JP2012107932A - Inspection method of cerebral infarction with clusterin protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of simply and efficiently inspecting cerebral infarction.SOLUTION: An inspection method for diagnosing cerebral infarction of an animal and an inspection method for diagnosing a type of cerebral infarction include the process of measuring the protein amount of Clusterin protein in a blood sample extracted from the inspection animal.

Description

  The present invention relates to a method for examining cerebral infarction, a method for evaluating cerebral infarction treatment or prevention effect, a method for screening for cerebral infarction preventive or therapeutic agent, and cerebral infarction examination, cerebral infarction treatment effect evaluation or cerebral infarction prevention / treatment. The present invention relates to a drug candidate compound screening kit.

  Stroke is a general term for cerebrovascular disorders such as cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage. Of these stroke types, cerebral infarction has been relatively increasing recently. Thrombolytic therapy, anticoagulant therapy, antiplatelet therapy, administration of cerebral protective drugs, etc. are used as drug therapy in the acute phase of cerebral infarction. Surgical surgery is performed for patients with severe or hemorrhagic cerebral infarction. Is done. Cerebral infarction symptoms are strongest in the acute phase and then gradually improve. This is because the necrotic brain tissue is swollen and the surrounding brain tissue is also compressed and damaged. As the swelling subsides, the surrounding tissues recover their function and the symptoms are fixed. However, since free radicals released from cerebral ischemic sites have the function of necrosing surrounding tissues, continuous treatment is required to improve the functional prognosis even after the acute phase.

The clinical types of cerebral infarction include lacunar infarction that develops when the intracerebral small artery is occluded, atherothrombotic cerebral infarction that develops when the intracerebral aorta is occluded by atheroma, and a thrombus in the heart becomes an obturator. Examples include cardiogenic cerebral embolism that develops by blocking an artery. Since appropriate treatment methods for the acute and chronic phases differ depending on these clinical types, a method for rapidly diagnosing the clinical type of patients with cerebral infarction has been demanded.
As a method of classifying clinical types of cerebral infarction, clinical symptom observation and echocardiography, MRI, MRA, cervical vascular echo, etc. are performed, and a flowchart of clinical diagnosis of cerebral infarction based on TOAST classification (Non-patent Document 1), etc. As a result of simultaneous measurement of molecular markers such as CRP, D-Dimer, RAGE, MMP-9, S100B, BNP within 24 hours of cerebral infarction, Although it has been disclosed that cardiogenic cerebral embolism can be classified as another disease type by setting a specific cutoff value with BNP and D-Dimer (Non-patent Document 2), etc. There was a need for molecular markers that could be diagnosed.

Lee, L.J. et al., Stroke, 1081-1089 (2000) Montaner et al., Stroke, 2280-2287 (2008)

  In view of the above problems, the present invention has an object to provide a method for quickly and surely inspecting cerebral infarction using serum or plasma of patients with cerebral infarction and the like, and a method for evaluating cerebral infarction treatment effects. To do.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have obtained the knowledge that the Clusterin protein in patient plasma differs between its concentrations in the plasma of patients with different disease types, and the amount of Clusterin protein Was found to be a useful index for the examination of cerebral infarction, and the present invention was completed.

That is, the gist of the present invention is as follows.
[1] A test method for diagnosing cerebral infarction in an animal, comprising a step of measuring the amount of Clusterin protein in a blood sample collected from the subject animal.
[2] The blood sample collected from the test animal is collected from the test animal at any time between immediately after the onset of cerebral infarction-like symptoms and the 20th day after the onset. [1] The examination method for diagnosis of cerebral infarction according to [1].
[3] A test method for diagnosing a cerebral infarction disease in the animal, comprising a step of measuring the amount of Clusterin protein in a blood sample collected from the test animal.
[4] The disease type diagnosis is one of lacunar infarction, atherothrombotic, cardiogenic cerebral embolism, aortic cerebral embolism, Branch atheromatous disease (BAD), arterial dissection, or unclassifiable The inspection method according to [3].
[5] The brain in the animal comprising the step of measuring the protein amount of the Clusterin protein in a blood sample collected from an animal in need of prevention or treatment of cerebral infarction administered with a cerebral infarction preventive or therapeutic agent A method for evaluating the effect of prevention or treatment of infarction.
[6] Screening of a prophylactic or therapeutic agent for cerebral infarction comprising a step of contacting a subject with a candidate compound for a prophylactic or therapeutic agent for cerebral infarction and then measuring the protein amount of Clusterin protein in the subject Method.
[7] A kit for screening for a cerebral infarction examination, a cerebral infarction therapeutic effect, or a cerebral infarction prophylactic / therapeutic drug candidate compound, comprising a reagent capable of measuring the protein mass of the Clusterin protein.

  According to the present invention, the clusterin protein in which the plasma concentration of a cerebral infarction patient is different from that in the plasma of a patient with a different disease type is a molecular marker for cerebral infarction examination (this is referred to as “Clusterin protein marker” in the present specification). May be provided). By using the Clusterin protein marker alone or in combination with other cerebral infarction molecular markers, it becomes possible to perform rapid and reliable diagnosis of cerebral infarction or classification of cerebral infarction pathology.

Hereinafter, embodiments of the present invention will be described in detail.
(1) Cerebral infarction examination method The first of the present invention is a method for examining cerebral infarction in an animal, comprising a step of measuring the amount of Clusterin protein in a blood sample collected from the subject animal.

  In the present specification, “cerebral infarction” is a disease in which the cerebral artery is blocked by a thrombus or embolus and becomes ischemic, and the brain tissue is necrotized and damaged, and a lacunar infarction that develops due to blocking of a small intracerebral artery. Atherothrombotic cerebral infarction caused by occlusion of intracerebral aorta due to atheroma, cardiogenic cerebral embolism caused by occlusion of intracerebral artery due to thrombosis in heart, from ascending aorta to aortic arch Aortic cerebral embolism, which is an embolism that develops when the obturator dissociates from the atherosclerotic lesion and occludes the intracerebral artery, and a plaque formed in the main artery obstructs the entrance of the penetrating branch “Branch atheromatous disease” (hereinafter sometimes referred to as “BAD”), an infarction of the penetrating branch region that occurs, arterial dissection where the intima of the cerebral blood vessel is damaged and blood enters the arterial wall to occlude the cerebral blood vessel Any of these are included. In addition, cerebral infarction includes any of acute phase, subacute phase, recovery phase (chronic phase) and the like.

  In the present specification, the “Clusterin protein” means a Clusterin protein which is a target for measuring the content in a blood sample in the test method of the present invention, and may be any protein derived from a test animal, but derived from a human Specifically, the protein represented by the specific amino acid sequence described below is exemplified. Furthermore, fragments, derivatives, and mutants of proteins having the same functions as these are also included.

  In the above examination method, the “test animal” may be any animal that may cause cerebral infarction, and specifically includes rodents such as humans, monkeys, and rats. Of these, the method for examining cerebral infarction of the present invention is particularly preferably performed in humans suspected of having cerebral infarction, humans after onset of cerebral infarction, or the like.

  Further, the “blood sample” collected from the test animal is not particularly limited as long as it contains Clusterin protein and can measure the concentration thereof. Specifically, EDTA plasma, citrate plasma, etc. Of these, any of plasma, serum, and whole blood may be used, but among these, EDTA plasma is preferably used because it can be easily collected, easily stored, and collected in a large amount. The timing for collecting the blood sample from the test animal may be any timing as long as the diagnosis of cerebral infarction is performed. Since cerebral infarction changes in symptoms and pathology from onset to onset, it will be described in detail below.

  In the test method of the present invention, the Clusterin protein, which is a target for measuring the content in a blood sample collected from a test animal, is a protein consisting of an amino acid sequence represented by Entry Name CLUS_HUMAN of UniProtKB in the case of humans. is there. For example, sequence information can be obtained by inputting the above Entry Name in a National Center for Biotechnology Information (NCBI) database or the like. The amino acid sequence is human, but when the test animal is different, a homologous protein derived from the animal becomes the protein to be measured.

  The method for measuring the content of the protein in the blood sample is not particularly limited as long as the content of the protein can be measured. For example, an existing immunoassay method using an antibody of the protein or a chromatographic method. All components captured on a chromatographic carrier (eg, cation exchanger, anion exchanger, hydrophobic chromatographic carrier, metal ion, etc.) under certain conditions using a combination of holographic techniques and time-of-flight mass spectrometry (TOF-MS) For example, a method of measuring the mass of the solution at once, a two-dimensional gel electrophoresis method, or the like is used. As the immunoassay, a method of quantitative detection according to an enzyme immunoassay, a method of measurement by a fluorescence immunoassay, a chemiluminescence immunoassay, or the like is preferable. The enzyme immunoassay method is a detection method using an enzyme as a labeling substance among the labeled immunoassay methods. It is particularly preferable to select an enzyme-linked immunosorbent assay (ELISA) method using an immunosolvent. Among the ELISA methods, the sandwich method is mentioned as one of the particularly preferable measurement modes in consideration of the convenience of operation, economic convenience, and general versatility in clinical examination. These measurement methods include, for example, the New Chemistry Laboratory (Edited by the Japanese Biochemical Society; Tokyo Kagaku Dojin), Molecular Cloning, A Laboratory Manual (T. Maniatis et al., Cold Spring Harbor Laboratory (2001)), Antibodies-A Laboratory. The method can be performed according to a method described in a general experiment such as Manual (E. Harlow, et al., Cold Spring Harbor Laboratory (1988)) or the like.

  Antibodies and the like (including antibody fragments) used in the above measurement can be obtained by a known method using the target Clusterin protein as an antigen. However, it is not necessary to be produced using the target Clusterin protein as an antigen, and any protein may be used as long as it exhibits at least cross-reactivity with the protein and can measure its content. As such an immunoassay and an antibody used therefor, for example, an antibody attached to Human Clusterin ELISA Kit (Catalog No. RD194034200R) manufactured by Biovendor Laboratory Medicine is preferably used.

  As a blood sample used in the method of the present invention, a blood sample immediately after being collected from a test animal is preferably used for the measurement, but a stored sample may be used. The method for storing the blood sample is not particularly limited as long as the amount of molecular marker for cerebral infarction examination does not change. For example, low temperature conditions such as 0 to 10 ° C. that do not freeze, dark conditions, and no vibration conditions. preferable. In the case of unavoidably freezing, a method capable of avoiding marker molecule decomposition, oxidation reaction, etc., such as deep freezing, is preferable.

  In the test method of the present invention, the content of the Clusterin protein marker in a blood sample collected from a test animal (in the present specification, this may be referred to as “specimen”) is measured, Can be used to examine cerebral infarction. In addition, it is a composite index that combines the contents of specimens such as CRP and D-Dimer, which are existing cerebral infarction markers, and links the observation of clinical symptoms with the results of echocardiography, MRI, MRA, cervical vascular echo, etc. It is possible to inspect more accurately by using.

  When the cerebral infarction test is performed using the content of the molecular marker for cerebral infarction test in the specimen as an index, a method may be used in which an appropriate cut-off value is defined for the test.

  The cut-off value is a value determined when determining a target disease group and a non-disease group (a group other than the target disease) by paying attention to a certain substance. When determining the target disease and non-disease, it is negative if it is below the cut-off value, positive if it is above the cut-off value, or positive if it is below the cut-off value, and if it is above the cut-off value The disease can be determined as negative (Kanai Masamitsu, Clinical Laboratory Proposal Kanehara Publishing Co., Ltd.).

  The index used for the purpose of evaluating the clinical usefulness of the cutoff value includes sensitivity and specificity. A certain population is determined using a cut-off value, and among the diseased patients, a (true positive) is positive in the determination, and b (false negative) is negative in the determination while being a disease patient. A / (a + b) when c (false positive) indicates that the patient is not a disease patient but is positive in the determination and d (true negative) indicates that the patient is not a disease patient and is negative in the determination. Can be represented as sensitivity (true positive rate) and d / (c + d) as specificity (true negative rate).

  The distribution of measured values between the target disease group and the non-disease group usually partially overlaps. Therefore, sensitivity and specificity change by raising or lowering the cutoff value. Decreasing the cut-off value increases the sensitivity, but the specificity decreases, and increasing the cut-off value decreases the sensitivity, but increases the specificity. As a determination method, it is preferable that both sensitivity and specificity are higher. A determination method in which the values of sensitivity and specificity do not exceed 0.5 is not recognized as useful.

  The method for setting the cut-off value is to set 95% of the distribution of the non-disease group as one of the values at both ends from the center as the cut-off value. When the distribution of the non-disease group shows a normal distribution And a method of setting an average value + 2 times standard deviation (SD) or an average value−2SD as a cut-off value.

  In general, in order to determine whether or not a diagnostic method is useful, the sensitivity and specificity vary depending on the cutoff value set as described above, so the sensitivity and specificity at a certain cutoff value are simply used. Rather than evaluating, it is desirable to evaluate with an index that maintains high sensitivity and specificity when the cut-off value is raised or lowered, for example, the AUC value of the ROC curve. The AUC value of the ROC curve becomes 1 when a cutoff value exists such that both sensitivity and specificity are 100%, and approaches 0.5 when the diagnostic performance is not good. Therefore, when the performance of a certain diagnostic method is judged by the AUC value of the ROC curve, it can be evaluated that the method is suitable as a diagnostic method if it is 0.7 or more. The Clusterin protein marker described in the present invention also has an AUC value of 0.7 or more on the ROC curve, and is therefore used for the cerebral infarction examination method described later.

(1-1) Cerebral infarction type examination method using Clusterin protein marker As a specific method of the cerebral infarction examination method using Clusterin protein marker of the present invention, an examination method for distinguishing the type of cerebral infarction is mentioned. It is done. The disease type means a clinically distinct disease type known per se, and specifically includes “lacuna infarction” (sometimes referred to herein as “lacuna”), “atherothrombosis” Cerebral infarction "(sometimes referred to herein as" atherothrombotic ")," cardiogenic cerebral embolism "(sometimes referred to herein as" cardiogenic cerebral embolism ") ), “Aortic cerebral embolism”, an embolism that develops when the obturator dissociates from the atherosclerotic lesion from the ascending aorta to the aortic arch and occludes the intracerebral artery, plaque formed in the main artery "Branch" is an infarction of the penetrating branch region caused by blocking the entrance of the penetrating branch
Atheromatous disease ”(hereinafter sometimes referred to as“ BAD ”), arterial dissection in which the intima of the cerebral blood vessels is damaged and blood enters the arterial wall to occlude the cerebral blood vessels, or none of them Type (hereinafter, this may be referred to as “non-classifiable type”).

  In order to distinguish the type of cerebral infarction by measuring the amount of Clusterin protein contained in the specimen, first, the cutoff value that can distinguish each type of disease in the amount of Clusterin protein in the blood sample is determined as described above. And the amount of Clusterin protein in the specimen is compared with the cut-off value to determine which disease type group it belongs to.

  The disease types that can be distinguished by the amount of Clusterin protein contained in the blood sample of the present invention are particularly those that can set a cut-off value that can be distinguished from each other by the Clusterin protein content between both disease type groups. Although there is no limitation, specifically, as shown in Table 1, for example, “atherothrombotic cerebral infarction” and “arterial dissection”, “cardiogenic cerebral embolism” and “BAD”, “lacuna infarction” and “ Arterial dissection, Aortic cerebral embolism and non-classifiable type, BAD and non-classifiable type, Arterial dissection and non-classifiable type, Atherothrombotic cerebral infarction and Aorta ”Genetic cerebral embolism”, “Atherothrombotic cerebral infarction” and “BAD”, “Lucuna infarction” and “BAD”, “BAD” and other types, “Arterial dissection” and other types Can be mentioned.

  The timing of obtaining the specimen when performing these measurements is not particularly limited immediately after the onset of cerebral infarction-like symptoms, but Table 1 shows preferred timings.

  In the measurement method of the present invention, the amount of Clusterin protein in the blood sample of a patient clinically determined to be the above-mentioned distinct disease type is measured in advance, and the above method is used to distinguish between both disease type groups. Set the value. The subject who collected the specimen for any disease type by measuring the content of the Clusterin protein marker in the collected specimen and comparing this absolute value with the cutoff value set by the above method It can be determined whether (animal) is included. The method for clinically confirming various cerebral infarctions is not particularly limited, but examples include methods for confirming from device diagnostic methods such as head X-ray CT, head MRI, MRA, cerebral angiography, cervical vascular echo, etc. It is done.

  In addition, when testing for the above-mentioned disease types, it is possible to combine the contents in specimens such as CRP and D-Dimer, which are existing cerebral infarction markers, and to observe clinical symptoms and echocardiography, MRI, MRA, cervical By using a composite index that correlates results such as blood vessel echoes, it is possible to inspect more accurately.

(2) Kit for measuring cerebral infarction The present invention further provides a kit for use in the above-described cerebral infarction examination, or evaluation of the prevention or treatment effect of cerebral infarction described below, or a screening method for a cerebral infarction prevention or treatment drug. The kit is also included. The contents of the kit are configured by a combination of equipment or reagents. However, the configuration or form of the kit is not limited as long as it includes a substance that is essentially the same as each component described below, or a substance that is essentially the same as a part thereof. Even if they are different, they are included in the kit of the present invention. Examples of the reagent include an anti-Clusterin protein marker antibody when the Clusterin protein marker is measured by an immunoassay. In addition, a biological sample diluent, an antibody-immobilized solid phase, a reaction buffer, a washing solution, a labeled secondary antibody or an antibody fragment thereof, a labeled detection reagent, a standard substance, and the like are also included as necessary. Examples of the diluted solution of the biological sample include an aqueous solution containing a protein such as BSA or casein in a surfactant or a buffer.

  As the antibody-immobilized solid phase, a material in which various polymer materials are shaped to suit the application and anti-molecular marker antibodies or antibody fragments thereof are solid-phased is used. Shapes are tubes, beads, plates, fine particles such as latex, sticks, etc., and materials are polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, polyethylene terephthalate, etc. Polymer materials, glass, ceramics and metals. Examples of the method for immobilizing an antibody include known methods such as a physical method and a chemical method, or a combination method thereof. For example, a 96-well polystyrene immunoassay microterplate prepared by subjecting an antibody or antibody fragment to a hydrophobic solid phase can be used.

  Any reaction buffer may be used as long as it provides a solvent environment for the binding reaction between the antibody on the antibody-immobilized solid phase and the antigen in the biological sample. However, surfactants, buffers, BSA, Reaction buffers containing proteins such as casein, preservatives, stabilizers, reaction accelerators and the like can be mentioned.

  As the labeled secondary antibody or antibody fragment thereof, the antibody or antibody fragment used in the present invention is labeled with a labeling enzyme such as horseradish peroxidase (HRP), bovine small intestine alkaline phosphatase, β-galactosidase, or a buffering agent. , BSA, casein and other proteins, preservatives and the like are used.

  As a reagent for detecting a labeled substance, for example, horseradish peroxidase according to the labeling enzyme, a substrate for absorption measurement such as tetramethylbenzidine or orthophenylenediamine, or a fluorescence such as hydroxyphenylpropionic acid or hydroxyphenylacetic acid. If the luminescent substrate such as a substrate or luminol is alkaline phosphatase, an absorbance measurement substrate such as 4-nitrophenyl phosphate, a fluorescent substrate such as 4-methylumbelliferyl phosphate, and the like can be given.

(3) Method for evaluating effect of prevention or treatment of cerebral infarction The second aspect of the present invention is blood collected from an animal in need of prevention or treatment of cerebral infarction administered with a cerebral infarction preventive or therapeutic agent. A method for evaluating the effect of preventing or treating cerebral infarction in an animal, comprising a step of measuring the amount of a Clusterin protein marker in a sample.

  Specifically, an animal that requires prevention or treatment of cerebral infarction is a human who exhibits symptoms of cerebral infarction or an animal described in (1) (these may be simply referred to as “subjects”), Examples include patients diagnosed with cerebral infarction by the method (1) above.

  The cerebral infarction preventive or therapeutic agent may be any as long as it is used as a cerebral infarction preventive or therapeutic agent. For example, thrombolytic agents such as urokinase and tissue plasminogen activator, antiparin such as heparin, etc. Examples include anticoagulants such as coagulants, cyclooxygenase inhibitors, phosphodiesterase inhibitors, thromboxen A2 (TXA2) synthesis inhibitors, and brain protective agents such as free radical scavengers.

  The clusterin protein marker level in blood samples taken from animals in need of prevention or treatment of cerebral infarction administered cerebral infarction preventive or therapeutic agent is increased or decreased compared to the amount before administration, or When approaching the amount in a control animal that does not develop cerebral infarction, it can be determined that there was a prophylactic or therapeutic effect.

  Since the cerebral infarction preventive or therapeutic agent to be treated varies depending on the disease type, it is possible to appropriately treat the patient if the disease type is diagnosed with the Clusterin protein marker. For example, if a lacunar infarction is diagnosed, it can be treated with a thromboxen A2 (TXA2) synthesis inhibitor and a cyclooxygenase inhibitor can prevent recurrence of cerebral infarction.

(4) Screening method for cerebral infarction preventive or therapeutic agent The third aspect of the present invention is to contact a subject with a candidate compound for a prophylactic or therapeutic agent for cerebral infarction, and then contact Clusterin in a blood sample of the subject. A screening method for a preventive or therapeutic agent for cerebral infarction, comprising a step of measuring the amount of a protein marker.

As the subject, a non-human animal individual, tissue, cell or the like in which the content of the Clusterin protein marker exhibits an abnormality similar to that of the cerebral infarction state is used. As an animal having a cerebral infarction symptom, for example, a cerebral infarction model non-human animal (Yuji Kuge, Kazuo Minematsu et al. Nylon Monofilament for Intraluminal Middle Cerebral Artery Occlusion in Rats. Stroke, 26, 1655 (1995)). In addition to the subject, it is also preferable to use an individual, tissue, cell, etc. of the same kind as the subject that has the same clusterin protein marker content (normal) as the normal state as a control.
Based on the Clusterin protein marker, if a pathological condition similar to a certain disease type in humans can be constructed in a non-human animal with cerebral infarction, an experimental system for developing a therapeutic agent in a patient with cerebral infarction of a certain disease type will be established be able to.

Examples of candidate compounds for preventing or treating cerebral infarction (hereinafter sometimes referred to as “candidate compounds”) to be brought into contact with these subjects include peptides, proteins, non-peptidic compounds, low-molecular synthetic compounds, and the like. These compounds may be novel compounds or known compounds. Moreover, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like containing these compounds may be used.
The dose, administration method, treatment time and the like may be appropriately selected according to the subject to be used.

  As a method for measuring the content of the Clusterin protein marker in the subject after contacting with the candidate compound, a method suitable for each subject can be used. For example, when the subject is a cell, it may be prepared as a cell extract according to a known method and used as a sample, or the cell may be immobilized on a culture plate or a slide glass and used as a sample. Also good. For example, when a cell extract is prepared and used as a sample, detection can be performed by ELISA or the like. It is preferable that these samples have the same number of cells used for extraction, or the amount of purified RNA or the amount of extracted protein so that the numerical values obtained as a result of detection can be compared and analyzed accurately.

If the amount of Clusterin protein marker in the subject contacted with the candidate compound is increased or decreased compared to the amount before contact, or approaches the amount in the control subject, the candidate compound is It can be determined to have a prophylactic or therapeutic effect.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Evaluation of Test Method for Discriminating Cerebral Infarction Type Using Clusterin Protein Marker Patients with a confirmed diagnosis for each type of cerebral infarction, specifically, “Lucuna Infarction” (in the table (Referred to as “lacuna”), “atherothrombotic cerebral infarction” (referred to as “atherothrombotic” in the table), “cardiogenic cerebral embolism” (referred to as “cardiogenic cerebral embolism” in the table) ), “Aortic cerebral embolism” (referred to as “aortic cerebral embolism” in the table), “Branch atheromatous disease” (referred to as “BAD” in the table), “arterial dissection” (in the table) , Referred to as “arterial dissection”, or patients with a disease type that does not fall into any of these (referred to as “non-classifiable type” in the table), immediately after the onset of cerebral infarction (described as “DAY0” in the table) ), Blood is collected at 7 days after the onset of disease (indicated as “DAY7” in the table). Zorehyo were obtained EDTA plasma from each number) described in 2. Clusterin protein concentration in EDTA plasma was measured by immunoassay using Human Clusterin ELISA Kit (Catalog No. RD194034200R) of Biovendor Laboratory Medicine. Specifically, a calibration curve was created using the standard Clusterin protein, and the concentration of Clusterin protein was calculated from this calibration curve.

Next, Fawcett, T. (2004) ROC Graphs:
The AUC value of the ROC curve was calculated according to the method described in Notes and Practical Considerations for Researchers. The results are shown in Table 2. At each blood sampling time point, a value exceeding 0.7 is shown to be useful as a test method for discriminating the target disease group from other disease groups.

  In other words, by comparing the concentrations of Clusterin protein in the plasma of patients with each type of disease immediately after the onset, a test is performed to distinguish between patients with cerebral infarction of the type of "Atherothrombotic cerebral infarction" and "Arterial dissection" In addition, in the same comparison, it is possible to distinguish between patients with cerebral infarction with the disease type of “cardiogenic cerebral embolism” and “Branch atheromatous disease”, with patients with cerebral infarction with the disease type of “lacuna infarction” and “arterial dissection”. Discriminating between patients with cerebral infarction with the types of “aortic cerebral embolism” and “non-classifiable cerebral infarction” , Distinguishing between cerebral infarction patients with "arterial dissection" and "unclassifiable cerebral infarction", "Atherothrombotic cerebral infarction" and "Branch atheromatous disease" with cerebral infarction, "Lucuna infarction" And “Branch aheromatous disease”, “Branch ath It was found that a test can be performed to discriminate between “eromatous disease” and other types of cerebral infarction patients, and “arterial dissection” and other types of cerebral infarction patients.

In addition, by comparing the concentrations of Clusterin protein in the plasma of patients with each disease type on the 7th day after the onset, patients with cerebral infarction with the disease types of “Atherothrombotic cerebral infarction” and “Aortic cerebral embolism” In the same comparison, in the same comparison, cerebral infarction patients with the types of "Atherothrombotic cerebral infarction" and "Branch atheromatous disease" can be identified, "Lachna infarction" and "Branch atheromatous disease" It was found that a test can be performed to discriminate between cerebral infarction patients with different disease types and “Branch atheromatous disease” and other types of cerebral infarction.

  According to the method of the present invention, diagnosis of cerebral infarction and evaluation of therapeutic / preventive effects can be performed, which is useful in the medical and diagnostic fields. Moreover, according to the method of the present invention, a therapeutic / prophylactic agent for cerebral infarction can be screened, which is also useful in the pharmaceutical field.

Claims (7)

  A test method for diagnosing cerebral infarction in an animal, comprising a step of measuring the amount of Clusterin protein in a blood sample collected from the subject animal.   The blood sample collected from the test animal is collected from the test animal at any time between immediately after the onset of cerebral infarction-like symptoms and 20 days after the onset. Test method for diagnosis of cerebral infarction as described in 1.   A test method for diagnosing a cerebral infarction disease in an animal, comprising a step of measuring a protein amount of a Clusterin protein in a blood sample collected from the test animal.   4. The diagnosis according to claim 3, wherein the disease type diagnosis is any one of lacunar infarction, atherothrombotic, cardiogenic cerebral embolism, aortic cerebral embolism, Branch atheromatous disease, arterial dissection, and unclassifiable. Inspection method.   Prevention of cerebral infarction in an animal comprising the step of measuring the amount of clusterin protein in a blood sample collected from an animal in need of prevention or treatment of cerebral infarction to which a prophylactic or therapeutic agent for cerebral infarction has been administered Or the evaluation method of therapeutic effect.   A method for screening a prophylactic or therapeutic agent for cerebral infarction, comprising the step of contacting a subject with a candidate compound for a prophylactic or therapeutic agent for cerebral infarction and then measuring the amount of Clusterin protein in the subject.   A kit for screening for a cerebral infarction test, a cerebral infarction therapeutic effect evaluation, or a cerebral infarction preventive / therapeutic drug candidate compound, comprising a reagent capable of measuring the protein amount of Clusterin protein.