Methods for detecting ischemic stroke

Abstract

The object of this invention is to provide a new method for detecting stroke. [Solution] The problem is solved by a method for detecting ischemic stroke, which includes the step of obtaining a measurement value of peroxiredoxin in a blood sample taken from a subject, and which suggests that the subject has an ischemic stroke when the measurement value is higher than a predetermined reference value.

Description

Technical Field

[0007] , , , ,

[0001] This specification discloses a method for detecting ischemic stroke.

Background Art

[0002] Patent Document 1 describes diagnosing cerebral infarction based on the measured value of peroxiredoxin.

[0003] Non-Patent Document 1 describes that peroxiredoxin 6 is strongly expressed in the cerebral infarction focus 12 hours after the onset of cerebral ischemia, and an increase in peroxiredoxin 6 is also observed in the extracellular fluid of the brain tissue of cerebral infarction patients.

[0004] Non-Patent Document 2 reports that the plasma level of heart-type fatty acid-binding protein (H-FABP) increases in the acute phase of ischemic stroke, and these increased levels are shown to be related to clinical severity.

Prior Art Documents

Patent Documents

[0008] The present invention includes the following embodiments. Section 1. The process includes obtaining a measurement of peroxiredoxin in a blood sample taken from a subject, If the measured value is higher than a predetermined reference value, it suggests that the subject has an ischemic stroke. A method for detecting ischemic stroke. Section 2. The method according to item 1, wherein the blood sample is a blood sample collected within 10 hours after the subject suffered an ischemic stroke. Section 3. The method according to claim 1, wherein the peroxiredoxin is peroxiredoxin 6. Section 4. A biomarker for diagnosing ischemic stroke, consisting of a polypeptide having the amino acid sequence of peroxiredoxin protein. Section 5. A diagnostic reagent for ischemic stroke, comprising an anti-peroxiredoxin antibody, for use in the method described in item 1. Section 6. A diagnostic kit for ischemic stroke used in the method described in item 1, comprising a first anti-peroxiredoxin antibody, a solid phase, a second anti-peroxiredoxin antibody, and a labeling substance. [Effects of the Invention]

[0009] This can provide a new method for detecting ischemic stroke. [Brief explanation of the drawing]

[0010] [Figure 1] This is a schematic diagram showing an example of a test kit. [Figure 2] This is a box plot showing the concentration of peroxiredoxin 6 in the blood of patients with cerebral infarction (cerebral thrombosis) and healthy individuals. [Figure 3] The ROC curve of the concentration of peroxiredoxin 6 in the blood of patients with cerebral infarction (cerebral thrombosis) is shown.

Mode for Carrying Out the Invention

[0011] 1. Method for Detecting Stroke One embodiment relates to a method for detecting stroke (hereinafter may be simply referred to as "method").

[0012] The method includes a step of obtaining a measured value of peroxiredoxin in a blood sample collected from a subject (hereinafter may be simply referred to as "measured value"). When the measured value is higher than a predetermined reference value, it suggests that the subject has ischemic stroke. When the measured value is higher than a predetermined reference value, it may be determined that the subject has ischemic stroke, and when the measured value is below the predetermined reference value, it may be determined that the subject does not have ischemic stroke.

[0013] In this specification, stroke may include ischemic stroke (also called cerebral infarction) such as cerebral embolism, cerebral thrombosis, transient ischemic attack, etc.; cerebral hemorrhage; subarachnoid hemorrhage; cerebral aneurysm, etc. The method is preferably used for detecting ischemic stroke.

[0014] The "subject" may be an individual with a history of stroke or other thrombosis (including arteriosclerosis), or an individual without a history of stroke or other thrombosis. Also, the subject may be an individual with subjective symptoms such as headache, paralysis of a part of the body, numbness of a part of the body, etc., or an asymptomatic individual. Furthermore, the subject includes a test body suspected of having stroke by medical interview, angiography, MRI examination, CT examination, etc.

[0015] In the present invention, the "individual" is not particularly limited, but includes humans and non-human mammals. Examples of non-human mammals include cows, horses, sheep, goats, pigs, dogs, cats, rabbits, monkeys, etc. Preferably, it is a human. Also, the age and gender of the individual are not limited.

[0016] The "blood sample" refers to blood (whole blood) collected from a subject, serum or plasma prepared from the blood. More preferably, it is serum or plasma, and even more preferably serum. The type of anticoagulant used when collecting plasma is not particularly limited. The blood sample of the subject used for measurement and the blood sample for determining a predetermined reference value may be of the same type or different types, but it is preferable that they are of the same type. Also, when plasma is used as the blood sample, the plasma for determining a predetermined reference value is preferably prepared from blood collected using the same anticoagulant as the plasma of the subject. Further, the blood sample may be fresh or preserved.

[0017] Preferably, the above blood sample is collected within 10 hours, preferably within 4 hours, more preferably within 3 hours, and even more preferably within 2 hours after the subject develops the above stroke, and even more preferably within 1 hour after the onset. When serum or plasma is used as the blood sample, it is preferably prepared from blood collected within 2 hours after the onset, and even more preferably from blood collected within 1 hour after the onset. The blood sample may be used for measurement immediately after collection, or may be used for measurement after being frozen and stored after collection.

[0018] Also, when storing the blood sample, it can be frozen and stored, but it is also possible to immediately separate the plasma or serum after blood collection from the subject and freeze and store the separated plasma or serum. <OO00105> Peroxiredoxin is an antioxidant enzyme having peroxidase activity that reduces hydrogen peroxide to water in the presence of a suitable electron donor. In the present invention, the peroxiredoxin to be measured is preferably, and in this specification, not limited as long as it is a member of the peroxiredoxin family present in mammalian cells. Preferably, it is peroxiredoxin group 1 (PRDX 1 variants 1-4), peroxiredoxin 5 (PRDX5), and peroxiredoxin 6 (PRDX6), and more preferably peroxiredoxin 6. Peroxiredoxin 6 (PRDX6) is a 25 kDa protein consisting of 224 amino acids (SEQ ID NO: 1).

[0020] In this specification, when referring specifically to the peroxiredoxin protein, the term "peroxiredoxin protein" may be used, and when referring specifically to the gene encoding peroxiredoxin, the term "peroxiredoxin gene" may be used.

[0021] "Peroxiredoxin protein measurement value" refers to a value that reflects the amount or concentration of peroxiredoxin protein. When the measurement value is expressed as "amount," it may be expressed as moles or mass, but it is preferable to express it as mass. When the value is expressed as "concentration," it may be expressed as molar concentration or as the ratio of mass per a certain volume of blood sample (mass / volume), but it is preferable to express it as mass / volume. In addition to the above, the intensity of signals such as fluorescence or luminescence may also be used as a value that reflects the amount or concentration.

[0022] "Prescribed reference value" refers to the reference value for peroxiredoxin measurement. This reference value can be determined based on the peroxiredoxin measurement in blood samples from individuals who have not suffered a stroke and / or the peroxiredoxin measurement in blood samples from individuals who have suffered a stroke.

[0023] For example, peroxiredoxin levels are measured using blood samples from multiple individuals who have experienced a stroke, and peroxiredoxin levels are measured using blood samples from multiple individuals who have not experienced a stroke. Based on these multiple values, the value that most accurately classifies positive and negative results can be set as the "reference value." Here, the "value that most accurately classifies results" can be appropriately set based on indicators such as sensitivity, specificity, positive predictive value, and negative predictive value, depending on the purpose of the test.

[0024] For example, in one embodiment, the lowest peroxiredoxin measurement value among the measurements obtained from blood samples from multiple individuals who have suffered a stroke may be used as the reference value. For example, in cases where it is desirable to reduce false positives as much as possible, such as in a screening test, this reference value can be suitably used.

[0025] Furthermore, in another embodiment, when determining a reference value based on peroxiredoxin measurements in blood samples from individuals who have not experienced a stroke, the highest peroxiredoxin measurement from blood samples of multiple individuals who have not experienced a stroke can be determined as the reference value. For example, if it is desirable to minimize false negatives, this reference value can be suitably used.

[0026] Alternatively, the reference value may be the peroxiredoxin measurement itself in a blood sample from an individual who has not suffered a stroke, or the mean, median, or mode of multiple peroxiredoxin measurements from an individual who has not suffered a stroke. Alternatively, the determination can be based on peroxiredoxin levels measured in multiple blood samples from individuals who have not experienced a stroke.

[0027] In this case, the reference value can be the average value of the peroxiredoxin measurements in the multiple blood samples, preferably the value obtained by subtracting the standard deviation of the peroxiredoxin measurements in the multiple blood samples by 1 from the average value, or more preferably the value obtained by subtracting the standard deviation by 2 from the average value.

[0028] Furthermore, as a reference value, past peroxiredoxin measurements (which may be a single value, or the mean, median, or mode of multiple values) obtained from the same subject before the subject suffered a stroke can also be used.

[0029] When determining a reference value based on peroxiredoxin levels in blood samples from individuals who have not experienced a stroke and peroxiredoxin levels in blood samples from individuals who have experienced a stroke, the average of the peroxiredoxin levels in a blood sample from one individual who has not experienced a stroke and the peroxiredoxin levels in a blood sample from one individual who has experienced a stroke can be used as the reference value. Alternatively, the reference value can be the average of the peroxiredoxin levels in multiple blood samples from individuals who have not experienced a stroke and the average of the peroxiredoxin levels in multiple blood samples from individuals who have experienced a stroke. In another embodiment, individuals who have not experienced a stroke and individuals who have experienced a stroke can be grouped together, and the median of the peroxiredoxin levels in the blood samples of this group can be used as the reference value.

[0030] In yet another embodiment, in the method for determining the above reference value, the measured value of peroxiredoxin in a blood sample of a healthy individual may be used instead of the measured value of peroxiredoxin in a blood sample of an individual that has not suffered a stroke. These reference values ​​may be determined when obtaining the peroxiredoxin measurement from the subject's blood sample, or they may be determined in advance.

[0031] A "healthy individual" is not particularly limited. Preferably, it refers to a human or non-human mammal as described in the "Individual" section, which does not show abnormal data in biochemical tests, blood tests, urine tests, serum tests, physiological tests, etc. There are no particular restrictions on the age or sex of a healthy individual.

[0032] "Individuals who have not suffered a stroke" are not particularly limited. Preferably, they are humans or non-human mammals as described in the "Individual" section, and who cannot be diagnosed as having suffered a stroke according to known diagnostic criteria.

[0033] "Multiple blood samples" refers to two or more, preferably five or more, and more preferably ten or more blood samples. These may be blood samples collected from different individuals, or they may be multiple blood samples from the same individual collected at different times. "Multiple values" refers to two or more, preferably five or more, and more preferably ten or more, measured values ​​of peroxiredoxin. "Multiple individuals" means two or more individuals, preferably five or more, and more preferably ten or more.

[0034] To determine the reference value, the individual from which the peroxiredoxin measurement is obtained does not necessarily have to be the same species, age, sex, etc. as the subject, but it is preferable that they be of the same species. Furthermore, it is preferable that the individual is of the same age and / or sex as the subject.

[0035] Peroxiredoxin protein levels can be measured using an antibody that specifically binds to peroxiredoxin protein, i.e., an anti-peroxiredoxin antibody. Alternatively, the measurement may be obtained using the "stroke test reagent containing anti-peroxiredoxin antibody" or "stroke test kit" described later. The order in which the blood sample and the anti-peroxiredoxin antibody are mixed is not particularly limited; they may be mixed substantially simultaneously or sequentially.

[0036] In this embodiment, a complex is formed first between the anti-peroxiredoxin antibody and the peroxiredoxin protein in the blood sample, and then the complex is immobilized on a solid phase. Alternatively, the anti-peroxiredoxin antibody can be immobilized on a solid phase beforehand, and then the immobilized anti-peroxiredoxin antibody and the peroxiredoxin protein in the blood sample can form a complex. More preferably, the complex is formed first, and then the complex is immobilized on the solid phase. The amount or concentration of peroxiredoxin protein contained in the blood sample can then be measured by detecting the immobilized complex on the solid phase or the complex formed on the solid phase using a method known in this art.

[0037] When first forming a complex between an anti-peroxiredoxin antibody and the peroxiredoxin protein in a blood sample, and then immobilizing the complex on a solid phase, the anti-peroxiredoxin antibody modified with biotin or the like is brought into contact with the peroxiredoxin protein in the blood sample to form the complex. By pre-binding avidins to the solid phase, the complex can be immobilized on the solid phase via the binding of biotin and avidins.

[0038] Furthermore, when immobilizing the anti-peroxiredoxin antibody on a solid phase in advance, the manner of immobilization of the anti-peroxiredoxin antibody on the solid phase is not particularly limited. For example, the anti-peroxiredoxin antibody and the solid phase may be directly bound, or they may be indirectly bound via another substance. Examples of direct binding include physical adsorption. Examples of indirect binding include binding via a combination of biotin and avidin or streptavidin (hereinafter also referred to as "avidins"). In this case, by modifying the anti-peroxiredoxin antibody with biotin in advance and pre-binding avidins to the solid phase, the anti-peroxiredoxin antibody and the solid phase can be indirectly bound via the binding of biotin and avidins. In this embodiment, it is preferable that the binding of the anti-peroxiredoxin antibody and the solid phase is indirect, via biotin and avidins.

[0039] The solid phase material is not particularly limited and can be selected from, for example, organic polymers, inorganic compounds, and biopolymers. Examples of organic polymers include latex, polystyrene, and polypropylene. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, and ferrite, etc.), silica, alumina, and glass. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, and cellulose. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited and can be, for example, particles, films, microplates, microtubes, or test tubes. Among these, particles are preferred, and magnetic particles are particularly preferred.

[0040] In this process, after the formation of the complex, preferably after complex formation and before detection of the labeled substance, B / F separation may be performed to remove unreacted free components that have not formed a complex. Unreacted free components refer to components that do not constitute a complex. Examples include anti-peroxiredoxin antibodies that did not bind to the peroxiredoxin protein. The means of B / F separation are not particularly limited, but if the solid phase is particles, B / F separation can be performed by recovering only the solid phase that has captured the complex by centrifugation. If the solid phase is a container such as a microplate or microtube, B / F separation can be performed by removing the liquid containing the unreacted free components. If the solid phase is magnetic particles, B / F separation can be performed by magnetically constraining the magnetic particles with a magnet and then aspirating and removing the liquid containing the unreacted free components with a nozzle. This method is preferred from the viewpoint of automation. After removing the unreacted free components, the solid phase that has captured the complex may be washed with a suitable aqueous medium such as PBS.

[0041] In this process, the detection of the complex can be performed using an anti-peroxiredoxin antibody labeled with a labeling substance, or using an unlabeled anti-peroxiredoxin antibody and an anti-immunoglobulin antibody labeled with a labeling substance that can bind to the unlabeled anti-peroxiredoxin antibody, but it is preferable to use a labeled anti-peroxiredoxin antibody. Furthermore, it is preferable that the epitope in the peroxiredoxin protein of the labeled anti-peroxiredoxin antibody is different from the epitope in the peroxiredoxin protein of the anti-peroxiredoxin antibody that binds to the solid phase.

[0042] The labeling substance used in labeled anti-peroxiredoxin antibodies or labeled anti-immunoglobulin antibodies is not particularly limited, as long as it produces a detectable signal. For example, it may be a substance that generates a signal itself (hereinafter also referred to as a "signal-generating substance"), or it may be a substance that generates a signal by catalyzing the reaction of other substances. Examples of signal-generating substances include fluorescent substances and radioisotopes. Examples of substances that generate a detectable signal by catalyzing the reaction of other substances include enzymes. Examples of enzymes include alkaline phosphatase, peroxidase, β-galactosidase, and luciferase. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor®, and fluorescent proteins such as GFP. Examples of radioisotopes include 125I, 14C, and 32P. Among these, enzymes are preferred as labeling substances, and alkaline phosphatase is particularly preferred.

[0043] Labeled anti-peroxiredoxin antibodies are obtained by labeling anti-peroxiredoxin antibodies with the above-mentioned labeling substance using a labeling method known in this technology. Alternatively, they may be labeled using a commercially available labeling kit. Labeled immunoglobulin antibodies may be obtained using the same method as for labeling anti-peroxiredoxin antibodies, or commercially available antibodies may be used.

[0044] In this process, the amount of peroxiredoxin in a blood sample can be measured by detecting the signal generated by the labeling substance of the labeled anti-peroxiredoxin antibody contained in the complex. Here, "detecting the signal" includes qualitatively detecting the presence or absence of a signal, quantitatively detecting the signal intensity, and semi-quantitatively detecting the signal intensity. Semi-quantitative detection means indicating the signal intensity in stages, such as "no signal," "weak," "medium," and "strong." In this process, it is preferable to detect the signal intensity quantitatively or semi-quantitatively.

[0045] The method for detecting the signal is publicly known in this technology. In this process, a measurement method can be appropriately selected according to the type of signal derived from the labeled substance. For example, if the labeled substance is an enzyme, the signal, such as light or color, generated by reacting the enzyme with a substrate can be measured using a known device such as a luminometer or spectrophotometer.

[0046] The enzyme substrate can be appropriately selected from known substrates depending on the type of enzyme. For example, when alkaline phosphatase is used as the enzyme, examples of substrates include chemiluminescent substrates such as CDP-Star® (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)trixyl[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium) and CSPD® (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, and p-nitrophenyl phosphate. CDP-Star® is particularly preferred. The luminescence of the substrate is preferably detected with a luminometer.

[0047] If the labeling material is a radioactive isotope, the radiation signal can be measured using a known device such as a scintillation counter. If the labeling material is a fluorescent substance, the fluorescence signal can be measured using a known device such as a fluorescence microplate reader. The excitation wavelength and fluorescence wavelength can be appropriately determined depending on the type of fluorescent substance used.

[0048] The signal detection results can be used as a measurement of peroxiredoxin protein. For example, when quantitatively detecting the signal intensity, the measured signal intensity itself or a value calculated from said measured signal intensity can be used as a measurement of peroxiredoxin protein. Examples of values ​​calculated from the measured signal intensity include the value obtained by subtracting the measured signal intensity of a negative control sample from the measured signal intensity, the value obtained by dividing the measured signal intensity by the measured signal intensity of a positive control sample, and combinations thereof. Examples of negative control samples include blood samples from healthy individuals. Examples of positive control samples include blood samples containing peroxiredoxin protein in a predetermined amount or concentration.

[0049] The "anti-peroxiredoxin antibody" is not limited in any way as long as it specifically binds to the peroxiredoxin protein. Polyclonal antibodies, monoclonal antibodies, and their fragments (e.g., Fab, F(ab)2, etc.) obtained by immunizing non-human animals with peroxiredoxin protein or a part thereof as an antigen can be used. Furthermore, the class and subclass of the immunoglobulin are not particularly limited. Chimeric antibodies may also be used. Additionally, scFv, etc., may be used.

[0050] The peroxiredoxin protein used as an antigen for producing anti-peroxiredoxin antibodies is preferably a peroxiredoxin protein derived from humans (e.g., GenBank:NP_004896.1 [SEQ ID NO: 1]), mice (e.g., GenBank:AAP21829.1), rats (e.g., NCBI Reference Sequence:NP_446028.1), etc., and more preferably the human peroxiredoxin protein represented by SEQ ID NO: 1. The peroxiredoxin protein used as an antigen may be extracted from mammalian cells according to known methods, or it may be a recombinant protein obtained by recombinant genetic engineering technology. When a part of the peroxiredoxin protein is used as the antigen, a fragment obtained by digesting the peroxiredoxin protein with an enzyme or the like may be used, or a peptide having the same amino acid sequence as a part of the peroxiredoxin protein may be used as the antigen. Such peptides can be synthesized by known methods.

[0051] Furthermore, commercially available anti-peroxiredoxin antibodies, such as peroxiredoxin 6 antibody (GTX115262; GeneTex) that reacts with human peroxiredoxin protein, can also be used.

[0052] Alternatively, to obtain measurements of peroxiredoxin protein, commercially available ELISA kits for detecting human peroxiredoxin protein can be used, such as the ELISA Kit for Peroxiredoxin 6 (catalog number MBS2020865) from MyBioSource (San Diego, California, USA). When obtaining measurements of peroxiredoxin in blood samples using a commercially available kit, the results can be obtained according to the protocol provided with the kit.

[0053] 2. Test reagents for detecting stroke containing anti-peroxiredoxin antibodies The present invention provides a diagnostic reagent for detecting stroke, comprising an anti-peroxiredoxin antibody used in the method described in 1. above. The anti-peroxiredoxin antibody described in item 1 above may be used.

[0054] The test reagent of this embodiment only needs to contain at least one type of anti-peroxiredoxin antibody. If the anti-peroxiredoxin antibody is a polyclonal antibody, it may be a polyclonal antibody obtained by immunity with one antigen, or it may be a polyclonal antibody obtained by immunity with two or more antigens in parallel in the same individual. Alternatively, polyclonal antibodies obtained by inoculating two or more antigens into separate animals may be mixed. If the anti-peroxiredoxin antibody is a monoclonal antibody, it may be a monoclonal antibody produced from one hybridoma, or it may be a monoclonal antibody produced from two or more hybridomas, and may contain two or more monoclonal antibodies, each recognizing the same or different epitopes. Alternatively, it may contain a mixture of one or more polyclonal antibodies and one or more monoclonal antibodies.

[0055] The form of the anti-peroxiredoxin antibody contained in the test reagent is not particularly limited and may be in a dry or liquid state, such as antiserum or ascites fluid containing the anti-peroxiredoxin antibody. Alternatively, the form of the anti-peroxiredoxin antibody may be in a dry or aqueous solution state, such as purified anti-peroxiredoxin antibody, an immunoglobulin fraction containing the anti-peroxiredoxin antibody, or an IgG fraction containing the anti-peroxiredoxin antibody.

[0056] If the above-mentioned form is an antiserum or ascites containing an anti-peroxiredoxin antibody in a dry or liquid state, it may further contain at least one of the following: a stabilizer such as β-mercaptoethanol or DTT; a protective agent such as albumin; a surfactant such as polyoxyethylene(20) sorbitan monolaurate or polyoxyethylene(10) octylphenyl ether; or a preservative such as sodium azide. Furthermore, if the form of the anti-peroxiredoxin antibody is a purified anti-peroxiredoxin antibody, an immunoglobulin fraction containing an anti-peroxiredoxin antibody, or an IgG fraction containing an anti-peroxiredoxin antibody in a dry or aqueous solution state, it may further contain at least one of the following: a buffer component such as phosphate buffer; a stabilizer such as β-mercaptoethanol or DTT; a protective agent such as albumin; a salt such as sodium chloride; a surfactant such as polyoxyethylene(20) sorbitan monolaurate or polyoxyethylene(10) octylphenyl ether; or a preservative such as sodium azide.

[0057] In the present invention, the anti-peroxiredoxin antibody may be unlabeled or labeled with biotin or the labeling substance described above, but it is preferable that it be labeled with biotin or the labeling substance described above. The labeling substance can be one of those exemplified in 1. above, but alkaline phosphatase is preferred. In addition, in the present invention, the anti-peroxiredoxin antibody may be provided in a state immobilized on a solid phase surface or the like. The solid phase and immobilization are as exemplified in section 1. above. Magnetic beads are preferred as the solid phase.

[0058] 3. Stroke screening kit The present invention is a stroke test kit used in a method for detecting stroke patients, comprising the above-mentioned "2. Test reagent for detecting stroke containing an anti-peroxiredoxin antibody".

[0059] More specifically, the present invention relates to a stroke test kit comprising a test reagent containing a biotin-labeled anti-peroxiredoxin antibody, and a test reagent containing an anti-peroxiredoxin antibody labeled with a labeling substance. The labeling substance is preferably alkaline phosphatase. This embodiment may further include a solid phase, preferably a solid phase conjugated with avidins, and more preferably magnetic beads conjugated with avidins. This embodiment may also include a substrate. The substrate is preferably CDP-Star®. The details of the labeling substance, solid phase, and substrate are as described in section 1 above, and that description may be incorporated herein.

[0060] The test kit of this embodiment preferably contains two types of anti-peroxiredoxin antibodies (first anti-peroxiredoxin antibody and second anti-peroxiredoxin antibody) that bind to different epitopes of the peroxiredoxin protein. In this case, a complex of the first anti-peroxiredoxin antibody, peroxiredoxin, the second anti-peroxiredoxin antibody, and a labeling substance is formed on the solid phase. Such a detection method is generally called sandwich ELISA. In this complex, the first anti-peroxiredoxin antibody is immobilized on the solid phase, and the second anti-peroxiredoxin antibody is directly or indirectly bound to the labeling substance. Here, indirect binding of the second anti-peroxiredoxin antibody and the labeling substance means that the labeling substance is bound to the second anti-peroxiredoxin antibody via an antibody or the like. For example, a labeling antibody that recognizes the second anti-peroxiredoxin antibody is bound to the second anti-peroxiredoxin antibody.

[0061] If the first anti-peroxiredoxin antibody is pre-bound to a solid phase, the test kit of this embodiment may include a solid phase immobilized with the first anti-peroxiredoxin antibody, a second anti-peroxiredoxin antibody, and a labeling substance. The second anti-peroxiredoxin antibody and the labeling substance may be housed in separate containers or in the same container. If the labeling substance is an enzyme and the test kit further includes a substrate, the enzyme and substrate must be housed in separate containers. When provided to the user, at least two of the solid phase, the second anti-peroxiredoxin antibody, and the labeling substance may be packaged together or separately.

[0062] If the first anti-peroxiredoxin antibody is not pre-bound to the solid phase, the test kit of this embodiment may include the solid phase, the first anti-peroxiredoxin antibody, the second anti-peroxiredoxin antibody, and a labeling substance. At least two of the first anti-peroxiredoxin antibody, the second anti-peroxiredoxin antibody, and the labeling substance may be contained in the same container or in separate containers. If the labeling substance is an enzyme and the test kit further contains a substrate, the enzyme and the substrate must be contained in separate containers. When provided to the user, the solid phase, the first anti-peroxiredoxin antibody, the second anti-peroxiredoxin antibody, and the labeling substance may be packaged together or in separate packaging.

[0063] The above-described test kit may be provided to the user as a kit as shown in Figure 1. The stroke test kit 50 includes an outer box 55, a first container 51 containing a plurality of magnetic particles as a solid phase, a second container 52 containing a first anti-peroxiredoxin antibody that can bind to the solid phase, a third container 53 containing an enzyme-labeled second anti-peroxiredoxin antibody, and an instruction manual 54 for the test kit. The instruction manual 54 may include instructions on how to handle the test kit, storage conditions, expiration date, etc. Containers containing substrate reagents, containers containing aqueous cleaning media, etc., may be included in the outer box 55. [Examples]

[0064] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0065] 1. Method Peroxiredoxin 6 levels were measured in nine patients hospitalized for cerebral infarction (cerebral thrombosis). These were collected at admission, serum was separated, and frozen at -80°C. Peroxiredoxin 6 levels were also measured in similarly processed frozen serum from 10 healthy individuals (NV). Peroxiredoxin 6 levels were measured using the Human Peroxiredoxin 6 (PRDX6) ELISA Kit from COSABIO. Statistical analysis was performed using the non-parametric Mann-Whitney method. Comparisons between the two groups were conducted using the Kulu-Wallis test, and AUC was calculated using judgment analysis.

[0066] 2.Results The median serum peroxiredoxin-6 concentration in patients with cerebral thrombosis was 318 ng / ml, compared to 53 pg / ml in healthy individuals (NV), showing significantly higher levels in patients with cerebral thrombosis compared to healthy individuals (p<0.01, Table 1, Figure 2). Furthermore, the AUC calculated by judgment analysis showed high specificity and sensitivity at 0.89 (Figure 3). These results indicate that measuring blood peroxiredoxin 6 levels is useful in diagnosing cerebral thrombosis.

[0067] [Table 1]

Claims

1. The process includes obtaining a measurement of peroxiredoxin in a blood sample taken from a subject, If the measured value is higher than a predetermined reference value, it suggests that the subject has an ischemic stroke. A method for detecting ischemic stroke.

2. The method according to claim 1, wherein the blood sample is a blood sample collected within 10 hours after the subject suffered an ischemic stroke.

3. The method according to claim 1, wherein the peroxiredoxin is peroxiredoxin 6.

4. A biomarker for diagnosing ischemic stroke, consisting of a polypeptide having the amino acid sequence of peroxiredoxin protein.

5. A diagnostic reagent for ischemic stroke used in the method according to claim 1, comprising an anti-peroxiredoxin antibody.

6. A diagnostic kit for ischemic stroke used in the method according to claim 1, comprising a first anti-peroxiredoxin antibody, a solid phase, a second anti-peroxiredoxin antibody, and a labeling substance.

Images