WO2012067152A1

WO2012067152A1 - Method for testing for cerebral infarction via endothelial protein c receptor

Info

Publication number: WO2012067152A1
Application number: PCT/JP2011/076416
Authority: WO
Inventors: 孝成北園; 正浩鴨打; 哲朗吾郷; 貴弘桑城; 裕清原; 淳秦; 秀人粟野; 啓一郎金谷; 輝章小林
Original assignee: 三菱化学株式会社; 国立大学法人九州大学; 一般社団法人久山生活習慣病研究所
Priority date: 2010-11-16
Filing date: 2011-11-16
Publication date: 2012-05-24
Also published as: JPWO2012067152A1

Abstract

A test method for diagnosing the type of or predicting the prognosis of a cerebral infarction in a subject animal, said method including a step wherein the amount of endothelial protein C receptor in a blood sample collected from said animal is measured.

Description

Endothelial Protein C Receptor protein for cerebral infarction

The present invention relates to a test method for diagnosing cerebral infarction and predicting prognosis.

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.
The prognosis of cerebral infarction is to evaluate the degree of movement disorder in daily life by the modified Rankin Scale (Non-patent Document 3) or Barthel Index (Non-patent Document 4) between 3 months or 1 year after onset. It is judged by. Predicting the prognosis in advance is important for early determination of a treatment method for cerebral infarction. Although it has been disclosed that death can be predicted by setting a specific cutoff value with BNP in plasma measured at the time of admission (Non-patent Document 5), a molecular marker for predicting the degree of injury when alive is required It was.
Patent Document 1 discloses the use of Endothelial Protein C Receptor protein for the diagnosis of cardiovascular diseases, particularly atherosclerosis. However, there is no disclosure or suggestion that Endothelial Protein C Receptor protein can be used for cerebral infarction disease diagnosis or prognosis prediction.

US Patent Application Publication No. 2005/0032140

In view of the above problems, an object of the present invention is to provide a method for quickly and surely examining the disease type and prognosis of cerebral infarction using serum or plasma of a cerebral infarction patient or the like.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that Endothelial Protein C Receptor protein in patient plasma differs between its concentrations in plasma of patients with different disease types and prognosis. As a result, the inventors have found that the amount of Endothelial-Protein-C Receptor protein is a useful index for cerebral infarction disease type and prognosis, and have completed the present invention.

That is, the gist of the present invention is as follows.
[1] A test method for diagnosing the pathological type of cerebral infarction in an animal, comprising measuring the protein amount of Endothelial Protein C Receptor protein in a blood sample collected from the subject animal.
[2] 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 [1].
[3] A test method for predicting the prognosis of cerebral infarction in an animal, comprising the step of measuring the amount of Endothelial Protein C Receptor protein in a blood sample collected from the subject animal.
[4] The prediction of the prognosis of cerebral infarction according to [3], wherein the blood sample collected from the test animal is collected from the test animal on the seventh day of the onset of cerebral infarction. Inspection method.

According to the present invention, Endothelial® Protein® C® Receptor protein whose plasma concentration in patients with cerebral infarction is different from that in plasma of patients with different disease types and prognosis is expressed as a molecular marker for cerebral infarction (in the present specification, (Sometimes referred to as “EPCR protein markers”). By using the EPCR protein marker alone or in combination with other cerebral infarction molecular markers, it becomes possible to classify the type of cerebral infarction and predict the prognosis.

Hereinafter, embodiments of the present invention will be described in detail.
The test method of the present invention is a method for testing the disease type and prognosis of cerebral infarction in the animal, including the step of measuring the amount of Endothelial Protein C Receptor protein in a blood sample collected from the test 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 cerebral aorta occlusion 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 and occludes 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 this specification, Endothelial Protein C Receptor protein (hereinafter referred to as “EPCR protein”) means an EPCR protein whose content in a blood sample is measured in the test method of the present invention, and is derived from a test animal. Any protein may be used, but specific examples of human-derived proteins include proteins represented by the specific amino acid sequences described below. 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 EPCR 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 Endothelial Protein C Receptor protein, which is a target for measuring the content in a blood sample collected from a test animal, is the amino acid represented by UniProtKB Entry Name EPCR # HUMAN in the case of humans. A protein consisting of a sequence. For example, sequence information can be obtained by inputting the above Entry Name in a National Center for Biotechnology Information (NCBI) database. 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. In addition, 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 are, for example, the New Chemistry Laboratory (Edited by the Japanese Biochemical Society; Tokyo Kagaku Dojin), MolecularMCloning, A Laboratory Manual (T. Maniatis et al., Cold Spring Harbor Laboratory (2001)), Antibodies-A Laboratory Manual (E. Harlow, et al., ColdSpring Harbor Laboratory (1988)) or the like can be carried out according to the method described in a general experiment or in conformity thereto.

Antibodies and the like (including antibody fragments) used in the above measurement can be obtained by a known method using the target EPCR protein as an antigen. However, it is not necessary to be produced using the target EPCR 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 EPCR Quantikine ELISA Kit (catalog No. DEPCR0) manufactured by R & D is preferably used.

The blood sample used in the method of the present invention is preferably used immediately after being collected from the test animal for the above 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, but there are, for example, a low temperature condition such as 0 to 10 ° C. that does not freeze, a dark condition, and no vibration condition. preferable. In the case of unavoidably freezing, a method capable of avoiding marker molecule decomposition or oxidation reaction such as deep freezing is preferable.

In the test method of the present invention, the content of the EPCR 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 the disease type and prognosis of 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.

(1) Cerebral infarction type inspection method using EPCR protein marker The type of cerebral infarction means a clinically distinct type known per se, and specifically, “lacuna infarction” ( (Sometimes referred to herein as “lacuna”), “atherothrombotic cerebral infarction” (sometimes referred to herein as “atherothrombotic”), “cardiogenic cerebral embolism” ( In this specification, it may be referred to as “cardiogenic cerebral embolism”), an embolism that develops when the obturator dissociates from the atherosclerotic nest formed from the ascending aorta to the aortic arch and occludes the intracerebral artery Aortic cerebral embolism, which is a symptom, and “Branch atheromatous disease” (hereinafter referred to as “BAD”), which is an infarction of the penetrating branch area caused by a plaque formed in the main artery blocking the entrance of the penetrating branch The inner lining of the cerebral blood vessels is damaged and blood enters the arterial wall. Aortic dissection or disease types that do not correspond to any of them that occlude a blood vessel (hereinafter sometimes referred to as "non-typeable") can be mentioned.

In order to distinguish the type of cerebral infarction by measuring the amount of EPCR protein contained in the specimen, first, in the amount of EPCR protein in the blood sample, a cut-off value that can distinguish each disease type is determined, This is done by comparing the amount of EPCR protein in the sample with the cut-off value to determine which disease type group it belongs to.

The cut-off value generally refers to a value determined when a target disease group and a non-disease group (group other than the target disease) are determined 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, if it is above the cut-off value The disease can be determined as negative (Kanai Masamitsu, Clinical Laboratory Proposal Kanehara Publishing Co., Ltd.).
In the present invention, a cut-off value is defined between the target disease type group and the other groups, and if the cut-off value or less, it falls under the target disease type. The disease type can be determined as not applicable to the target disease type if it is below the value, or as applicable if it is above the cutoff value.

Sensitivity and specificity can be cited as an index used for the purpose of evaluating the clinical usefulness of the cutoff value. A certain population is determined using a cut-off value, and among the patients with cerebral infarction of the target disease type, a (true positive) that is positive in the determination is determined while being a cerebral infarction patient of the disease type B (false negative) for negative, c (false positive) for positive in spite of not being the type of cerebral infarction When expressed as d (true negative), the value represented by a / (a + b) is the sensitivity (true positive rate), and the value represented by d / (c + d) is the specificity (true negative rate). ).

The distribution of measured values between the target disease type group and other disease type groups 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.

As a method for setting the cut-off value, 95% of the distribution of other disease type groups including 95% of the distribution at the both ends from the center is set as the cut-off value, and the distribution of other disease type groups is normal. In the case of showing the distribution, a method of setting an average value + 2 standard deviation (SD) or an average value−2SD as a cut-off value may be used.

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. Since the EPCR protein marker also has an ROC curve AUC value of 0.7 or more, it is used in the cerebral infarction pathological examination method described later.

As a disease type that can be distinguished by the amount of EPCR protein contained in the blood sample of the present invention, as long as it can set a cut-off value that can be distinguished from each other by the EPCR protein content between both disease type groups, Although there is no limitation, specifically, for example, as shown in Table 1, "Atherothrombotic cerebral infarction" and "Aortic cerebral embolism", "Atherothrombotic cerebral infarction" and "Classification not possible", " Atherothrombotic cerebral infarction and arterial dissection, Cardiogenic cerebral embolism and arterial dissection, Lacunar infarction and aortic cerebral embolism, Lacunar infarction and arterial dissection, Examples include “lacuna infarction” and “non-classifiable type”, “BAD” and “non-classifiable type”, “arterial dissection” and other disease types, “non-classifiable type” and other disease types, and the like.

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 EPCR 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 EPCR 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) Test method for predicting cerebral infarction using EPCR protein marker As a specific method of cerebral infarction test method using EPCR protein marker of the present invention, predicting the prognosis of cerebral infarction in a subject (animal) Inspection method for the above. The prognosis of cerebral infarction is indicated by the degree of injury due to cerebral infarction of the patient after a certain period of time after the onset of cerebral infarction. The degree of failure can be assessed using an already established indicator known per se. Indicators include, for example, Japan's modified Rankin Scale (mRS) criteria (Yukito Shinohara et al. MRS Reliability Study Group, research on reliability of modified Rankin Scale-introduction of Japanese criteria and questionnaire, stroke 2007; 29: 6-13) etc. are used.

After a certain period of time, it may be any time after onset, but from the viewpoint of assessing the state of rehabilitation of a subject (animal), for example, it is generally evaluated from 3 months to 1 year after the onset Is. In predicting the prognosis, taking into consideration the usefulness of the prediction method, the amount of EPCR protein in the blood sample obtained from the subject for about 2 weeks immediately after the onset, and the state of the subject 3 months after the onset The method of predicting is preferable.

In the measurement method of the present invention, the amount of EPCR protein in a blood sample at the time of measurement is measured in advance for each patient who has clinically determined the above-mentioned mRS at the time of prediction. Then, a cutoff value for distinguishing both mRS groups is set by the above-described method. More specifically, for example, when distinguishing between patients with mRS 0-1 after 3 months and patients with 2-5, patients with mRS 0-5 3 months after the onset (for prediction purposes) 7 days after the onset of the disease (at the time of measurement), the amount of EPCR protein in the blood sample is measured, and the cut-off between the measured value of patients whose mRS is 0 to 1 and the measured value of patients 2 to 5 after 3 months Set the value. After that, the sample for the measurement purpose is collected, the content of the EPCR protein marker in the sample is measured, and this absolute value is compared with the cutoff value set by the above method, so that The state of the subject (animal) can be predicted.

In the prediction method of the present invention, particularly preferably, the subject in the vicinity of about 3 months later by measuring the amount of EPCR protein in a sample collected from a subject about 2 weeks after onset, more preferably 7 days after onset. And a method for predicting whether the mRS is 2 to 5 or 1 or less.

In addition, another preferred prediction method of the present invention is to measure the amount of EPCR protein in a sample collected from a subject about 2 weeks from immediately after onset, more preferably 7 days after onset, by measuring the amount of EPCR protein around 3 months later. A method for predicting whether a subject's mRS will be 3-5 or less than 2 or less, about 2 weeks immediately after onset, more preferably EPCR protein amount in a sample collected from a subject 7 days after onset A preferable example is a method of predicting whether the mRS of the subject in the vicinity of 3 months after the measurement will be 4 to 5 or 3 or less.

Thus, a subject (animal) diagnosed with cerebral infarction by the method of the present invention has a very good prognostic course and therapeutic effect by receiving a treatment suitable for each disease.

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 EPCR protein marker Patients with 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) EDTA plasma was collected from each (number of people listed in Table 2). It was. The EPCR protein concentration in EDTA plasma was measured by an immunoassay using the R & D Human EPCR Quantikine ELISA Kit (Catalog No. DEPCR0). Specifically, a standard curve was prepared using the standard EPCR protein, and the concentration of the EPCR protein was calculated from this standard curve.

Next, in order to evaluate the discrimination performance at each blood sampling time point, the AUC value of the ROC curve was calculated according to the method described in Fawcett, T. (2004) ROC Graphs: NotesNoteand 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 between a target disease and other diseases.

In other words, by comparing the concentration of EPCR protein in the plasma of each disease-type patient immediately after the onset, to distinguish between patients with cerebral infarction of the disease type of “Atherothrombotic cerebral infarction” and “Aortogenic cerebral embolism” In addition, in the same comparison, discrimination of cerebral infarction patients with "Atherothrombotic cerebral infarction" and "Unclassifiable cerebral infarction", "Atherothrombotic cerebral infarction" and "Arterial dissection" Cerebral infarction patients with different types of disease, cerebral infarction patients with "cardiogenic cerebral embolism" and "arterial dissection" types, "Lachna infarction" and "aortogenic cerebral embolism" Discrimination of cerebral infarction patients, discrimination of cerebral infarction patients with `` Lucuna infarction '' and `` arterial dissection '', discrimination of cerebral infarction patients with `` Lucuna infarction '' and `` unclassifiable cerebral infarction '', `` Branch atheromatous Distinguishing between patients with cerebral infarction of the disease type `` disease '' and `` unclassifiable cerebral infarction '', `` arterial dissection '' Determination of other disease type cerebral infarction patients, and were found to perform the "non-typeable cerebral infarction" as the inspection for the other disease types the discrimination of stroke patients.

Example 3 Evaluation of a test method for predicting the prognosis of cerebral infarction using an EPCR protein marker The prognosis of cerebral infarction is indicated by the degree of injury due to cerebral infarction of the patient after a certain period of time after the onset of cerebral infarction. It is. Japanese version modified Rankin Scale (mRS) criteria (Yukito Shinohara et al. MRS Reliability Study Group, reliability of modified Rankin Scale-Japanese version criteria and questionnaire) Blood was collected 7 days after the onset of cerebral infarction (indicated as “DAY7” in the table) for patients classified as 0-5 using the introduction of Stroke 2007; 29: 6-13) (each in Table 3) EDTA plasma was obtained.
The concentration of EPCR protein in the plasma was measured in the same manner as in Example 1, and the AUC value of the ROC curve was calculated from the measured values in the same manner as in Example 1. The results are shown in Table 3. It is shown that a value exceeding 0.7 at each time of blood collection is useful as a test method for determining the prognosis of a cerebral infarction patient.

Specifically, on the degree of injury (prognosis) 3 months after the onset of cerebral infarction, by comparing the concentration of EPCR protein in the plasma on the 7th day after the onset of the patients who become each rank after 3 months, the onset 3 months later, rank 2-5 patients, rank 0-1 patients, rank 3-5 patients, rank 0-2 patients, rank 4-5 patients, rank 0-3 patients It turned out that the test | inspection for discrimination can be performed.

Reference Example 1 Evaluation of cerebral infarction type using CD40 ligand protein marker and examination method for prognosis prediction Blood was collected from 111 cerebral infarction patients and 91 healthy subjects immediately after the onset of cerebral infarction and 7 days later. from Rules Based Medicine, Inc. ask the immunoassay (human Map ^TM v1.6 method) the amino acid sequence as shown in Entry Name CD40L_HUMAN of human CD40 ligand (UniProtKB in EDTA plasma with the then obtaining the EDTA plasma The protein concentration was measured. Next, if the measured value falls below the lower limit of detection, the value half of the lower limit of detection is complemented, and if the measured value exceeds the upper limit of detection, a value twice the upper limit of detection is complemented. In order to know whether there is a difference between cerebral infarction patients and healthy individuals, AUC values of ROC curves were calculated according to the method described in Fawcett, T. (2004) ROC Graphs: Notes and Practical Considerations for Researchers. The results are shown in Table 4. Since it exceeded 0.7 at each blood sampling time point, it was shown that CD40 ligand is useful as a test method for discriminating between cerebral infarction patients and healthy subjects.

Next, blood was collected from 173 patients with cerebral infarction immediately after the onset of cerebral infarction and 7 days later with the above CD40 ligand, and after obtaining EDTA plasma, an immunoassay method (Human Map ^TM v1 was requested from Rules Based Medicine). .6) was used to measure the protein concentration of human CD40 ligand (consisting of the amino acid sequence indicated by Entry Name CD40L_HUMAN of UniProtKB) in EDTA plasma. Next, if the measured value falls below the lower limit of detection, the value half of the lower limit of detection is complemented, and if the measured value exceeds the upper limit of detection, a value twice the upper limit of detection is complemented. In order to know whether there is a difference between cerebral infarction patients and healthy individuals, AUC values of ROC curves were calculated according to the method described in Fawcett, T. (2004) ROC Graphs: Notes and Practical Considerations for Researchers.
Regarding the measured values immediately after the onset of cerebral infarction, the analysis of the disease type diagnosis was performed in the grouping corresponding to Table 2 above. As a result, AUC was found in all of the ^seven group combinations ((2 ⁷ -2) ÷ 2 = 63). The value was below 0.7. From this, it was found that some biomarkers that are different between cerebral infarction and healthy subjects are not used for diagnosis of disease type.
Similarly to Table 4, blood was collected 7 days after the onset of cerebral infarction and analyzed for prognosis. As a result, the AUC value did not exceed 0.7 for the three combinations. From this, it was found that some biomarkers that are different between cerebral infarction and healthy subjects are not used for prognosis diagnosis.

According to the method of the present invention, diagnosis of cerebral infarction and prognosis prediction can be performed, which is useful in the medical and diagnostic fields.

Claims

A test method for diagnosing a cerebral infarction pathology in the animal, comprising measuring the amount of Endothelial Protein C receptor protein in a blood sample collected from the test animal.
2. The diagnosis according to claim 1, wherein the disease type diagnosis is one of lacunar infarction, atherothrombotic, cardiogenic cerebral embolism, aortic cerebral embolism, Branch 、 atheromatous disease, arterial dissection, and unclassifiable. Inspection method.
A test method for predicting the prognosis of cerebral infarction in an animal, comprising the step of measuring the amount of Endothelial Protein C receptor protein in a blood sample collected from the test animal.
The test method for predicting the prognosis of cerebral infarction according to claim 3, wherein the blood sample collected from the test animal is collected from the test animal on the seventh day of the onset of cerebral infarction. .