effects associated with cardiac intervention, the method according to the present invention would be beneficial to the health system in that it saves resources.

  Of course, in the context of the above-described method according to the present invention, a subject diagnosed as suffering from MI, and thus a subject who has recently developed MI may undergo cardiac intervention.

  As used herein, the term “identifying” means assessing whether a subject is capable of undergoing cardiac intervention. As will be appreciated by those skilled in the art, such an assessment is usually not intended to be correct for all (i.e. 100%) of the subjects to be identified. However, the term requires that a statistically significant fraction of subjects can be identified (eg, a cohort in a cohort study). Those skilled in the art can use various well-known statistical evaluation means, for example, determination of confidence intervals, determination of p-values, Student's t test, Mann-Whitney test, etc. It is possible to determine whether a part is statistically significant. Detailed content can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in the population can be suitably identified by the method according to the invention.

  The term “cardiac intervention” encompasses treatment regimens for MI deemed appropriate by those skilled in the art. The term encompasses surgery, microsurgery, other invasive therapies that affect the cardiovascular system (preferably the heart), as well as interventions with conservative (non-surgical) treatment methods. Conservative methods are known in the art and include non-pharmacological and pharmacological methods. A pharmacological method means administration of a therapeutically effective amount of a pharmaceutical (eg, heparin, acetylsalicylic acid, clopidogrel). Preferably, cardiac intervention as used herein is a treatment regimen that aims to restore the proper oxygen supply of the heart. This is preferably accomplished by restoring blood flow across the blood vessels that maintain the heart, ie the coronary vessels. Such blood vessels can be damaged, for example, by thrombotic plaques or atherosclerotic plaques. Thus, cardiac intervention should preferably include the destruction and / or removal of such plaque and vascular recovery, if necessary. Preferred cardiac interventions according to the present invention include percutaneous coronary angioplasty, percutaneous transluminal coronary balloon angioplasty, laser angioplasty, coronary stenting, bypass grafting, or blood flow recovery, blood vessels Selected from the group consisting of endoluminal techniques aimed at patency, plaque stabilization, and / or reduction of intracoronary thrombus load.

Furthermore, the present invention relates to a method for determining possible treatments for a subject suffering from acute coronary syndrome and having a myocardial troponin level lower than that which is detectable but is considered an indicator of myocardial infarction. This method has the following steps:
(A) determining the amount of H-FABP in the sample of the subject;
(B) comparing the amount of H-FABP determined in step (a) with at least one reference amount; and (c) based on the information obtained in steps (a) and (b) Including initiating an intervention or avoiding a cardiac intervention.

  In one embodiment of the above-described method according to the invention, additionally, the amount of myoglobin in the sample of the subject is determined in step (a) and compared with at least one reference amount of myoglobin in step (b). Thus, in step (c), the determined amount of H-FABP and myoglobin, comparison of the amount of myoglobin with at least one reference amount of myoglobin, and the amount of H-FABP and at least one reference amount of H-FABP Based on the comparison, the initiation of cardiac intervention or avoidance of cardiac intervention is performed.

  Further included in the present invention is a method according to the present invention comprising means for determining the amount of H-FABP in a sample of a subject and means for comparing the amount with at least one reference amount. A kit or device for carrying out the method. Preferably, the kit or device comprises means for determining the amount of myoglobin in the sample of the subject and means for comparing the amount to at least one reference amount.

  The term “kit” as used herein means a group of the above-mentioned means, preferably provided individually or in a single container. The kit may additionally include a means for determining the amount of cardiac troponin. Preferably, the kit may additionally comprise a user manual for interpreting the results of any measurement relating to the diagnosis of MI in a subject as defined in the present invention. Specifically, such a manual may include information regarding what determined amount corresponds to what type of diagnosis. This is outlined in detail elsewhere in this specification. In addition, such user manuals may provide instructions for accurately using the components of the kit to determine the amount of each biomarker.

  As used herein, the term “device” refers to a system of means comprising at least the means described above operatively linked to each other so as to be able to diagnose MI or to identify a subject capable of performing cardiac intervention. means. The device invention may additionally include means for determining the amount of cardiac troponin. Preferred means for determining the amount of myoglobin and H-FABP and means for performing the comparison are disclosed above in connection with the method according to the invention. How the means are operatively linked will depend on the type of means incorporated into the device. For example, when a means for automatically determining the amount of peptide is applied, the data obtained by the automatic actuating means can be processed by, for example, a computer program so as to obtain a desired result. Preferably, in such cases, the means are contained within a single device. Thus, the device may include an analysis unit for measuring the amount of peptide or polypeptide in the applied sample and a computer unit for processing the data obtained to perform the evaluation. As another option, when using means such as a test strip to determine the amount of peptide or polypeptide, the means for comparison includes a control strip or control table that assigns the determined amount to a reference amount. sell. The test strip is preferably bound to a ligand that specifically binds to a peptide or polypeptide referred to herein. The strip or device preferably includes a means for detecting binding of the peptide or the polypeptide to the ligand. Preferred means for detection are disclosed above in connection with embodiments relating to the method according to the invention. In such a case, the means are operatively linked in the sense that the user of the system matches the measurement result of the quantity with its diagnostic or prognostic value based on the instructions and interpretations described in the manual. The means may exist as a separate device in such embodiments, but is preferably packaged together as a kit. The person skilled in the art will understand without difficulty how to connect the means. Preferred devices are those that can be applied without special knowledge of a specialist clinician, for example, test strips or electronic devices that simply need to be filled with a sample. The results may be given as raw data output that requires interpretation by the clinician. However, the output of the device is preferably processed or evaluated raw data that does not require a clinician for interpretation. Further preferred devices are analytical units / devices (eg biosensors, arrays, solid carriers bound to ligands that specifically recognize myoglobin or H-FABP, plasmon surface resonance devices, NMR spectrometers, mass spectrometers, etc.) Or an evaluation unit / device referred to above which is compatible with the method according to the invention.

  The invention also provides the use of H-FABP and preferably in addition to it myoglobin, and / or the amount and preferably in addition to H-FABP, for the manufacture of a diagnostic composition for diagnosing myocardial infarction in a subject. It relates to the use of means for determining the amount of myoglobin and / or to the use of means for comparing the amount of H-FABP and preferably in addition to that the amount of myoglobin with at least one reference amount.

  All references cited in this specification are hereby incorporated by reference with respect to their entire disclosure content, and the disclosure content is expressly stated herein. .

  The following examples merely illustrate the invention. None of these are considered to limit the scope of the present invention.

Example 1: Myoglobin, H-FABP, and troponin T in patients with acute coronary syndrome
Sixty-nine patients with characteristic symptoms of ACS (eg chest pain) were examined. Blood samples were obtained within the first 2 hours after the onset of symptoms. The patient was examined by ECG for diagnosis of ST-elevated MI. In addition, the troponin T concentration was determined using a troponin T assay with a detection limit of 0.01 ng / ml. Additional blood samples were obtained from patients who could not be diagnosed with STEMI or NSTEMI (TnT concentrations greater than 0.01 ng / ml but less than 0.1 ng / ml, and therefore a level indicative of necrosis). Troponin T levels higher than 0.1 ng / ml in samples taken at least 6 hours after symptom onset were considered as an indicator of recently developed MI (MI converter), otherwise diagnosed as UAP (non-MI conversion) Person).

For later analysis, a sensitive TnT assay with a detection limit of 0.002 ng / ml, Tina-Quant® Myoglobin Test System (Roche Diagnostics) and H-FABP ELISA test kit (HBT for human heart-type fatty acid binding protein) ELISA test kits; HyCult Biotechnology, Uden, The Netherlands) were used to determine the concentrations of TnT, myoglobin, and H-FABP in samples from patients who could not be diagnosed with STEMI or NSTEMI (first sample) TnT in it is detectable and is higher than 0.01 ng / ml but less than 0.1 ng / ml). The results are shown in the table below.

Example 2: Myoglobin, H-FABP, and troponin T in patients with stable coronary heart disease
Myoglobin, H-FABP, and sensitive troponin T were measured in blood samples from a total of 234 patients with stable coronary heart disease. The patient was clearly not suffering from an acute coronary event. H-FABP was measured as specified above. Troponin T was measured by a sensitive troponin T test with a detection limit of 0.002 ng / ml. Patients were subjected to detailed cardiological studies including echocardiography and coronary angioplasty. Coronary heart disease was subdivided into 1 branch, 2 branch, or 3 branch lesions. In this case, there should be more than 50% stenosis per vessel. The results are shown in the table below.

Claims (22)

A method for diagnosing myocardial infarction in a subject suffering from acute coronary syndrome (ACS) and having a myocardial troponin level lower than a level that is detectable but is considered an indicator of myocardial infarction (MI), comprising: Steps:
(A) determining the amount of heart-type fatty acid binding protein (H-FABP) in the sample of the subject;
(B) comparing the amount of H-FABP determined in step (a) with at least one reference amount; and (c) based on the information obtained in steps (a) and (b) A method as described above, comprising the step of diagnosing.   The method according to claim 1, wherein the sample is obtained within 4 hours after the onset of ACS symptoms.   3. The method of claim 1 or 2, wherein the subject already had a myocardial troponin level at the onset of symptoms of ACS that is detectable but lower than a level considered to be an indicator of myocardial infarction (MI).   4. The amount of H-FABP is compared to a reference amount for excluding a recent onset of MI and / or a reference amount for determining a recent onset of MI. The method described in 1.   The method according to any one of claims 1 to 4, wherein the reference amount for determining the recent onset of MI is 5700 pg / ml.   6. The method according to any one of claims 1 to 5, wherein an amount of H-FABP that is greater than a reference amount for determining a recent onset of MI is indicative of a recent onset of MI.   The method according to any one of claims 1 to 6, wherein the reference amount for excluding the recent onset of MI is 2500 pg / ml.   The method according to any one of claims 1 to 7, wherein an amount of H-FABP less than a reference amount for excluding the recent onset of MI indicates that MI did not develop.   Additionally, the step (aa) of determining the amount of myoglobin is performed and the determined amount of myoglobin is compared with the reference amount of at least one myoglobin in step (bb). 2. The method according to item 1.   The amount of myoglobin and the amount of H-FABP is a reference amount of myoglobin and H-FABP to rule out the recent onset of MI and / or a reference amount of myoglobin to determine the recent onset of MI and / or The method of claim 9, wherein the method is compared to a reference amount of H-FABP.   10. The method of claim 9, wherein the reference amount for determining the recent onset of MI is 77 ng / ml for myoglobin and 5700 pg / ml for H-FABP.   The method according to claim 11 or 12, wherein an amount of myoglobin and an amount of H-FABP that are higher than a reference amount for determining a recent onset of MI are indicative of a recent onset of MI.   11. The method of claim 10, wherein the reference amount for excluding the recent onset of MI is 55 ng / ml for myoglobin and 2500 pg / ml for H-FABP.   14. The method according to claim 10 or 13, wherein a myoglobin amount less than a reference amount for excluding the recent onset of MI indicates that MI did not develop.   15. The method according to any one of claims 1 to 14, wherein the cardiac troponin is cardiac troponin T.   16. The method of any one of claims 1-15, wherein the subject has a troponin level in the range of 0.002 ng / ml to 0.1 ng / ml.   A method of identifying a subject capable of undergoing cardiac intervention, wherein the subject has an acute coronary syndrome and has a myocardial troponin level that is lower than a level that is detectable but is considered an indicator of myocardial infarction. And (c) performing steps (a) and (b) and optionally steps (aa) and (bb) according to any one of claims 1 to 16, and (c) a heart based on the information obtained. The method as described above, comprising the step of identifying a subject who can perform an intervention.   17. A method for determining possible treatments for a subject suffering from acute coronary syndrome and having a myocardial troponin level lower than a level that is detectable but is considered an indicator of myocardial infarction, comprising: Whether to recommend starting cardiac intervention based on the information obtained, and (c) performing steps (a) and (b) and optionally steps (aa) and (cc) as described in any one of the paragraphs Or the method comprising the step of avoiding cardiac intervention.   A kit or device for carrying out the method according to any one of claims 1 to 18, comprising means for determining the amount of H-FABP in a sample of the subject, and optionally the subject's A kit or device as described above, comprising means for determining the amount of myoglobin in the sample, and means for comparing the amount of H-FABP and optionally the amount of myoglobin with at least one reference amount.   20. A kit or device according to claim 19 additionally comprising means for determining the amount of cardiac troponin. A method of excluding myocardial infarction in a subject suffering from acute coronary syndrome and having a myocardial troponin level that is detectable but lower than a level considered as an indicator of myocardial infarction (MI), comprising the following steps:
(A) determining the amount of heart-type fatty acid binding protein (H-FABP) in the sample of the subject;
(B) comparing the amount of H-FABP determined in step (a) with a reference amount for excluding myocardial infarction; and (c) a reference amount for determining the amount of H-FABP to exclude myocardial infarction. Excluding myocardial infarction if less than,
Including the above method.   24. The method of claim 21, wherein the subject already had a myocardial troponin level at the onset of ACS symptoms that was detectable but lower than a level considered to be an indicator of myocardial infarction (MI).

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