Reagent and method for diagnosing arteriosclerotic disease

Abstract

The present invention relates to a reagent for use in the diagnosis, assessment of severity, prediction of onset or aggravation, or assessment of onset risk or aggravation risk of an arteriosclerotic disease. The reagent includes: an antibody to a complex, which is bonded to a neutrophil-derived protein or a corresponding binding molecule thereof; or an antigen-binding fragment of said antibody.

Description

Reagents and methods for diagnosing arteriosclerotic diseases

[0001] This patent application claims priority to Japanese Patent Application No. 2023-040611, the entire contents of which are incorporated herein by reference. The present invention relates to reagents and methods for diagnosing arteriosclerotic diseases.

[0002] Atherosclerosis, also known as atherosclerosis, is a condition in which the arterial lumen narrows and loses elasticity due to the accumulation of cholesterol in the blood beneath the intima of the arterial wall. It commonly occurs in coronary arteries, cerebral blood vessels, carotid arteries, aorta, and popliteal arteries. Cholesterol-deposited plaques on the inner surface of the arterial wall can impair blood flow and potentially lead to life-threatening conditions due to plaque rupture. Serious atherosclerotic diseases include acute coronary syndrome, cerebrovascular disease, and aortic disease. These lifestyle-related diseases are not only major causes of death, but also reduce quality of life (QOL). Atherosclerotic plaque formation is thought to be caused by a combination of factors, such as dyslipidemia, hypertension, and lifestyle changes, including diet, exercise, smoking, and alcohol use.

[0003] Acute coronary syndrome is a collective term for acute myocardial infarction (AMI), unstable angina, and sudden cardiac death due to ischemia caused by coronary artery plaque rupture. In recent years, the widespread use of highly sensitive troponin assays has enabled the detection of minute myocardial damage due to coronary artery ischemia, and troponin is being used to diagnose non-ST elevation myocardial infarction (NSTEMI). AMI is a disease caused by coronary artery plaque rupture, which blocks or subblocks the coronary arteries, resulting in myocardial necrosis due to interruption of blood flow to the myocardium. However, troponin is a marker of myocardial tissue injury and cannot determine the state of plaque instability, which is the cause of AMI. Furthermore, blood levels of troponin are not specific to AMI, as they are also elevated due to myocardial injury caused by various conditions other than AMI.

[0004] Several attempts have been made to diagnose arteriosclerosis using biomarkers. A typical example involves measuring blood cholesterol, which is related to the formation of arteriosclerosis (Patent Documents 1 and 2). However, while blood cholesterol levels are related to the gradual progression of arteriosclerosis, they cannot determine the state of vulnerable plaques. Furthermore, attempts to capture proteins shed from arteriosclerotic plaques into the blood using antibodies obtained from arteriosclerotic tissue itself as an antigen (Patent Document 3), as well as attempts to capture cardiovascular events using markers that increase with systemic acute inflammation (Patent Document 4), have also failed to determine plaque instability.

[0005] In recent years, neutrophils, which are involved in acute inflammation, have been reported to be involved in chronic inflammatory diseases such as diabetes, nonalcoholic steatohepatitis, atherosclerosis, autoimmune diseases, and chronic obstructive pulmonary disease (Non-Patent Document 1). Neutrophils are a type of white blood cell that play a major role in defending the body against bacterial and fungal infections. In humans, they account for approximately 45-75% of all white blood cells in the blood. It has been revealed that neutrophils control infection during acute inflammation not only by phagocytosis of invading pathogens but also by releasing chromatin fibers called neutrophil extracellular traps (NETs) (Non-Patent Document 2). When neutrophils are activated at sites of pathogenic inflammation, they capture pathogens by releasing chromatin fibers containing DNA and histones, and then kill them using myeloperoxidase, lactoferrin, elastase, and other enzymes contained in the granules. This series of cell death-mediated infection control mechanisms is considered to be a special type of cell death known as NETosis. Incidentally, there is a report that cerebral infarction caused by the rupture of atherosclerotic plaque in the carotid artery is associated with NETs (Non-Patent Document 3). There are also reports that ST-segment elevation myocardial infarction caused by coronary artery occlusion due to the progression of atherosclerotic plaque is associated with NETs (Non-Patent Documents 4, 5, 6, and 7). These reports investigated the concentration of myeloperoxidase-DNA complexes released by neutrophils. Non-Patent Document 7 confirmed the relationship between neutrophil elastase levels and the onset of heart failure by blood sampling after the onset of myocardial infarction.

[0006] As such, it has been difficult to evaluate the state of coronary artery plaque using conventional technology. If it becomes possible to measure the state of vulnerable plaque, where the fibrous cap of the plaque is thin and vulnerable to rupture, using blood biomarkers, it is believed that diagnostic accuracy will improve, prognosis for high-risk patients will be improved, and medical resources will be allocated appropriately.

[0007] JP 9-203736, JP 2009-288219, JP 61-130238, JP 2002-48790

[0008] Andrea Herrero-Cervera,et al.,Neutrophils in chronic inflammatory diseases. Cell Mol Immunol. 2022 Feb;19(2):177-191.Volker Brinkmann,et al.,Neutrophil Extracellular Traps Kill Bacteria. Science. 2004 Mar 5;303(5663):1532-5.de Vries JJ,et al.,Association between plaque vulnerability and neutrophil extracellular traps (NETs) levels: The Plaque At RISK study. PLoS One. 2022; 17(6):e0269805.Mangold A,et al.,Correction to: Coronary Neutrophil Extracellular Trap Burden and Deoxyribonuclease Activity in ST-Elevation Acute Coronary Syndrome Are Predictors of ST-Segment Resolution and Infarct Size.Circulation Research. 2015;116:1182-1192.Jing Liu,et al.,Neutrophil extracellular traps and dsDNA predict outcomes among patients with ST-elevation myocardial infarction. Sci Rep. 2019 Aug 12;9(1):11599.Dimitrios A Stakos,et al.,Expression of functional tissue factor by neutrophil extracellular traps in culprit artery of acute myocardial infarction. Eur Heart J. 2015 Jun 7;36(22):1405-14. Julian I Borissoff, et al. , Elevated Levels of Circulating DNA and Chromatin Are Independently Associated with Severe Coronary Atherosclerosis and a Prothrombotic State. Arterioscler Thromb Vasc Biol. 2013 Aug; 33(8): 2032-2040. Shibata, M.: Changes in blood granulocyte elastase levels and acute prognosis in the microcytic phase of myocardial infarction. Non-emergency medicine start 1994; 5: 161-9;

[0009] An object of the present invention is to provide a reagent and a method for diagnosing arteriosclerotic diseases, assessing their severity, predicting their onset or progression, or assessing the risk of their onset or progression. Another object of the present invention is to provide a reagent and a method for assessing the state of atherosclerosis.

[0010] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have discovered that the blood concentration of a neutrophil-derived protein correlates with the onset of arteriosclerotic disease and the progression of atherosclerosis, and that the higher the risk of developing arteriosclerotic disease and the more advanced the atherosclerosis, the higher the blood concentration of the neutrophil-derived protein. This finding led to the completion of the present invention. Specifically, the present invention includes the following aspects: [1-1] A reagent for use in diagnosing, assessing the severity, predicting the onset or progression of arteriosclerotic disease, or assessing the risk of onset or progression of arteriosclerotic disease, comprising an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule. [1-2] The reagent according to [1-1], wherein the arteriosclerotic disease is at least one selected from the group consisting of acute coronary syndrome, cerebrovascular disease, and aortic disease. [1-3] The reagent according to [1-2], wherein the acute coronary syndrome is at least one selected from the group consisting of acute myocardial infarction, unstable angina, and sudden cardiac death due to ischemia. [1-4] The reagent according to [1-3], wherein the acute coronary syndrome is acute myocardial infarction. [1-5] The reagent according to [1-4], wherein the acute myocardial infarction is non-ST elevation myocardial infarction. [1-6] The reagent according to [1-2], wherein the cerebrovascular disease is at least one selected from the group consisting of cerebral aneurysm, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic cerebral embolism. [1-7] The reagent according to [1-2], wherein the aortic disease is at least one selected from the group consisting of aortic aneurysm and aortic dissection. [1-8] The reagent according to any of [1-1] to [1-7], wherein the neutrophil-derived protein is neutrophil elastase. [1-9] The reagent according to any of [1-1] to [1-8], wherein the reagent is a reagent for immunoassay. [1-10] The reagent according to [1-9], wherein the immunoassay reagent is a reagent for use in at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody assay, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry.[2-1] A reagent for evaluating a state of atherosclerosis, comprising an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule. [2-2] The reagent according to [2-1], wherein the evaluation of the state of atherosclerosis is evaluation of the state of at least one type of plaque selected from the group consisting of vulnerable plaque, ruptured plaque, plaque prone to erosion, plaque that has undergone erosion, plaque with internal bleeding, plaque with nodular calcification, and plaque exhibiting severe stenosis. [2-3] The reagent according to [2-1] or [2-2], wherein the neutrophil-derived protein is neutrophil elastase. [2-4] The reagent according to any of [2-1] to [2-3], wherein the reagent is a reagent for immunological assay. [2-5] The reagent according to [2-4], wherein the immunoassay reagent is a reagent for use in at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody technique, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [3-1] A method for diagnosing an arteriosclerotic disease, assessing its severity, predicting the onset or progression of an arteriosclerotic disease, or assessing the risk of onset or progression of an arteriosclerotic disease, comprising: (1) contacting a sample with an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule, and (2) measuring the binding state between the antibody or its antigen-binding fragment and the complex bound to the neutrophil-derived protein or its corresponding binding molecule. [3-2] The method according to [3-1], wherein the arteriosclerotic disease is at least one selected from the group consisting of acute coronary syndrome, cerebrovascular disease, and aortic disease. [3-3] The method according to [3-2], wherein the acute coronary syndrome is at least one selected from the group consisting of acute myocardial infarction, unstable angina, and sudden cardiac death due to ischemia. [3-4] The method according to [3-3], wherein the acute coronary syndrome is acute myocardial infarction.[3-5] The method according to [3-4], wherein the acute myocardial infarction is non-ST elevation myocardial infarction. [3-6] The method according to [3-2], wherein the cerebrovascular disease is at least one selected from the group consisting of cerebral aneurysm, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic cerebral embolism. [3-7] The method according to [3-2], wherein the aortic disease is at least one selected from the group consisting of aortic aneurysm and aortic dissection. [3-8] The method according to any of [3-1] to [3-7], wherein the neutrophil-derived protein is neutrophil elastase. [3-9] The method according to any one of [3-1] to [3-8], wherein the method for measuring the binding state is at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody technique, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [3-10] The method according to any one of [3-1] to [3-9], wherein the sample is at least one selected from the group consisting of plasma, serum, and whole blood. [4-1] A method for assessing the state of atherosclerosis, comprising: (1) contacting a sample with an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or its antigen-binding fragment and the complex bound to the neutrophil-derived protein or its corresponding binding molecule. [4-2] The method according to [4-1], wherein the evaluation of the atherosclerotic state is evaluation of the state of at least one type of plaque selected from the group consisting of vulnerable plaque, ruptured plaque, plaque prone to erosion, plaque that has undergone erosion, plaque that has caused internal bleeding, plaque with nodular calcification, and plaque exhibiting severe stenosis. [4-3] The method according to [4-1] or [4-2], wherein the neutrophil-derived protein is neutrophil elastase.[4-4] The method according to any one of [4-1] to [4-3], wherein the method for measuring the binding state is at least one immunological assay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [4-5] The method according to any one of [4-1] to [4-4], wherein the sample is at least one selected from the group consisting of plasma, serum, and whole blood. [5-1] A method for diagnosing an arteriosclerotic disease, assessing its severity, predicting the onset or progression of an arteriosclerotic disease, or assisting in the assessment of the risk of onset or progression of an arteriosclerotic disease, comprising: (1) contacting a sample with an antibody or its antigen-binding fragment against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or its antigen-binding fragment and the complex bound to the neutrophil-derived protein or its corresponding binding molecule. [5-2] The method according to [5-1], wherein the arteriosclerotic disease is at least one selected from the group consisting of acute coronary syndrome, cerebrovascular disease, and aortic disease. [5-3] The method according to [5-2], wherein the acute coronary syndrome is at least one selected from the group consisting of acute myocardial infarction, unstable angina, and sudden cardiac death due to ischemia. [5-4] The method according to [5-3], wherein the acute coronary syndrome is acute myocardial infarction. [5-5] The method according to [5-4], wherein the acute myocardial infarction is non-ST elevation myocardial infarction. [5-6] The method according to [5-2], wherein the cerebrovascular disease is at least one selected from the group consisting of cerebral aneurysm, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic cerebral embolism. [5-7] The method according to [5-2], wherein the aortic disease is at least one selected from the group consisting of aortic aneurysm and aortic dissection. [5-8] The method according to any of [5-1] to [5-7], wherein the neutrophil-derived protein is neutrophil elastase.[5-9] The method according to any one of [5-1] to [5-8], wherein the method for measuring the binding state is at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody technique, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [5-10] The method according to any one of [5-1] to [5-9], wherein the sample is at least one selected from the group consisting of plasma, serum, and whole blood. [6-1] A method for assisting in the assessment of atherosclerosis, comprising: (1) contacting a sample with an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or its antigen-binding fragment and the complex bound to the neutrophil-derived protein or its corresponding binding molecule. [6-2] The method according to [6-1], wherein the evaluation of the atherosclerotic state is evaluation of at least one type of plaque state selected from the group consisting of vulnerable plaque, ruptured plaque, erosion-prone plaque, eroded plaque, plaque with internal bleeding, plaque with nodular calcification, and plaque exhibiting severe stenosis. [6-3] The method according to [6-1] or [6-2], wherein the neutrophil-derived protein is neutrophil elastase. [6-4] The method according to any one of [6-1] to [6-3], wherein the method for measuring the binding state is at least one immunological assay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [6-5] The method according to any one of [6-1] to [6-4], wherein the sample is at least one selected from the group consisting of plasma, serum, and whole blood.[7-1] A method for collecting data to diagnose an arteriosclerotic disease, assess its severity, predict the onset or progression of an arteriosclerotic disease, or assess the risk of onset or progression of an arteriosclerotic disease, comprising: (1) contacting a sample with an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or its antigen-binding fragment and the complex bound to the neutrophil-derived protein or its corresponding binding molecule. [7-2] The method according to [7-1], wherein the arteriosclerotic disease is at least one selected from the group consisting of acute coronary syndrome, cerebrovascular disease, and aortic disease. [7-3] The method according to [7-2], wherein the acute coronary syndrome is at least one selected from the group consisting of acute myocardial infarction, unstable angina, and sudden cardiac death due to ischemia. [7-4] The method according to [7-3], wherein the acute coronary syndrome is acute myocardial infarction. [7-5] The method according to [7-4], wherein the acute myocardial infarction is non-ST elevation myocardial infarction. [7-6] The method according to [7-2], wherein the cerebrovascular disease is at least one selected from the group consisting of cerebral aneurysm, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic cerebral embolism. [7-7] The method according to [7-2], wherein the aortic disease is at least one selected from the group consisting of aortic aneurysm and aortic dissection. [7-8] The method according to any one of [7-1] to [7-7], wherein the neutrophil-derived protein is neutrophil elastase. [7-9] The method according to any one of [7-1] to [7-8], wherein the method for measuring the binding state is at least one immunological assay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [7-10] The method according to any one of [7-1] to [7-9], wherein the sample is at least one selected from the group consisting of plasma, serum, and whole blood.[8-1] A method for collecting data for assessing the state of atherosclerosis, comprising: (1) contacting a sample with an antibody or its antigen-binding fragment against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the state of binding between the antibody or its antigen-binding fragment and the complex bound to the neutrophil-derived protein or its corresponding binding molecule. [8-2] The method according to [8-1], wherein the assessment of the state of atherosclerosis is assessment of the state of at least one type of plaque selected from the group consisting of vulnerable plaque, ruptured plaque, erosion-prone plaque, eroded plaque, plaque with internal bleeding, plaque with nodular calcification, and plaque exhibiting severe stenosis. [8-3] The method according to [8-1] or [8-2], wherein the neutrophil-derived protein is neutrophil elastase. [8-4] The method according to any one of [8-1] to [8-3], wherein the method for measuring the binding state is at least one immunological assay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [8-5] The method according to any one of [8-1] to [8-4], wherein the sample is at least one selected from the group consisting of plasma, serum, and whole blood. [9-1] A method for detecting or quantifying a complex bound to a neutrophil-derived protein or its corresponding binding molecule in a sample from a subject for whom an assessment of the state of atherosclerosis is required, the method comprising: (1) contacting the sample with an antibody or an antigen-binding fragment thereof against the complex bound to the neutrophil-derived protein or its corresponding binding molecule, and (2) measuring the binding state between the antibody or its antigen-binding fragment and the complex bound to the neutrophil-derived protein or its corresponding binding molecule. [9-2] The method according to [9-1], wherein the arteriosclerotic disease is at least one selected from the group consisting of acute coronary syndrome, cerebrovascular disease, and aortic disease.[9-3] The method according to [9-2], wherein the acute coronary syndrome is at least one selected from the group consisting of acute myocardial infarction, unstable angina, and sudden cardiac death due to ischemia. [9-4] The method according to [9-3], wherein the acute coronary syndrome is acute myocardial infarction. [9-5] The method according to [9-4], wherein the acute myocardial infarction is non-ST elevation myocardial infarction. [9-6] The method according to [9-2], wherein the cerebrovascular disease is at least one selected from the group consisting of cerebral aneurysm, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic cerebral embolism. [9-7] The method according to [9-2], wherein the aortic disease is at least one selected from the group consisting of aortic aneurysm and aortic dissection. [9-8] The method according to any of [9-1] to [9-7], wherein the neutrophil-derived protein is neutrophil elastase. [9-9] The method according to any one of [9-1] to [9-8], wherein the method for measuring the binding state is at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [9-10] The method according to any one of [9-1] to [9-9], wherein the sample is at least one selected from the group consisting of plasma, serum, and whole blood. [10-1] A method for detecting or quantifying a complex bound to a neutrophil-derived protein or its corresponding binding molecule in a sample from a subject for whom an assessment of the state of atherosclerosis is required, the method comprising: (1) contacting the sample with an antibody or an antigen-binding fragment thereof against the complex bound to the neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or its antigen-binding fragment and the complex bound to the neutrophil-derived protein or its corresponding binding molecule.[10-2] The method according to [10-1], wherein the evaluation of the atherosclerotic state is evaluation of the state of at least one type of plaque selected from the group consisting of vulnerable plaque, ruptured plaque, plaque prone to erosion, plaque that has undergone erosion, plaque that has caused internal bleeding, plaque with nodular calcification, and plaque exhibiting severe stenosis. [10-3] The method according to [10-1] or [10-2], wherein the neutrophil-derived protein is neutrophil elastase. [10-4] The method according to any one of [10-1] to [10-3], wherein the method for measuring the binding state is at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody technique, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [10-5] The method according to any one of [10-1] to [10-4], wherein the sample is at least one selected from the group consisting of plasma, serum, and whole blood. [11-1] Use of an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule, for the manufacture of a reagent for diagnosing, assessing the severity of, predicting the onset or progression of, or assessing the risk of onset or progression of an arteriosclerotic disease. [11-2] The use according to [11-1], wherein the arteriosclerotic disease is at least one selected from the group consisting of acute coronary syndrome, cerebrovascular disease, and aortic disease. [11-3] The use according to [11-2], wherein the acute coronary syndrome is at least one selected from the group consisting of acute myocardial infarction, unstable angina, and sudden cardiac death due to ischemia. [11-4] The use according to [11-3], wherein the acute coronary syndrome is acute myocardial infarction. [11-5] The use according to [11-4], wherein the acute myocardial infarction is non-ST elevation myocardial infarction. [11-6] The use according to [11-2], wherein the cerebrovascular disease is at least one selected from the group consisting of cerebral aneurysm, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic cerebral embolism.[11-7] The use according to [11-2], wherein the aortic disease is at least one selected from the group consisting of aortic aneurysm and aortic dissection. [11-8] The use according to any of [11-1] to [11-7], wherein the neutrophil-derived protein is neutrophil elastase. [11-9] The use according to any of [11-1] to [11-8], wherein the reagent is an immunoassay reagent. [11-10] The use according to [11-9], wherein the immunoassay reagent is a reagent for use in at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. [12-1] Use of an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule, for the manufacture of a reagent for assessing the state of atherosclerosis. [12-2] The use according to [12-1], wherein the assessment of the state of atherosclerosis is assessment of the state of at least one type of plaque selected from the group consisting of vulnerable plaque, ruptured plaque, plaque prone to erosion, plaque that has undergone erosion, plaque with internal bleeding, plaque with nodular calcification, and plaque exhibiting severe stenosis. [12-3] The use according to [12-1] or [12-2], wherein the neutrophil-derived protein is neutrophil elastase. [12-4] The use according to any of [12-1] to [12-3], wherein the reagent is a reagent for immunoassay. [12-5] The use according to [12-4], wherein the immunoassay reagent is a reagent for use in at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry.

[0011] According to the present invention, it is possible to diagnose arteriosclerotic diseases, evaluate their severity, predict the onset or aggravation of arteriosclerotic diseases, or evaluate the risk of onset or aggravation of arteriosclerotic diseases, thereby making it possible to particularly predict the onset and aggravation of arteriosclerotic diseases at an early stage.Furthermore, according to the present invention, it is possible to evaluate the state of atherosclerosis, thereby making it possible to early confirm the presence or absence of unstable plaques that are prone to rupture, particularly in NSTEMI patients.

[0012] Fig. 1 shows the standard curve for the neutrophil elastase ELISA measurement system constructed in Example 1. Fig. 2 shows the neutrophil elastase concentration in each serum sample measured in Example 2 using the reagent constructed in Example 1.

[0013] As used herein, "neutrophil" refers to a cell that contains proteolytic enzymes such as myeloperoxidase (MPO), neutrophil elastase, and cathepsin G in its cytoplasmic granules and that eliminates pathogens in response to stimuli through phagocytosis, degranulation, and production of reactive oxygen species (ROS) and inflammatory cytokines. Neutrophil extracellular traps (NETs) are known as an important biological defense function of neutrophils.

[0014] As used herein, "neutrophil-derived protein" refers to a protein released into the blood from neutrophils. Examples include proteins released into the body by NETs, ​​specifically neutrophil elastase, azurocidin, lactoferrin, myeloperoxidase, peptidylarginine deiminase 4 (hereinafter also referred to as "PAD4"), etc. Preferably, neutrophil elastase is used. Furthermore, as used herein, "a complex of a neutrophil-derived protein bound to a corresponding binding molecule" refers to a complex of the above-mentioned "neutrophil-derived protein" bound to its corresponding binding molecule. Specific examples of the complex include, for example, a complex of neutrophil elastase and α １ Antitrypsin (A1AT), α １ Antichymotrypsin (ACT), or α ２In particular, neutrophil elastase and α- １ Antitrypsin (A1AT) or α １ and complexes with antichymotrypsin (ACT). Hereinafter, unless otherwise specified, "neutrophil-derived proteins" and "complexes of neutrophil-derived proteins bound to corresponding binding molecules" will be collectively referred to as "neutrophil-derived proteins."

[0015] As used herein, the term "antibody or antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule" is not particularly limited as long as it specifically binds to the neutrophil-derived protein or its corresponding binding molecule. Examples of antibodies or antigen-binding fragments capable of specifically binding to a "neutrophil-derived protein" include antibodies or fragments thereof capable of recognizing and binding to a portion of the neutrophil-derived protein as an epitope. Examples of antibodies or antigen-binding fragments capable of specifically binding to a "complex" of a neutrophil-derived protein and a binding molecule include antibodies or antigen-binding fragments thereof capable of recognizing and binding to a portion of the neutrophil-derived protein present on the surface of the complex as an epitope, and antibodies or antigen-binding fragments thereof capable of recognizing and binding to a portion of the binding molecule as a partial or complete epitope. The "antibody" may be a monoclonal or polyclonal antibody. The antibody may be commercially available or may be produced by known methods such as cell fusion technology, genetic recombination technology, or phage display using a neutrophil-derived protein or a portion thereof as an antigen. Furthermore, the above-mentioned "antigen-binding fragment of an antibody" refers to a fragment of the above-mentioned antibody that has the ability to bind to a neutrophil-derived protein, and examples thereof include Fab fragments obtained by partial digestion of the above-mentioned antibody with papain or the like, F(ab')2 fragments obtained by partial digestion with pepsin or the like, and Fab' fragments obtained by reducing the F(ab')2 fragments. Preferably, the example includes a monoclonal antibody or an antigen-binding fragment thereof. Hereinafter, unless otherwise specified, the above-mentioned "antibody" and the above-mentioned "antibody antigen-binding fragment" will be collectively referred to as "antibody."

[0016] As used herein, "arteriosclerotic disease" includes arteriosclerosis itself, as well as ischemic heart disease, such as myocardial infarction and angina pectoris, which develop due to arteriosclerosis-induced vascular stenosis in the heart or brain, cerebrovascular disease, cerebral hemorrhage, aortic aneurysm, aortic dissection, nephrosclerosis in the renal artery and resulting renal failure, and peripheral arteriosclerosis obliterans. Preferably, at least one disease selected from the group consisting of acute coronary syndrome, cerebrovascular disease, and aortic disease is included. Examples of the "acute coronary syndrome" include at least one disease selected from the group consisting of acute myocardial infarction, unstable angina, and sudden cardiac death due to ischemia. Preferable examples include acute myocardial infarction, particularly non-ST elevation myocardial infarction. Examples of the "cerebrovascular disease" include at least one disease selected from the group consisting of cerebral aneurysm, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic cerebral embolism. Examples of the "aortic disease" include at least one disease selected from the group consisting of aortic aneurysm and aortic dissection.

[0017] "Atherosclerosis" as used herein refers to the formation of plaque-like growths on the inside of arteries, and can occur in all large and medium-sized arteries, including major arteries such as coronary arteries, cerebral arteries, and the aorta. As plaques grow, they impair blood flow, and if they suddenly rupture, platelets gather there, forming a blood clot and causing atherothrombosis, which blocks the blood vessel. Examples of serious diseases caused by this series of mechanisms include the following: Coronary artery plaque: acute coronary syndrome (unstable angina, acute myocardial infarction, sudden cardiac death due to ischemia); Cerebral artery plaque: cerebrovascular disease (cerebral aneurysm, lacunar infarction, atherothrombotic cerebral infarction, cardiogenic cerebral embolism); Aortic plaque: aortic disease (aortic aneurysm, aortic dissection). Furthermore, the above-mentioned plaque may be at least one type selected from the group consisting of coronary artery plaques that are prone to rupture, ruptured plaques, plaques that are prone to erosion, plaques that have eroded, plaques that have caused internal bleeding, plaques with nodular calcification, and plaques that exhibit severe stenosis.

[0018] As used herein, "diagnosis" refers to determining the current or future condition of a disease. As used herein, "severity" includes mild, moderate, severe, etc., and "progression" includes progression from mild to moderate or severe, and from moderate to severe. As used herein, "onset" refers to the appearance of symptoms of a disease.

[0019] As used herein, "assessing the state of atherosclerosis" refers to obtaining information related to the onset and severity of acute disease due to arterial occlusion or subocclusion by evaluating the progression of atherosclerosis, which is characterized by patchy intimal plaques growing toward the lumen of medium- and large-sized arteries, including coronary arteries, carotid arteries, cerebral arteries, aorta, aortic branches, and major arteries of the limbs, from the concentration of blood protein markers. This evaluation includes, for example, evaluating the state of coronary artery plaque, cerebral artery plaque, and aortic plaque, and particularly includes evaluating the state of at least one type of coronary plaque selected from the group consisting of vulnerable plaque, ruptured plaque, erosion-prone plaque, eroded plaque, plaque with internal bleeding, plaque with nodular calcification, and plaque exhibiting severe stenosis.

[0020] As used herein, the term "subject" refers to a living organism to be tested, particularly an animal (e.g., a mammal such as a human, mouse, rat, hamster, guinea pig, monkey, cow, pig, horse, rabbit, sheep, goat, cat, or dog), and particularly to a human (also referred to as a "test subject" in this case). Furthermore, the term "test subject" (or "test subject" in the case of a human) as used herein includes a subject (patient) suffering from a disease, a subject (test subject) suspected of having the disease, a subject (test subject) at risk of having the disease, and a subject (healthy individual) who is not suffering from a disease.

[0021] The "sample" used herein is not particularly limited as long as it is a sample that can contain the above-mentioned neutrophil-derived proteins, and examples thereof include blood samples prepared from blood collected from a human subject. The term "blood sample" used herein refers to a sample containing at least a portion of blood components, and may be any of whole blood, serum, and plasma, or any of these diluted forms. The blood sample is preferably serum or plasma. Preparation of the blood sample can be carried out by known methods.

[0022] [Diagnosis of arteriosclerotic disease, assessment of severity, prediction of onset or aggravation, or assessment of risk of onset or aggravation] In one embodiment, the present invention provides a reagent for use in diagnosing arteriosclerotic disease, assessing severity, predicting onset or aggravation, or assessing risk of onset or aggravation, the reagent comprising an antibody or an antigen-binding fragment thereof against a complex bound to the above-mentioned neutrophil-derived protein or its corresponding binding molecule.

[0023] The composition, shape, state, etc. of the reagent are not particularly limited. Furthermore, the antibody or antigen-binding fragment thereof in the reagent may be one type or two or more types. When two or more types of antibodies or antigen-binding fragments thereof are used, they may be, for example, two or more types of antibodies or antigen-binding fragments thereof that bind to different epitopes, or two or more types of antibodies or antigen-binding fragments thereof that bind to the same epitope. Furthermore, when neutrophil-derived proteins can exist in a sample both in a free form and in a complex, at least one of the two or more types of antibodies may be an antibody or antigen-binding fragment thereof against the complex. Examples of such combinations include a combination of two or more antibodies or antigen-binding fragments thereof that can recognize and bind to different portions of a neutrophil-derived protein as an epitope; a combination of at least one antibody that can recognize and bind to a portion of a neutrophil-derived protein as an epitope and an antigen-binding fragment of at least one antibody that can recognize and bind to an epitope different from the epitope of the antibody; a combination of at least one antibody or antigen-binding fragment thereof that can recognize and bind to a portion of a neutrophil-derived protein as an epitope and at least one antibody or antigen-binding fragment thereof that can recognize and bind to a portion of a complex as a partial or complete epitope; and a combination of at least one antibody or antigen-binding fragment thereof that can recognize and bind to a portion of a neutrophil-derived protein as an epitope and an antibody or antigen-binding fragment thereof that can recognize and bind to a portion of a binding molecule as a partial or complete epitope. More specifically, for example, the neutrophil-derived protein is human neutrophil elastase and the binding molecule is human α １ In the case of antitrypsin (A1AT), examples include a combination of an anti-human neutrophil elastase antibody and an anti-human A1AT antibody.

[0024] In the reagent, the antibody may be immobilized on a support. The support can be appropriately selected depending on the method in which the reagent is used. Examples of the support include well plates (e.g., 96-well microplates), membranes (e.g., nitrocellulose membranes, polyvinylidene fluoride membranes), slide glasses, magnetic beads, latex particles, etc. The antibody can be immobilized on the support by a known method selected depending on the material of the support.

[0025] The antibody may also be labeled. The labeling substance used for labeling is not particularly limited, and any known substance may be used. Examples of the labeling substance include enzyme labels such as peroxidase and alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); radioisotope labels such as iodine-125; electrochemiluminescent labels such as ruthenium complexes; biotin; and metal nanoparticles. Labeling of the antibody can be performed using a known method selected according to the type of labeling substance.

[0026] The reagent may be in the form of a kit. The kit may contain other elements in addition to the antibody. Examples of the other elements include a detection reagent for a labeled substance, a standard sample of a neutrophil-derived protein, a reagent for preparing a blood sample, a diluent, a buffer, a support, and instructions for use.

[0027] Examples of the reagent include reagents for use in immunoassays (reagents for immunoassays), specifically reagents for use in enzyme immunoassays, fluorescent enzyme immunoassays, chemiluminescent enzyme immunoassays, chemiluminescent immunoassays, electrochemiluminescent immunoassays, fluorescent antibody assays, radioimmunoassays, Western blotting, immunoblotting, latex agglutination, immunochromatography, nephelometry, etc. Preferred are reagents for use in latex agglutination or immunochromatography. These may be commercially available or may be prepared according to known methods. More specifically, for example, a reagent for use in latex agglutination comprises: (i) an antibody-sensitized latex solution containing latex particles to which at least one antibody or its antigen-binding fragment capable of recognizing and binding to a portion of a neutrophil-derived protein (e.g., neutrophil elastase) as an epitope; and (ii) a binding molecule (e.g., α １ Examples of such reagents include a reagent comprising an antibody-sensitized latex solution containing latex particles to which an antibody or an antigen-binding fragment thereof is bound, which is capable of recognizing and binding to a part of the antigen-binding fragment (antitrypsin) as part or all of an epitope.

[0028] The above reagent can detect neutrophil-derived proteins in a sample (especially a blood sample) and can measure the amount (concentration) of neutrophil-derived proteins. Here, the blood concentration of neutrophil-derived proteins shows a positive correlation with the progression of atherosclerotic disease from onset to severity. That is, the blood concentration of neutrophil-derived proteins increases as atherosclerotic disease progresses from onset to severity. Therefore, the blood concentration of neutrophil-derived proteins can be used as an indicator for diagnosing atherosclerotic disease. Therefore, the above reagent can be used for diagnosing atherosclerotic disease, assisting in the diagnosis, collecting data for the diagnosis, etc.

[0029] Furthermore, the severity of arteriosclerotic disease can be evaluated based on the blood concentration of neutrophil-derived proteins. For example, a reference value or cutoff value (pathological condition identification value) (hereinafter collectively referred to as "reference value / cutoff value") can be determined by statistically processing data obtained from multiple healthy individuals and patients, and the severity of arteriosclerotic disease can be evaluated based on this value. For example, a value below a first reference value / cutoff value can be evaluated as mild, a value between the first reference value / cutoff value and a second reference value / cutoff value can be evaluated as moderate, and a value above the second reference value / cutoff value can be evaluated as severe. The first reference value / cutoff value can be an upper reference limit determined based on multiple healthy individuals, and the second reference value / cutoff value can be calculated by multiplying the upper reference limit by a specific number. Therefore, the above-mentioned reagent can be used to evaluate the severity of arteriosclerotic disease, assist in that evaluation, collect data for that evaluation, etc.

[0030] Furthermore, based on the blood concentration of neutrophil-derived proteins, it is possible to predict whether or not arteriosclerotic disease will develop or whether or not arteriosclerotic disease will worsen. For example, a reference value / cutoff value can be determined by statistically processing data obtained from multiple healthy individuals or patients, and based on this value, it is possible to predict whether or not arteriosclerotic disease will develop or whether or not arteriosclerotic disease will worsen. For example, if the blood concentration is equal to or greater than a predetermined reference value / cutoff value, it can be predicted that arteriosclerotic disease will develop, and if the blood concentration is less than the reference value / cutoff value, it can be predicted that arteriosclerotic disease will not develop. Similarly, if the blood concentration is equal to or greater than a predetermined reference value / cutoff value, it can be predicted that arteriosclerotic disease will worsen, and if the blood concentration is less than the reference value / cutoff value, it can be predicted that arteriosclerotic disease will not worsen. Therefore, the above-mentioned reagent can be used to predict the onset or worsening of arteriosclerotic disease, assist in such prediction, collect data for such prediction, etc.

[0031] In addition, the presence or absence, or high or low, of the risk of developing or worsening arteriosclerotic disease can be determined based on the blood concentration of neutrophil-derived proteins. For example, a reference value / cutoff value can be determined by statistically processing data obtained from multiple patients, and the presence or high or low risk of developing or worsening arteriosclerotic disease can be determined based on this value. For example, if the reference value / cutoff value is equal to or greater than a predetermined reference value / cutoff value, the risk of developing arteriosclerotic disease can be determined to be present or high. Conversely, if the reference value / cutoff value is less than the predetermined reference value / cutoff value, the risk of developing or worsening arteriosclerotic disease can be determined to be absent or low. Similarly, if the reference value / cutoff value is equal to or greater than a predetermined reference value / cutoff value, the risk of developing or worsening arteriosclerotic disease can be determined to be present or high. Conversely, if the reference value / cutoff value is less than the predetermined reference value / cutoff value, the risk of developing or worsening arteriosclerotic disease can be determined to be absent or low. Therefore, the above-mentioned reagent can be used to evaluate the risk of developing or worsening arteriosclerotic disease, assist in such evaluation, collect data for such evaluation, etc.

[0032] The sample to be measured using the above-mentioned reagent may be a blood sample prepared from blood collected from a human subject (subject), and the subject may not have been confirmed to have an arteriosclerotic disease, or may have been confirmed to have an arteriosclerotic disease. The presence of the disease can be confirmed by known methods. For example, confirmation can be made by antibody testing, PCR testing, biomarker testing, or a doctor's diagnosis.

[0033] Furthermore, one embodiment of the present invention is a method for diagnosing arteriosclerotic disease, assessing its severity, predicting the onset or progression of arteriosclerotic disease, or assessing the risk of onset or progression of arteriosclerotic disease, comprising: (1) contacting a sample with an antibody or antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the complex bound to the neutrophil-derived protein or its corresponding binding molecule and an antibody or antigen-binding fragment thereof against the complex bound to the neutrophil-derived protein or its corresponding binding molecule.

[0034] Furthermore, one embodiment of the present invention is a method for assisting in the diagnosis of arteriosclerotic disease, assessment of its severity, prediction of the onset or progression of arteriosclerotic disease, or assessment of the risk of onset or progression of arteriosclerotic disease, comprising: (1) contacting a sample with an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or antigen-binding fragment thereof and the complex bound to the neutrophil-derived protein or its corresponding binding molecule.

[0035] Furthermore, one embodiment of the present invention is a method for collecting data for diagnosing arteriosclerotic disease, assessing its severity, predicting the onset or progression of arteriosclerotic disease, or assessing the risk of onset or progression of arteriosclerotic disease, comprising: (1) contacting a sample with an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or antigen-binding fragment thereof and the complex bound to the neutrophil-derived protein or its corresponding binding molecule.

[0036] The sample used in the above-mentioned methods for diagnosing arteriosclerotic diseases, methods for assisting in diagnosis, and methods for collecting data for diagnosis includes samples to be measured using the above-mentioned reagents. The antibodies used in the above-mentioned methods are the same as those used in the above-mentioned reagents, and may be one type or two or more types. The above-mentioned methods can be carried out using one or two or more types of the above-mentioned reagents.

[0037] The above step (1) is a step of contacting a sample with an antibody to bind the neutrophil-derived protein contained in the sample to the antibody. Therefore, the step is not particularly limited as long as it is performed under conditions that allow the neutrophil-derived protein to bind to the antibody. For example, the step can be performed in accordance with a known immunological assay.

[0038] The step (2) is a step of measuring the state of binding between the neutrophil-derived protein obtained in the step (1) and the antibody. The measurement of the state of binding is not particularly limited, and known qualitative or quantitative methods can be used. Specific examples include known immunoassays, such as enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. The above-mentioned measurement methods can detect, for example, the neutrophil-derived protein in a sample and quantify the amount (concentration) of the neutrophil-derived protein in the sample.

[0039] The above-mentioned method for diagnosing, etc., an arteriosclerotic disease may include a step (3) of diagnosing an arteriosclerotic disease, assessing its severity, predicting the onset or aggravation of the disease, or assessing the risk of onset or aggravation of the disease, based on the amount (concentration) of the neutrophil-derived protein obtained in step (2). Step (3) includes, for example, making a diagnosis using the amount (concentration) of the neutrophil-derived protein as an index, as explained in the above-mentioned reagent, or determining a reference value / cutoff value and assessing the severity, predicting the onset or aggravation, or assessing the risk of onset or aggravation based on the reference value / cutoff value.

[0040] The above-mentioned method for assisting in the diagnosis of arteriosclerotic diseases may further include steps such as determining a reference value / cut-off value that serves as an indicator for the diagnosis, etc., based on the amount (concentration) of the neutrophil-derived protein obtained in step (2), as described above for the reagent, and providing the results of comparison with the reference value / cut-off value.

[0041] The method for collecting data for diagnosing or otherwise treating arteriosclerotic diseases may further include steps such as collecting data for determining a reference value / cut-off value that serves as an indicator for the diagnosis or the like, based on the amount (concentration) of the neutrophil-derived protein obtained in step (2), as described above for the reagent, and providing data compared with the reference value / cut-off value.

[0042] In one embodiment, the present invention provides a method for detecting or quantifying a complex bound to a neutrophil-derived protein or its corresponding binding molecule in a sample from a subject for whom an atherosclerotic state needs to be evaluated, the method comprising: (1) contacting the sample with an antibody or antigen-binding fragment thereof directed to a complex bound to the neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or its antigen-binding fragment and the complex bound to the neutrophil-derived protein or its corresponding binding molecule. The sample in this method includes samples that are the subject of measurement for the reagents and methods used in the diagnosis of arteriosclerotic diseases described above. The antibodies used in this method are the same as those used in the reagents and methods used in the diagnosis of arteriosclerotic diseases described above, and may be one type or two or more types. This method can be performed using one or more of the reagents used in the diagnosis of arteriosclerotic diseases described above. Steps (1) and (2) in this method are the same as steps (1) and (2) in the method for diagnosing arteriosclerotic diseases described above.

[0043] Furthermore, as one embodiment, the present invention includes use of an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule for the manufacture of a reagent for diagnosing an arteriosclerotic disease, assessing its severity, predicting the onset or progression of an arteriosclerotic disease, or assessing the risk of onset or progression of an arteriosclerotic disease. The antibody in this embodiment is the same as the reagent used for diagnosing an arteriosclerotic disease described above, and may be one type or two or more types.

[0044] [Evaluation of atherosclerotic status] In one embodiment, the present invention provides a reagent for evaluating atherosclerotic status, comprising an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule. The composition, form, state, etc. of the reagent are similar to those of the reagents used for diagnosing atherosclerotic diseases.

[0045] The reagent can detect neutrophil-derived proteins in a sample (especially a blood sample) and measure the amount (concentration) of neutrophil-derived proteins. Here, the blood concentration of neutrophil-derived proteins shows a positive correlation with the progression of atherosclerosis. That is, the blood concentration of neutrophil-derived proteins increases as atherosclerosis progresses. Therefore, the blood concentration of neutrophil-derived proteins can be used as an indicator for diagnosing atherosclerosis-related diseases (e.g., arteriosclerotic diseases). Therefore, the reagent can be used to evaluate the state of atherosclerosis, assist in that evaluation, collect data for that evaluation, and the like.

[0046] Furthermore, the progression of atherosclerosis can be assessed based on the blood concentration of neutrophil-derived proteins. For example, a reference value / cutoff value can be determined by statistically processing data obtained from multiple healthy individuals or patients, and the progression of atherosclerosis can be assessed based on this value. For example, a value below a first reference value / cutoff value can be assessed as mild, a value between the first reference value / cutoff value and a second reference value / cutoff value can be assessed as moderate, and a value above the second reference value / cutoff value can be assessed as severe. The first reference value / cutoff value can be an upper reference limit determined by multiple healthy individuals, and the second reference value / cutoff value can be calculated by multiplying the upper reference limit by a specific number. Therefore, the above-mentioned reagent can be used to assess the progression of atherosclerosis, assist in such assessment, collect data for such assessment, etc.

[0047] Furthermore, the severity of atherosclerosis-related diseases can be evaluated based on the blood concentration of neutrophil-derived proteins. For example, a reference value / cutoff value (pathological condition identification value) can be determined by statistically processing data obtained from multiple healthy individuals or patients, and the severity of the disease can be evaluated based on this value. For example, a value below a first reference value / cutoff value can be evaluated as mild, a value between the first reference value / cutoff value and a second reference value / cutoff value can be evaluated as moderate, and a value above the second reference value / cutoff value can be evaluated as severe. The first reference value / cutoff value can be an upper reference limit determined by multiple healthy individuals, and the second reference value / cutoff value can be calculated by multiplying the upper reference limit by a specific number. Therefore, the above-mentioned reagent can be used to evaluate the severity of atherosclerosis-related diseases, assist in such evaluation, and collect data for such evaluation.

[0048] Furthermore, based on the blood concentration of neutrophil-derived proteins, it is possible to predict whether or not an atherosclerosis-related disease will develop or whether or not the disease will worsen. For example, a reference value / cutoff value can be determined by statistically processing data obtained from multiple healthy individuals or patients, and based on this value, it is possible to predict whether or not the disease will develop or whether or not the disease will worsen. For example, if the reference value / cutoff value is equal to or greater than a predetermined reference value / cutoff value, it is possible to predict whether or not the disease will develop, and if the reference value / cutoff value is less than the predetermined reference value / cutoff value, it is possible to predict whether or not the disease will worsen. Similarly, if the reference value / cutoff value is equal to or greater than a predetermined reference value / cutoff value, it is possible to predict whether or not the disease will worsen, and if the reference value / cutoff value is less than the predetermined reference value / cutoff value, it is possible to predict whether or not the disease will worsen. The first reference value / cutoff value can be an upper reference limit determined by multiple healthy individuals, and the second reference value / cutoff value can be obtained by multiplying the upper reference limit by a specific number. Therefore, the above-mentioned reagent can be used to predict the onset or worsening of atherosclerosis-related disease, assist in such prediction, collect data for such prediction, etc.

[0049] In addition, the presence or absence, or high or low, of the risk of developing or worsening atherosclerosis-related disease can be determined based on the blood concentration of neutrophil-derived proteins. For example, a reference value / cutoff value can be determined by statistically processing data obtained from multiple healthy individuals or patients, and the presence or high or low risk of developing or worsening a disease can be determined based on this value. For example, if the reference value / cutoff value is equal to or greater than a predetermined reference value / cutoff value, the risk of developing the disease can be determined to be present or high. Conversely, if the reference value / cutoff value is less than the predetermined reference value / cutoff value, the risk can be determined to be absent or low. Similarly, if the reference value / cutoff value is equal to or greater than a predetermined reference value / cutoff value, the risk of worsening a disease can be determined to be present or high. Conversely, if the reference value / cutoff value is less than the predetermined reference value / cutoff value, the risk can be determined to be absent or low. Therefore, the above-mentioned reagent can be used to assess the risk of developing or worsening atherosclerosis-related disease, assist in such assessment, collect data for such assessment, etc.

[0050] The sample to be measured using the above-mentioned reagent may be a blood sample prepared from blood collected from a human subject (subject), and the subject may or may not have been confirmed to have atherosclerosis. Furthermore, the subject may or may not have been confirmed to have an atherosclerosis-related disease. The presence of the disease can be confirmed by known methods. For example, confirmation can be made by antibody testing, PCR testing, biomarker testing, or a doctor's diagnosis.

[0051] Furthermore, as one embodiment, the present invention includes a method for assessing the state of atherosclerosis, comprising: (1) contacting a sample with an antibody or antigen-binding fragment thereof directed against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the complex bound to the neutrophil-derived protein or its corresponding binding molecule and the antibody or antigen-binding fragment thereof directed against the complex bound to the neutrophil-derived protein or its corresponding binding molecule.

[0052] Furthermore, in one embodiment, the present invention includes a method for assisting in the assessment of atherosclerosis status, comprising: (1) contacting a sample with an antibody or antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state of the antibody or antigen-binding fragment thereof to the complex bound to the neutrophil-derived protein or its corresponding binding molecule.

[0053] Furthermore, in one embodiment, the present invention includes a method for collecting data for assessing the state of atherosclerosis, comprising: (1) contacting a sample with an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or antigen-binding fragment thereof and the complex bound to the neutrophil-derived protein or its corresponding binding molecule.

[0054] The sample used in the above-mentioned methods for assessing the state of atherosclerosis, the methods for assisting assessment, and the methods for collecting data for assessment includes samples to be measured using the above-mentioned reagents. The antibodies used in the above-mentioned methods are the same as those used in the above-mentioned reagents, and may be one type or two or more types. Furthermore, the above-mentioned methods can be carried out using one or two or more types of the above-mentioned reagents.

[0055] The above step (1) is a step of contacting a sample with an antibody to bind the neutrophil-derived protein contained in the sample to the antibody. Therefore, the step is not particularly limited as long as it is performed under conditions that allow the neutrophil-derived protein to bind to the antibody. For example, the step can be performed in accordance with a known immunological assay.

[0056] The step (2) is a step of measuring the state of binding between the neutrophil-derived protein obtained in the step (1) and the antibody. The measurement of the state of binding is not particularly limited, and known qualitative or quantitative methods can be used. Specific examples include known immunoassays, such as enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry. The above-mentioned measurement methods can detect, for example, the neutrophil-derived protein in a sample and quantify the amount (concentration) of the neutrophil-derived protein in the sample.

[0057] The method for evaluating the state of atherosclerosis may include step (3) of evaluating the stage of atherosclerosis based on the amount (concentration) of the neutrophil-derived protein obtained in step (2). Step (3) may include, for example, evaluating the stage of atherosclerosis using the amount (concentration) of the neutrophil-derived protein as an index, as described above for the reagent, or determining a reference value / cutoff value and evaluating the stage of atherosclerosis based on the reference value / cutoff value.

[0058] The method for evaluating the state of atherosclerosis may also include a step (3) of diagnosing an atherosclerosis-associated disease, assessing its severity, predicting the onset or aggravation of the disease, or assessing the risk of onset or aggravation of the disease, based on the amount (concentration) of the neutrophil-derived protein obtained in step (2). Step (3) may include, for example, making a diagnosis using the amount (concentration) of the neutrophil-derived protein as an indicator, or determining a reference value / cutoff value and assessing the severity, predicting the onset or aggravation, or assessing the risk of onset or aggravation based on the reference value / cutoff value, as described above for the reagent.

[0059] The above-mentioned method for assisting in the assessment of the state of atherosclerosis may further comprise steps of determining a reference value / cut-off value that serves as an indicator for assessing the degree of progression, etc., based on the amount (concentration) of the neutrophil-derived protein obtained in step (2), as described above for the reagent, and providing the results of comparison with the reference value / cut-off value.

[0060] The method for collecting data to assess the state of atherosclerosis may further include steps of collecting data for determining a reference value / cut-off value that serves as an index for assessing the degree of progression of atherosclerosis, based on the amount (concentration) of the neutrophil-derived protein obtained in step (2), as described above for the reagent, and providing data compared with the reference value / cut-off value.

[0061] In one embodiment, the present invention provides a method for detecting or quantifying a complex bound to a neutrophil-derived protein or its corresponding binding molecule in a sample from a subject for whom an atherosclerotic condition needs to be assessed, the method comprising: (1) contacting the sample with an antibody or antigen-binding fragment thereof directed to a complex bound to the neutrophil-derived protein or its corresponding binding molecule; and (2) measuring the binding state between the antibody or antigen-binding fragment thereof and the complex bound to the neutrophil-derived protein or its corresponding binding molecule. The sample used in this method includes samples that are the subject of measurement using the reagents and methods used to assess atherosclerotic conditions described above. The antibodies used in this method are the same as those used in the reagents and methods used to assess atherosclerotic conditions described above, and may be one type or two or more types. This method can be performed using one or more of the reagents used to assess atherosclerotic conditions described above. Furthermore, steps (1) and (2) in this method are the same as steps (1) and (2) in the method for assessing atherosclerotic conditions described above.

[0062] Furthermore, in one embodiment, the present invention includes use of an antibody or an antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule for the manufacture of a reagent for assessing the state of atherosclerosis. The antibody in this embodiment is the same as that used in the reagent for assessing the state of atherosclerosis described above, and may be one type or two or more types.

[0063] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0064] Example 1 Production of Monoclonal Antibodies Antibodies A and B were obtained according to the following procedure. Human leukocyte-derived purified elastase (Elastin Products, Cat. No. CK828) was suspended in adjuvant (Complete Freund's adjuvant: CFA) and immunized with BALB / c mice (5-week-old, female) at 2-week intervals. Partial blood samples were taken midway through the immunization, and the degree of titer increase at the antiserum level was confirmed using reactivity with the immunogen, human leukocyte-derived purified elastase, as an indicator. After confirming sufficient antibody titer increase through multiple immunizations, antibody-producing cells were collected from the spleen and fused with myeloma cells (P3X63Ag8.653, ECACC). Cell fusion was performed using the PEG method, and the fused cells were seeded onto culture plates. The cells were then cultured in a 37°C carbon dioxide incubator. Next, the hybridoma culture supernatant was collected, and the antibody titer was confirmed using the reactivity with purified elastase derived from human leukocytes as an index. The antibody-producing clone cells were then subcultured and cloned by limiting dilution. Cells derived from the resulting single colony were obtained as anti-human neutrophil elastase monoclonal antibody-producing hybridomas. The anti-human A1AT antibody-producing hybridomas were also cloned using human plasma-derived α １Antitrypsin (Merck, Cat. No. A6150-25MG) was used to obtain the hybridomas by a similar method. The two monoclonal antibody-producing hybridomas obtained were then cultured in large quantities. Each hybridoma was intraperitoneally administered to BALB / c mice that had been pre-fed and given pristane intraperitoneally. The mice were then bred for 10 to 25 days to allow ascites to accumulate. The ascites fluid was then collected, and the obtained ascites antibodies were subjected to affinity purification to obtain antibody A, an anti-human neutrophil elastase monoclonal antibody, and antibody B, an anti-human A1AT antibody.

[0065] <Construction of Neutrophil Elastase ELISA Measurement System> Enzyme immunoassay was performed by sandwich enzyme immunoassay using a kit composed of the following reagents. (1) Standard Reagent: Antibody A and antibody B were bound to HiTrap NHS-activated HP Columns (Cytiva, Cat. No. 17071601), respectively, to obtain an antibody A-binding affinity column and an antibody B-binding affinity column. Next, 30 mL of plasma from a healthy subject was diluted 10-fold with 20 mM phosphate buffer, and the filtrate was passed through a 0.45 μm filter and collected. The filtrate was passed through the antibody A-binding affinity column, and the adsorbed protein components were eluted with 100 mM glycine-HCl buffer, pH 2.7, to obtain the eluted fraction. The eluted fraction was buffer-exchanged with 20 mM phosphate buffer and then passed through an antibody B-binding affinity column. The adsorbed protein components were eluted with 100 mM glycine-HCl buffer, pH 2.7, to obtain the eluted fraction. The eluted fraction was buffer-exchanged with 20 mM phosphate buffer to obtain a standard reagent stock solution. A dilution series of the standard reagent stock solution was prepared with 1% BSA-containing PBS buffer, with 1% BSA-containing PBS buffer alone designated as standard reagent 0. (2) Sample dilution solution: 1% BSA-containing PBS buffer. Use as is. (3) Enzyme-labeled antibody solution: Horseradish peroxidase (HRP)-labeled enzyme-labeled antibody B diluted 16,000-fold with 1% BSA-containing PBS buffer. Use as is. (4) Reaction plate: Antibody A was immobilized on a 96-well microplate and then blocked. One well was used per measurement. (5) Washing solution: 0.05% Tween 20, PBS buffer. Use as is. (6) Substrate solution: A solution containing tetramethylbenzidine (3,3',5,5'-tetramethyl benzidine stabilized substrate: TMB, Agilent). Use the substrate solution as is. 100 μL of the substrate solution is used per measurement. (7) Reaction stop solution: 3N sulfuric acid. Use as is. 100 μL of the reaction stop solution is used per measurement.

[0066] A standard curve was prepared according to the following procedure. First, the standard reagent was appropriately diluted with the sample dilution solution to prepare neutrophil elastase solutions of various concentrations. Then, 100 μL of each diluted standard solution was added to the reaction plate, stirred in a mixer, and incubated at room temperature for 1 hour. The reaction solution was then removed from each well using an ELISA washer, and 0.3 mL of washing solution prepared by diluting the stock washing solution was added to each well for washing. This washing process was repeated three times, after which the remaining washing solution in the wells was removed using a paper towel or similar. Next, 100 μL of enzyme-labeled antibody solution was added, and the wells were incubated at room temperature for 1 hour. The reaction solution was then removed from each well using an ELISA washer, and 0.3 mL of washing solution prepared by diluting the stock washing solution was added to each well for washing. This washing process was repeated three times, after which the remaining washing solution in the wells was removed using a paper towel or similar. Next, 100 μL of substrate solution was added, and the wells were incubated at room temperature for exactly 30 minutes in the dark. Thereafter, 100 μL of a reaction stop solution was added and the mixture was quickly stirred in a mixer to stop the enzyme reaction. The absorbance at 450 nm was measured for each well, and a standard curve was created ( FIG. 1 ).

[0067] Example 2 Measurement of human serum samples using an ELISA measurement system Eleven serum samples collected from patients diagnosed with acute coronary syndrome (unstable angina: 2 cases, non-ST elevation myocardial infarction: 4 cases), cerebrovascular disease (cerebral aneurysm: 3 cases), and aortic disease (thoracic aortic aneurysm rupture: 2 cases), as disease groups with unstable plaque, and three serum samples collected from healthy subjects (Table 1 below), were measured for neutrophil elastase concentration using the reagent constructed in Example 1. The results are shown in Figure 2. If the upper limit of the measurement results for the healthy subject samples is taken as the reference upper limit, all patients with unstable plaque showed values ​​more than twice the reference upper limit.

Claims

1. A reagent for use in diagnosing an arteriosclerotic disease, assessing the severity, predicting the onset or aggravation of the disease, or assessing the onset risk or aggravation risk, comprising: A reagent comprising an antibody or antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule.

2. 2. The reagent according to claim 1, wherein the arteriosclerotic disease is at least one selected from the group consisting of acute coronary syndrome, cerebrovascular disease, and aortic disease.

3. 3. The reagent according to claim 2, wherein the acute coronary syndrome is at least one selected from the group consisting of acute myocardial infarction, unstable angina, and sudden cardiac death due to ischemia.

4. The reagent according to claim 3, wherein the acute coronary syndrome is acute myocardial infarction.

5. The reagent according to claim 4, wherein the acute myocardial infarction is non-ST elevation myocardial infarction.

6. 3. The reagent according to claim 2, wherein the cerebrovascular disease is at least one selected from the group consisting of cerebral aneurysm, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic cerebral embolism.

7. The reagent according to claim 2, wherein the aortic disease is at least one selected from the group consisting of aortic aneurysm and aortic dissection.

8. A reagent for assessing the state of atherosclerosis, comprising: A reagent comprising an antibody or antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule.

9. The reagent according to claim 1 , wherein the reagent is a reagent for immunoassay.

10. The reagent according to claim 9, wherein the immunoassay reagent is a reagent for use in at least one immunoassay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry.

11. A reagent described in any one of claims 1 to 10, wherein the neutrophil-derived protein is neutrophil elastase.

12. A method for diagnosing an arteriosclerotic disease, assessing its severity, predicting its onset or aggravation, or assessing its onset risk or aggravation risk, comprising: (1) contacting a sample with an antibody or antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; (2) measuring the binding state between the antibody or its antigen-binding fragment and a complex bound to the neutrophil-derived protein or its corresponding binding molecule; A method comprising:

13. The method according to claim 12, wherein the arteriosclerotic disease is at least one selected from the group consisting of acute coronary syndrome, cerebrovascular disease, and aortic disease.

14. 1. A method for assessing atherosclerotic status, comprising: (1) contacting a sample with an antibody or antigen-binding fragment thereof against a complex bound to a neutrophil-derived protein or its corresponding binding molecule; (2) measuring the binding state between the antibody or its antigen-binding fragment and a complex bound to the neutrophil-derived protein or its corresponding binding molecule; A method comprising:

15. The method according to claim 12, wherein the method for measuring the binding state is at least one immunological assay selected from the group consisting of enzyme immunoassay, fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent antibody assay, radioimmunoassay, Western blotting, immunoblotting, latex agglutination, immunochromatography, and nephelometry.

16. 13. The method of claim 12, wherein the sample is at least one selected from the group consisting of plasma, serum, and whole blood.

17. A method according to any one of claims 12 to 16, wherein the neutrophil-derived protein is neutrophil elastase.