Methods, kits, biomarkers, and biomarker sets to assist in the diagnosis of neurodegenerative diseases.

Abstract

To provide a method for assisting in the diagnosis of neurodegenerative diseases, and a method for assisting in the differentiation between Alzheimer's disease and Parkinson's disease; a diagnostic kit for neurodegenerative diseases, and a differential diagnosis kit for Alzheimer's disease and Parkinson's disease; and a biomarker for the diagnosis of neurodegenerative diseases, a set of biomarkers for the diagnosis of neurodegenerative diseases, and a set of biomarkers for the differentiation between Alzheimer's disease and Parkinson's disease. [Solution] A method to assist in the diagnosis of neurodegenerative diseases, comprising detecting the amount of CD41 or CD61 in extracellular vesicles in a test sample, wherein an indicator including the amount of CD41 or CD61 is lower than that of subjects without neurodegenerative diseases, which suggests a neurodegenerative disease.

Description

Technical Field

[0001] The present invention relates to a method for assisting in the diagnosis of neurodegenerative diseases using an index including the amount of CD41 or the amount of CD61 in extracellular vesicles. The present invention further relates to a method for assisting in the discrimination between Alzheimer's disease and Parkinson's disease using the ratio of the amount of CD41 to the amount of CD9 or the ratio of the amount of CD61 to the amount of CD9 in extracellular vesicles. The present invention further relates to a diagnostic kit for neurodegenerative diseases and a discrimination kit for Alzheimer's disease and Parkinson's disease. The present invention further relates to a biomarker for diagnosing neurodegenerative diseases, a biomarker set for diagnosing neurodegenerative diseases, and a biomarker set for discriminating between Alzheimer's disease and Parkinson's disease.

Background Art

[0002] Parkinson's disease (PD) is a neurodegenerative disease in which neurons existing in the substantia nigra in the brain degenerate and dopamine in the striatum is lacking, making it difficult to move smoothly. The morbidity rate of Parkinson's disease is as high as 100 to 150 per 100,000 people, and the morbidity rate increases with aging. Therefore, the number of patients is rapidly increasing with the aging of the population.

[0003] Currently, there is no radical treatment for Parkinson's disease. However, it is known that the administration of dopamine precursor therapeutic agents such as levodopa (L-Dopa) and dopamine agonists can supplement the reduced dopamine action in the brain and improve motor symptoms.

[0004] As a biomarker for obtaining objective data on the pathogenesis of Parkinson's disease, for example, Patent Document 1 describes a method for assisting in the diagnosis of Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanin as an index.

[0005] Patent Document 2 describes that the amount of extravalent protein (CD41) detected by the sandwich enzyme-linked immunosorbent assay (sandwich ELISA) method using solid-phase Tim4 protein-labeled anti-CD41 antibody is increased in blood samples from patients with neurodegenerative diseases compared to healthy individuals. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2021 / 107155 publication [Patent Document 2] Japanese Patent Publication No. 2021-12189 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a method to assist in the diagnosis of neurodegenerative diseases and a method to assist in differentiating between Alzheimer's disease and Parkinson's disease. The present invention further aims to provide a diagnostic kit for neurodegenerative diseases and a kit for differentiating between Alzheimer's disease and Parkinson's disease. The present invention further aims to provide a biomarker for the diagnosis of neurodegenerative diseases, a set of biomarkers for the diagnosis of neurodegenerative diseases, and a set of biomarkers for differentiating between Alzheimer's disease and Parkinson's disease. [Means for solving the problem]

[0008] The inventors have found that by detecting the amount of CD41 in extracellular vesicles in a test sample using a sandwich ELISA method with Tim4 protein and an anti-CD41 antibody, healthy individuals and Parkinson's disease patients can be differentiated. Furthermore, the inventors have found that by the ratio of the amount of CD41 to the amount of CD9 in extracellular vesicles in a test sample, Alzheimer's disease patients and Parkinson's disease patients can be differentiated. This invention was completed based on the above findings.

[0009] The present invention provides the following: <1> A method for assisting the diagnosis of neurodegenerative diseases, comprising detecting the amount of CD41 or CD61 in extracellular vesicles in a test sample, wherein an indicator including the amount of CD41 or CD61 is lower than that of subjects without neurodegenerative diseases, which suggests the presence of a neurodegenerative disease. <2> The above detection further includes detecting the amount of CD9 in extracellular vesicles, wherein the index including the amount of CD41 or CD61 is the ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9. <1> Methods used. <3> The above extracellular vesicles are extracellular vesicles containing phosphatidylserine. <1> or <2> Methods used. <4> The above test sample is a blood sample or cerebrospinal fluid. <1> from <3> The method described in any one of the following. <5> The amount of CD41-positive extracellular vesicles or the amount of CD61-positive extracellular vesicles are used as the amount of CD41 or CD61, respectively. <1> from <4> The method described in any one of the following. <6> The neurodegenerative disease is Parkinson's disease or Alzheimer's disease. <1> from <5> The method described in any one of the following. <7> A method to assist in differentiating between Alzheimer's disease and Parkinson's disease, comprising detecting the amount of CD41 or CD61 in extracellular vesicles and the amount of CD9 in extracellular vesicles in a test sample, wherein a lower ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9, than that of subjects other than those with Parkinson's disease, suggests Parkinson's disease. <8> The above extracellular vesicles are extracellular vesicles containing phosphatidylserine. <7> Methods used. <9> The above test sample is a blood sample or cerebrospinal fluid. <7> or <8> Methods used. <10> The amounts of CD41-positive extracellular vesicles, CD61-positive extracellular vesicles, or CD9-positive extracellular vesicles are used as the amounts of CD41, CD61, or CD9, respectively. <7> from <9> The method described in any one of the following. <11> A diagnostic kit for neurodegenerative diseases containing a substance that binds to CD41 or a substance that binds to CD61. <12> Furthermore, it contains a substance that binds to CD9, <11> The kit described above. <13> Furthermore, it contains a substance that binds to phosphatidylserine, <11> or <12> The kit described above. <14> A diagnostic kit for differentiating between Alzheimer's disease and Parkinson's disease, containing substances that bind to CD41, or substances that bind to CD61, and substances that bind to CD9. <15> Furthermore, it contains a substance that binds to phosphatidylserine, <14> The identification kit described above. <16> A diagnostic biomarker for neurodegenerative diseases, comprising extracellular vesicles containing CD41 or CD61. <17> Extracellular vesicles having CD41, or extracellular vesicles having CD61, and A set of diagnostic biomarkers for neurodegenerative diseases, containing extracellular vesicles with CD9. <18> Extracellular vesicles having CD41, or extracellular vesicles having CD61, and A set of biomarkers containing extracellular vesicles with CD9, used to differentiate between Alzheimer's disease and Parkinson's disease.

[0010] Use of substances that bind to CD41 or CD61 for the manufacture of diagnostic kits for neurodegenerative diseases. The use of substances that bind to CD41, or substances that bind to CD61, and substances that bind to CD9, for the manufacture of a differential diagnosis kit for Alzheimer's disease and Parkinson's disease. <c>Use of extracellular vesicles containing CD41 or CD61 as diagnostic biomarkers for neurodegenerative diseases. <d>Use of extracellular vesicles containing CD41, or extracellular vesicles containing CD61, and extracellular vesicles containing CD9 as a set of diagnostic biomarkers for neurodegenerative diseases. <e>Use of extracellular vesicles containing CD41, or extracellular vesicles containing CD61, and extracellular vesicles containing CD9 as a set of biomarkers for differentiating Alzheimer's disease from Parkinson's disease. [Effects of the Invention]

[0011] According to the present invention, the diagnosis of neurodegenerative diseases can be assisted with high accuracy. According to the present invention, the differentiation between Alzheimer's disease and Parkinson's disease can be assisted with high accuracy. [Brief explanation of the drawing]

[0012] < / e> < / d> < / c> [Figure 1] Figure 1 shows a box-and-whisker plot created based on the sample measurement values in Example 1 and Comparative Example 1. [Figure 2] Figure 2 shows a box-and-whisker plot created based on the sample measurement values in Comparative Example 2 and Comparative Example 3. [Figure 3] Figure 3 shows a box-and-whisker plot created based on the corrected sample measurement values in Example 2 and Example 3. [Figure 4] Figure 4 shows a box-and-whisker plot created based on the corrected sample measurement values in Example 4 and Example 5. [Figure 5] Figure 5 shows a box-and-whisker plot created based on the sample measurement values in Example 6. [Figure 6] Figure 6 shows a box-and-whisker plot created based on the sample measurement values in Example 7. [Figure 7] Figure 7 shows a box-and-whisker plot created based on the sample measurement values in Example 8. [Figure 8] Figure 8 shows a box-and-whisker plot created based on the sample measurement values in Example 9.

Embodiments for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail. In this specification, "~" indicates a range including the numerical values described before and after it as the minimum value and the maximum value, respectively.

[0014] Extracellular vesicles are small membrane vesicles derived from cells and composed of a lipid bilayer. Examples of extracellular vesicles include those with a diameter of 20 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 50 nm to 500 nm, and particularly preferably 50 nm to 200 nm. Extracellular vesicles can be classified in various ways depending on their developmental origin and size, as described in, for example, Nature Reviews Immunology 9, 581-593 (August 2009) and "Obesity Research" Vol. 13 No. 2 2007 Topics Naoto Aoki et al. Specifically, examples include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, apoptotic bodies, and adiposomes, with exosomes and microvesicles being preferred, and exosomes being more preferred.

[0015] Exosomes are small membrane vesicles composed of a lipid bilayer, derived from cells. For example, they may have a diameter of 50 nm to 200 nm, preferably 50 nm to 150 nm, and more preferably 50 nm to 100 nm. Exosomes are thought to originate from late endosomes.

[0016] Microvesicles are small membrane vesicles derived from cells, composed of a lipid bilayer. Examples include those with a diameter of 100 nm to 1000 nm, preferably 100 nm to 800 nm, and more preferably 100 nm to 500 nm. Microvesicles are thought to originate from the cell membrane.

[0017] As the test sample, a biological sample derived from the subject can be used. Extracellular vesicles may be contained in a biological sample derived from a subject, or they may be isolated from a biological sample derived from a subject, with those isolated from a biological sample derived from a subject being preferred.

[0018] Any biological sample derived from a subject may contain extracellular vesicles, such as blood-derived samples including serum, plasma, whole blood, and buffy coat; and body fluid samples such as cerebrospinal fluid, urine, saliva, semen, pleural exudate, tears, sputum, mucus, lymph, ascites, pleural fluid, amniotic fluid, bladder lavage fluid, and bronchoalveolar lavage fluid. Blood samples or cerebrospinal fluid are preferred, serum, plasma, or cerebrospinal fluid are more preferred, serum or plasma are even more preferred, and plasma is particularly preferred. Furthermore, blood-derived samples are more useful because they place less burden on the subject during sample collection. Biological samples derived from subjects may be, for example, directly collected from subjects, or may have undergone pretreatment such as recovery, concentration, purification, isolation, dilution with buffer solutions, or sterilization by filtration. These pretreatments should be carried out appropriately according to standard procedures. Hereinafter, biological samples derived from subjects may be abbreviated as "biological samples."

[0019] Methods for isolating extracellular vesicles from biological samples can be carried out according to conventional methods and are not particularly limited. Examples of methods for isolating extracellular vesicles from biological samples include affinity methods (e.g., phosphatidylserine (PS) affinity method), fractionation centrifugation (e.g., pellet-down method, sucrose cushion method, ultracentrifugation such as density gradient centrifugation), immunoprecipitation, chromatography (e.g., ion exchange chromatography, gel permeation chromatography), density gradient methods (e.g., sucrose density gradient method), electrophoresis (e.g., organelle electrophoresis), magnetic separation (e.g., magnetically activated cell sorting (MACS) method), ultrafiltration concentration (e.g., nanomembrane ultrafiltration concentration method), Percoll gradient isolation, methods using microfluidic devices, and polyethylene glycol (PEG) precipitation. Affinity methods are preferred because they yield extracellular vesicles with high purity, or fractionation centrifugation is preferred because it theoretically allows for unbiased recovery. Affinity methods or ultracentrifugation are more preferred, and affinity methods are particularly preferred. Among affinity methods, the PS affinity method, which is affinity purification for phosphatidylserine, is preferred. Affinity methods and fractionation centrifugation can be carried out, for example, in accordance with the method described in WO2016 / 088689. These isolation methods may be performed using only one method or in combination of two or more methods. Furthermore, isolation using a single method may be repeated two or more times.

[0020] Examples of neurodegenerative diseases include, but are not limited to, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar degeneration, frontotemporal lobar degeneration, Lewy body dementia, multiple system atrophy, Huntington's disease, progressive supranuclear palsy, corticobasal degeneration, and Down syndrome. The neurodegenerative disease is preferably Parkinson's disease or Alzheimer's disease, and more preferably Parkinson's disease.

[0021] The subjects are not particularly limited and may include individuals suspected of having or being at risk of developing neurodegenerative diseases such as Parkinson's disease, as well as individuals whose status or risk of developing such neurodegenerative diseases is unknown. Examples of subjects include individuals diagnosed as potentially having a neurodegenerative disease such as Parkinson's disease based on diagnostic criteria or other methods, individuals diagnosed as being at risk of developing such neurodegenerative diseases such as Parkinson's disease based on diagnostic criteria or other methods, and other individuals suspected of having or being at risk of developing such neurodegenerative diseases such as Parkinson's disease; individuals who have not been diagnosed as potentially having a neurodegenerative disease such as Parkinson's disease based on diagnostic criteria or other methods; and individuals whose status or risk of developing such neurodegenerative diseases is unknown. Subjects may be individuals suspected of having or being at risk of developing Parkinson's disease or Alzheimer's disease. For example, this could include individuals diagnosed as potentially having Parkinson's disease or Alzheimer's disease based on diagnostic criteria or other methods, or individuals diagnosed as being at risk of developing Parkinson's disease or Alzheimer's disease based on diagnostic criteria or other methods.

[0022] Diagnostic criteria include, for example, medical history, metaiodobenzylguanidine (MIBG) myocardial scintigraphy, dopamine transporter scintigraphy, and other tests related to biomarkers recommended in Parkinson's disease treatment guidelines. On the other hand, tests related to candidate biomarkers for Parkinson's disease, such as alpha-synuclein seeds, are also included. Hereinafter, biomarkers for neurodegenerative diseases such as Parkinson's disease and candidate biomarkers may be collectively referred to as "other biomarkers for neurodegenerative diseases."

[0023] <Methods to assist in the diagnosis of neurodegenerative diseases, and methods to assist in differentiating between Alzheimer's disease and Parkinson's disease> The method for assisting the diagnosis of neurodegenerative diseases according to the present invention (hereinafter also referred to as the ND diagnostic assistance method) is a method for assisting the diagnosis of neurodegenerative diseases, comprising detecting the amount of CD41 or CD61 in extracellular vesicles in a test sample, wherein a lower level of an indicator including the amount of CD41 or CD61 than that of a subject without a neurodegenerative disease suggests a neurodegenerative disease. The ND diagnostic assistance method can assist in differentiating between neurodegenerative diseases and subjects without a neurodegenerative disease (preferably healthy individuals).

[0024] The present invention provides a method for assisting in the differentiation between Alzheimer's disease and Parkinson's disease (hereinafter also referred to as the AD / PD differentiation assistance method), which includes detecting the amount of CD41 or CD61 in extracellular vesicles and the amount of CD9 in extracellular vesicles in a test sample, wherein a ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9, that is lower than that of subjects other than those with Parkinson's disease suggests Parkinson's disease.

[0025] Detection in this invention includes qualitative, semi-quantitative, and quantitative methods, with quantitative detection being preferred.

[0026] In the context of ND diagnostic support methods and AD / PD differential diagnosis support methods, "quantity" can refer to mass, concentration, or number. Furthermore, "quantity" may also include measured values ​​that correlate with mass, concentration, or number (e.g., absorbance, change in absorbance, transmitted light, change in transmitted light, fluorescence intensity, change in fluorescence intensity, luminescence, change in luminescence, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, change in reflectance, refractive index, change in refractive index, etc.).

[0027] In the methods for assisting ND diagnosis and AD / PD differentiation, the extracellular vesicles are preferably extracellular vesicles containing phosphatidylserine.

[0028] In the ND diagnostic support method, detection further includes detecting the amount of CD9 in extracellular vesicles, and the index including the amount of CD41 or CD61 may be the ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9.

[0029] Specific examples of indicators that include the amount of CD41 or CD61 include: (1) The amount of CD41 or CD61 in extracellular vesicles; or (2) The ratio of the amount of CD41 or CD61 to the amount of CD9 in extracellular vesicles; We can list some examples.

[0030] In the ND diagnostic support method, the amount of CD41-positive extracellular vesicles or the amount of CD61-positive extracellular vesicles may be used as the amount of CD41 or CD61, respectively. In AD / PD differential diagnosis aids, the amount of CD41-positive extracellular vesicles, the amount of CD61-positive extracellular vesicles, or the amount of CD9-positive extracellular vesicles may be used as the amount of CD41, the amount of CD61, or the amount of CD9, respectively.

[0031] Methods for detecting the amount of CD41 or CD61 in extracellular vesicles in ND diagnostic aids, and methods for detecting the amount of CD41 or CD61 in extracellular vesicles and the amount of CD9 in extracellular vesicles in AD / PD differential diagnosis aids, are not particularly limited, as long as they are methods commonly used in this field.

[0032] Detection can be performed using, for example, immunological assays, mass spectrometry, or a combination thereof. In immunological assays, substances that bind to CD41, or substances that bind to CD61, and optionally substances that bind to CD9 and / or phosphatidylserine can be used. Among the above, immunological assays are preferred.

[0033] Examples of substances that bind to CD41, CD61, and CD9 include antibodies that bind to CD41, CD61, or CD9, proteins that bind to CD41, CD61, or CD9, such as lectins that bind to the sugar chains of CD41, CD61, or CD9, and nucleic acids that bind to CD41, CD61, or CD9. Among these, proteins that bind to CD41, CD61, or CD9 are preferred, and antibodies that bind to CD41, CD61, or CD9 are more preferred. The substances that bind to CD41, CD61, and CD9 are preferably substances that specifically bind to CD41, CD61, or CD9, respectively, and the same applies to preferred specific examples. The substances that bind to CD41, CD61, and CD9 may each be used individually or in combination of two or more, but it is preferable to use only one.

[0034] Examples of substances that bind to phosphatidylserine include proteins that bind to phosphatidylserine and nucleic acids that bind to phosphatidylserine, with proteins that bind to phosphatidylserine being preferred. Examples of proteins that bind to phosphatidylserine include antibodies that bind to phosphatidylserine and phosphatidylserine-affinity proteins, with phosphatidylserine-affinity proteins being preferred. Examples of antibodies that bind to phosphatidylserine include anti-phosphatidylserine antibody 1H6 (Merck KGaA). Examples of phosphatidylserine affinity proteins include Tim proteins such as Tim1 (T-cell immunoglobulin-mucin-domain 1), Tim2 (T-cell immunoglobulin-mucin-domain 2), Tim3 (T-cell immunoglobulin-mucin-domain 3), and Tim4 (T-cell immunoglobulin-mucin-domain 4); anti-phosphatidylserine antibodies; Annexin V; and MFG-E8. Tim proteins and anti-phosphatidylserine antibodies are preferred, with Tim proteins being more preferred. Among the Tim proteins, Tim1 and Tim4 are preferred, with Tim4 being more preferred. The substance that binds to phosphatidylserine is preferably a substance that specifically binds to phosphatidylserine, and the same applies to specific examples of preferred substances. The substance that binds to phosphatidylserine may be one type or two or more types, but it is preferable to use only one type.

[0035] The substances that bind to CD41, CD61, CD9, and phosphatidylserine may be commercially available products or those prepared appropriately by conventional methods.

[0036] Antibodies that bind to CD41, CD61, CD9, and phosphatidylserine may be polyclonal or monoclonal antibodies, and may be used individually or in appropriate combinations. Furthermore, antibodies that bind to CD41, CD61, CD9, and phosphatidylserine may be not only intact immunoglobulin molecules themselves, but also fragment antibodies such as Fab, F(ab')2, F(ab'), single-chain antibodies (single-chain Fv), diabodies, triabodies, tetrabodies, etc., which are fragments of immunoglobulin molecules that have the ability to bind to antigens, or synthetic antibodies. When preparing these antibodies, the method may be followed, for example, as described in "Immunoassay Methods" (edited by the Biochemical Measurement Research Group, Kodansha, 2014).

[0037] The substances that bind to CD41, CD61, CD9, and phosphatidylserine may be labeled with a labeling substance. Examples of labeling substances include enzymes such as peroxidase, microperoxidase, and alkaline phosphatase; radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P, and 35S; fluorescent substances such as fluorescein, fluorescein isothiocyanate (FITC), 4-methylumbelliferone, HiLyte, Alexa, CyDye, or rhodamine, or derivatives thereof; luminescent substances such as luciferin, luminol, and ruthenium complexes; substances that absorb in the ultraviolet region such as phenol, naphthol, or anthracene, or derivatives thereof; substances that have properties as spin labeling agents, such as compounds having an oxyl group, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl; and nanoparticles such as gold colloid and quantum dots. Enzymes and fluorescent substances are preferred. The method for labeling substances that bind to CD41, CD61, CD9, or phosphatidylserine with the above-mentioned labeling substances is not particularly limited and may be carried out according to known labeling methods. The measurement of these labeling substances may be carried out according to known measurement methods appropriate to the labeling substance.

[0038] A substance that binds to CD41, a substance that binds to CD61, a substance that binds to CD9, and a substance that binds to phosphatidylserine may be used as a primary affinity substance, or any one of these, and a secondary affinity substance (e.g., a secondary antibody) that specifically binds to the primary affinity substance may be used further. The secondary affinity substance may be labeled with a labeling substance, and is preferred, with a secondary antibody labeled with a labeling substance being more preferred. The labeling substance and labeling method are the same as those described above, and the preferences are also the same.

[0039] Furthermore, labeling with a labeling substance may utilize the binding of avidins and biotins by using a substance that binds to CD41, a substance that binds to CD61, a substance that binds to CD9, and a substance that binds to phosphatidylserine, to which one of avidins and biotins is bound, or by using a labeling substance to which the other of avidins and biotins is bound. Examples of biotins include biotin, iminobiotin, desthiobiotin, biocitin, and biotin sulfoxide, with biotin being preferred. Examples of avidins include avidin, tamavidin, tamavidin 2, and streptavidin, with streptavidin being preferred. Methods for binding one of avidins and biotins to a substance that binds to CD41, a substance that binds to CD61, a substance that binds to CD9, and a substance that binds to phosphatidylserine, and methods for binding the other of avidins and biotins to a labeling substance may be carried out according to conventional methods.

[0040] The substances that bind to CD41, CD61, CD9, and phosphatidylserine may be immobilized on a solid phase, and it is preferable that the substance that binds to phosphatidylserine is immobilized on a solid phase.

[0041] Examples of solid phases include synthetic polymer compounds such as latex, polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber, as well as inorganic substances such as porous glass, frosted glass, ceramics, alumina, silica gel, activated carbon, and metal oxides. Furthermore, two or more of these may be used in combination.

[0042] The shape of the solid phase is not particularly limited and can include, for example, microtiter plates (ELISA plates), beads, tubes (microtubes), particles, a dedicated tray with multiple tubes molded together, disc-shaped pieces, test tubes, etc.

[0043] The method for immobilizing substances that bind to CD41, CD61, CD9, and / or phosphatidylserine onto a solid phase is not particularly limited as long as it is a method commonly used in this field, and can be carried out according to conventional methods.

[0044] The combination of substances that bind to CD41, substances that bind to CD61, substances that bind to CD9, and / or substances that bind to phosphatidylserine is not particularly limited, but a combination of proteins that bind to CD41, proteins that bind to CD61, proteins that bind to CD9, and / or proteins that bind to phosphatidylserine is preferred.

[0045] For example, a combination of an antibody that specifically binds to CD41 or CD61, an antibody that specifically binds to CD9, and an antibody that specifically binds to Tim protein or phosphatidylserine could be used. A combination of an anti-CD41 antibody or anti-CD61 antibody, an anti-CD9 antibody, and Tim protein is more preferable. A combination of anti-CD41 antibody, anti-CD9 antibody, and Tim protein is particularly preferred.

[0046] Specific methods for measuring the amount of CD41 or CD61 in extracellular vesicles, and the amount of CD9 in extracellular vesicles, include, for example, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme fluorescence immunoassay (FEIA), enzyme fluorescence immunoassay (FIA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), immunocomplex transfer assay, immunochromatography (ICA), luminescent oxygen channeling immunoassay (LOCI), and liquid-phase binding assay-electrokinetic analyte transport. Examples of known immunoassays and mass spectrometry methods include assays (LBA-EATA method), capillary electrophoresis methods such as lectin electrophoresis, Western blotting, immunohistochemistry (NIA method) such as latex immunohistochemistry, immunoturbidiometry (TIA method) such as latex immunoturbidiometry, immunoaggregation methods such as microparticle counting immunoaggregation assay (PCIA method), assays that detect the presence of the target molecule using surface plasmon resonance (SPR method), AlphaLISA method, fluorescence resonance energy transfer (FRET), or bioluminescence resonance energy transfer (BRET), flow cytometry, and other known immunoassays and mass spectrometry methods. Immunological assays are preferred, and enzyme-linked immunosorbent assay (ELISA method) is more preferred.

[0047] The method for detecting the amount of CD41 or CD61 in extracellular vesicles, and the principle for measuring the amount of CD9 in extracellular vesicles, are not particularly limited. Examples include the sandwich method and the competitive method, with the sandwich method being preferred. Alternatively, the homodianism method and the heterogeneous method may be used as the measurement principle, with the heterogeneous method being preferred.

[0048] The measurement of the amount of CD41 or CD61 in extracellular vesicles, and the amount of CD9 in extracellular vesicles, may be carried out specifically in accordance with the method described in WO2016 / 088689, and all descriptions in WO2016 / 088689 are incorporated herein by reference.

[0049] A preferred method for measuring the amount of CD41 or CD61 in extracellular vesicles, and the amount of CD9 in extracellular vesicles, specifically includes, for example, (1) contacting extracellular vesicles containing CD41, CD61, or CD9 in a biological sample with a substance that binds to CD41, a substance that binds to CD61, or a substance that binds to CD9 to form a complex containing the extracellular vesicles containing CD41, CD61, or CD9 in the biological sample and the substance that binds to CD41, a substance that binds to CD61, or a substance that binds to CD9 (hereinafter sometimes abbreviated as the "complex formation step"), and (2) measuring the amount of the above complex (hereinafter sometimes abbreviated as the "complex amount measurement step").

[0050] Specific and preferred examples of substances that bind to CD41, CD61, or CD9 are the same as those described above.

[0051] In the complex formation process, the substance that binds to phosphatidylserine may be brought into contact with the biological sample. When a substance that binds to phosphatidylserine is brought into contact with a biological sample, there is no particular order in which the substance that binds to phosphatidylserine and the substance that binds to CD41, CD61, or CD9 are brought into contact with the biological sample. However, it is preferable to bring the biological sample into contact with the substance that binds to phosphatidylserine first, and then with the substance that binds to CD41, CD61, or CD9.

[0052] In other words, the complex formation step preferably includes a first step of contacting a biological sample with a substance that binds to phosphatidylserine to form a first complex composed of extracellular vesicles in the biological sample and the substance that binds to phosphatidylserine, and a second step of contacting the first complex with a substance that binds to CD41, a substance that binds to CD61, or a substance that binds to CD9 to form a second complex composed of the first complex with the substance that binds to CD41, a substance that binds to CD61, or a substance that binds to CD9.

[0053] Specifically, the complex formation step involves, for example, contacting a biological sample with an antibody or Tim protein (preferably Tim1 or Tim4, more preferably Tim4) that binds to phosphatidylserine immobilized on a solid phase, to form a first complex between the antibody or Tim protein that binds to phosphatidylserine and extracellular vesicles having phosphatidylserine and CD41, CD61, or CD9 in the biological sample. Then, contacting the first complex with an antibody that binds to CD41, an antibody that binds to CD61, or an antibody that binds to CD9, to form a second complex between the first complex and the antibody.

[0054] After the complex has formed, it is preferable to perform a washing operation (B / F separation) at least before the complex quantity measurement step. Specifically, for example, the washing operation may be performed after the formation of the first complex and / or after the formation of the second complex in the above method, and it is preferable to perform the washing operation (B / F separation) after the formation of the first complex and then the washing operation (B / F separation) after the formation of the second complex.

[0055] The complex quantity measurement step is a step of measuring the amount of the complex obtained in the complex formation step, and any method that can measure the amount of the complex may be used. More specifically, the complex amount measurement step involves detecting the labeling substance in a complex obtained in the above complex formation step, which includes an antibody or Tim protein (preferably Tim1 or Tim4, more preferably Tim4) that specifically binds to phosphatidylserine immobilized on a solid phase, an extracellular vesicle containing phosphatidylserine and CD41, CD61, or CD9, and an antibody that specifically binds to a labeling substance (preferably enzymes or fluorescent substances). This can be done by, for example, (1) using an antibody that specifically binds to CD41, CD61, or CD9 and is labeled with a labeling substance, (2) using a labeled secondary antibody that specifically binds to the "antibody that specifically binds to CD41, CD61, or CD9" and is labeled with a labeling substance, or (3) using an antibody that specifically binds to CD41, CD61, or CD9 and is conjugated with one of avidins or biotins, and a labeling substance that is conjugated with the other of avidins or biotins.

[0056] In the complex amount measurement process, it is preferable to perform a washing operation (B / F separation) before detecting the labeled substance.

[0057] The measurement of the amount of CD41, CD61, or CD9 is, more specifically, performed by contacting a biological sample with an antibody or Tim protein (preferably Tim1 or Tim4, more preferably Tim4) that specifically binds to phosphatidylserine immobilized on a solid-phase plate, to form a first complex between the antibody or Tim protein that specifically binds to phosphatidylserine and extracellular vesicles containing phosphatidylserine and CD41, CD61, or CD9 in the biological sample; if necessary, after B / F separation, (1) contact the first complex with an antibody that specifically binds to CD41, CD61, or CD9 labeled with a labeling substance to form a second complex between the first complex and an antibody that specifically binds to CD41, CD61, or CD9 labeled with a labeling substance; (2) contact the first complex with an antibody that specifically binds to CD41, CD61, or CD9, and the first complex with CD41, CD61 or (1) A second complex is formed with an antibody that specifically binds to CD9, and if necessary, after B / F separation, the second complex is brought into contact with a labeled secondary antibody labeled with a labeling substance that specifically binds to the "antibody that specifically binds to CD41, CD61, or CD9", to form a third complex between the second complex and the labeled secondary antibody, or (3) the first complex is brought into contact with an antibody that specifically binds to CD41, CD61, or CD9, to which one of avidins and biotins is bound, to form a second complex with an antibody that specifically binds to CD41, CD61, or CD9, to which one of avidins and biotins is bound, and if necessary, after B / F separation, a third complex is formed between the second complex and a labeling substance (enzymes, fluorescent substances are preferred) to which the remaining one of avidins and biotins is bound, and after B / F separation, the labeling substance of the obtained second or third complex can be detected.

[0058] The amount of biological sample, the amount (concentration) of protein in the biological sample, the amount (concentration) of phosphatidylserine and CD41, CD61, or CD9 in extracellular vesicles in the biological sample, the amount (concentration) of substances that bind to CD41, CD61, or CD9 and substances that bind to phosphatidylserine to be reacted with these, the labeling substance and labeling method, etc., should be appropriately set according to the type of biological sample, the required measurement sensitivity, the measurement method and measuring device used, etc. Furthermore, the amount (concentration) of CD41, CD61, or CD9 in extracellular vesicles in biological samples may be calculated by creating a calibration curve using standards. Examples of such standards include CD41, CD61, or CD9, or extracellular vesicles containing CD41, CD61, or CD9.

[0059] In diagnostic support methods for neurodegenerative diseases, assistance in the diagnosis and differential diagnosis can be provided using indicators that include the amount of CD41 or CD61. In the ND diagnostic support method, assistance in the diagnosis and differentiation of neurodegenerative diseases includes using an index containing the amount of CD41 or CD61, and further using a value obtained by detecting a biomarker related to neurodegenerative diseases in the test sample (preferably the amount of other biomarkers for the neurodegenerative diseases), and it is preferable to use only an index containing the amount of CD41 or CD61.

[0060] As an indicator including the amount of CD41 or CD61, the ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9, may be used. The ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9, can be calculated by dividing the amount of CD41 or CD61 (A) by the amount of CD9 (B) ((A) / (B)).

[0061] Using the ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9, as an index that includes the amount of CD41 or CD61, involves using the ratio of the amount of CD9 to the amount of CD41 or the ratio of the amount of CD61 to the amount of CD9. The ratio of the amount of CD9 to the amount of CD41 or the ratio of the amount of CD9 to the amount of CD61 can be calculated by dividing the amount of CD9 (B) by the amount of CD41 or CD61 (A) ((B) / (A)).

[0062] Assisting in the diagnosis and differential diagnosis of neurodegenerative diseases includes, for example, obtaining data suggestive of neurodegenerative diseases, such as the prevalence of neurodegenerative diseases and the risk of developing them. Specifically, this includes whether the subject is likely to have a neurodegenerative disease, whether the subject is likely to have a neurodegenerative disease, whether the subject is at high risk of developing a neurodegenerative disease, and whether the subject is at risk of developing a neurodegenerative disease. Preferably, this includes the subject's disease status.

[0063] In the diagnostic support method for neurodegenerative diseases, assistance in differentiating neurodegenerative diseases may be provided, for example, by comparing an index containing the amount of CD41 or CD61 in extracellular vesicles in a biological sample derived from a subject, obtained by detecting the amount of CD41 or CD61, with an index containing the amount of CD41 or CD61 in subjects other than those with neurodegenerative diseases (preferably healthy individuals) or a predetermined reference value (cutoff value).

[0064] In AD / PD differential diagnosis assistance methods, assistance in differentiating Parkinson's disease may be performed, for example, by comparing the ratio of the amount of CD41 or CD61 to the amount of CD9, calculated based on the detection of the amount of CD41 or CD61 and the amount of CD9, with the ratio of the amount of CD41 or CD61 to the amount of CD9 in Alzheimer's disease or a predetermined reference value (cutoff value).

[0065] Comparisons of indicators including the amount of CD41 or CD61 include comparisons using only indicators including the amount of CD41 or CD61, such as the amount of CD41 or CD61 relative to the amount of CD9; and comparisons using the amount of CD41 or CD61 relative to the amount of CD9, and values ​​obtained by subjecting the detection of biomarkers related to neurodegenerative diseases in the test sample (preferably the amount of other biomarkers for the neurodegenerative diseases) to multivariate analyses such as multiple logistic regression analysis, discriminant analysis, Poisson regression analysis, multiple regression analysis, Cox proportional hazards model, and path analysis (values ​​derived from the subjects).

[0066] In the ND diagnostic support method, specifically, if (1) the amount of CD41 or CD61, or (2) the ratio of the amount of CD41 or CD61 to the amount of CD9 is lower than a predetermined reference value, data suggesting Person's disease may be obtained, such as "the subject is likely to have a neurodegenerative disease, or the subject may have a neurodegenerative disease, or the subject is at high risk of developing a neurodegenerative disease, or the subject is at risk of developing a neurodegenerative disease."

[0067] Furthermore, if (1) the amount of CD41 or CD61, or (2) the ratio of the amount of CD41 or CD61 to the amount of CD9 is above a predetermined reference value, data that does not suggest Person's disease, such as "the subject is unlikely to have a neurodegenerative disease or is not likely to have a neurodegenerative disease, or the subject has a low risk of developing a neurodegenerative disease or is not at risk of developing a neurodegenerative disease," may be obtained.

[0068] In the AD / PD differentiation support method, specifically, if the ratio of the amount of CD41 or CD61 to the amount of CD9 is lower than a predetermined reference value, data suggesting Parkinson's disease, such as "the subject is likely to have Parkinson's disease, or the subject may have Parkinson's disease, or the subject is at high risk of developing Parkinson's disease, or the subject is at risk of developing Parkinson's disease," may be obtained, or data not suggesting Alzheimer's disease, such as "the subject is unlikely to have Alzheimer's disease, or the subject is not likely to have Alzheimer's disease, or the subject is at low risk of developing Alzheimer's disease, or the subject is not at risk of developing Alzheimer's disease."

[0069] Furthermore, if the ratio of the amount of CD41 or CD61 to the amount of CD9 is above a predetermined reference value, data that does not suggest Parkinson's disease, such as "the subject is unlikely to have Parkinson's disease, or the subject is unlikely to have Parkinson's disease, or the subject has a low risk of developing Parkinson's disease, or the subject has no risk of developing Parkinson's disease," or data that suggests Alzheimer's disease, such as "the subject is likely to have Alzheimer's disease, or the subject may have Alzheimer's disease, or the subject has a high risk of developing Alzheimer's disease, or the subject has a risk of developing Alzheimer's disease," may be obtained.

[0070] Specific examples of predetermined reference values ​​(cutoff values) in ND diagnostic support methods include those set in advance to distinguish between subjects with neurodegenerative diseases and subjects without neurodegenerative diseases. (1) The amount of CD41 or CD61; or (2) The ratio of the amount of CD41 or CD61 to the amount of CD9; or (3) Values ​​derived from the above subjects These are some examples.

[0071] The method for determining the predetermined reference values ​​is not particularly limited. For example, the amount of CD41 or CD61 contained in biological samples obtained from patients with neurodegenerative diseases and healthy individuals can be measured, and the obtained amounts of CD41 or CD61 can be used to determine the reference values ​​through statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis). When setting the predetermined reference values, it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, etc. The predetermined reference values ​​can be set so that the sensitivity is 60% or higher, preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher. For example, the specificity can be set so that it is 60% or higher, preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher.

[0072] A specific example of a predetermined reference value (cutoff value) in an AD / PD differential diagnosis method is the ratio of the amount of CD41 or CD61 to the amount of CD9, which is predetermined to distinguish between Parkinson's disease and Alzheimer's disease. The method for determining the predetermined reference value is not particularly limited. For example, the amount of CD41 or CD61 and the amount of CD9 in biological samples obtained from patients with Parkinson's disease and healthy individuals can be detected, the ratio of the amount of CD41 or CD61 to the amount of CD9 can be calculated, and the obtained ratio of the amount of CD41 or CD61 to the amount of CD9 can be used to determine the value through statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis). When setting the above predetermined reference value, it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, etc. The predetermined reference values ​​mentioned above can be set, for example, so that the sensitivity is 60% or higher, preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher. For example, the specificity can be set to 60% or higher, preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher.

[0073] When using "the ratio of the amount of CD41 or CD61 to the amount of CD41 or CD61 in extracellular vesicles" as "the ratio of the amount of CD9 to the amount of CD41 or CD61 in extracellular vesicles," as will be clear from the examples described later, a value greater than or equal to that of subjects other than neurodegenerative diseases is a method that suggests neurodegenerative diseases.

[0074] <Diagnostic kits for neurodegenerative diseases, and kits for differentiating between Alzheimer's disease and Parkinson's disease> The diagnostic kit for neurodegenerative diseases of the present invention comprises a substance that binds to CD41 or a substance that binds to CD61. The diagnostic kit for neurodegenerative diseases of the present invention may further comprise a substance that binds to CD9. The diagnostic kit for neurodegenerative diseases of the present invention may further comprise a substance that binds to phosphatidylserine.

[0075] The Alzheimer's disease and Parkinson's disease differentiation kit of the present invention comprises a substance that binds to CD41, or a substance that binds to CD61, and a substance that binds to CD9. The Alzheimer's disease and Parkinson's disease differentiation kit of the present invention may further comprise a substance that binds to phosphatidylserine.

[0076] The diagnostic kit for neurodegenerative diseases and the differential diagnosis kit for Alzheimer's disease and Parkinson's disease according to the present invention are collectively referred to as the "kit of the present invention."

[0077] In the kit of the present invention, the substances that bind to CD41, CD61, CD9, and phosphatidylserine are the same as those described above in the ND diagnostic support method and the AD / PD differential diagnosis support method, and the preferred substances are also the same. The substances that bind to CD41, CD61, CD9, and phosphatidylserine may be in solution, frozen, dried, or lyophilized state. Furthermore, the substances that bind to CD41, CD61, CD9, and phosphatidylserine may be included in the kit as a single reagent or as separate reagents.

[0078] The combinations of substances that bind to CD41, CD61, CD9, and phosphatidylserine in the kit of the present invention include the same combinations as those described above in the ND diagnostic support method and the AD / PD differential diagnosis support method, and the preferred combinations are also the same.

[0079] The substances that bind to CD41, CD61, CD9, and / or phosphatidylserine in the kit of the present invention may be immobilized on a solid phase or labeled with a labeling substance. The solid phase, the method of immobilization to the solid phase, the labeling substance, and the labeling method are the same as those described above in the ND diagnostic support method and the AD / PD differential diagnosis support method, and the preferred ones are also the same.

[0080] The kit of the present invention may further include a secondary affinity substance (e.g., a secondary antibody) that specifically binds to a substance that binds to CD41, a substance that binds to CD61, a substance that binds to CD9, and / or a substance that binds to phosphatidylserine. The secondary affinity substance in the kit of the present invention is the same as that described above in the ND diagnostic support method and the AD / PD differentiation support method, and the preferred substances are also the same. Furthermore, the secondary affinity substance in the kit of the present invention may be labeled with a labeling substance, and the labeling substance and labeling method are the same as those described above in the ND diagnostic support method and the AD / PD differentiation support method, and the preferred substances are also the same.

[0081] The substances that bind to CD41, CD61, CD9, and / or phosphatidylserine in the kit of the present invention may be avidins and biotins to which either has been bound. The avidins and biotins are the same as those described above in the ND diagnostic support method and the AD / PD differential diagnosis support method, and the preferred ones are also the same. The kit of the present invention may further contain a labeled substance to which either avidins or biotins are bound. The labeled substance and labeling method are the same as those described above in the ND diagnostic support method and AD / PD differential diagnosis support method, and preferred labeling methods are also the same.

[0082] The concentrations (amounts) of the substances that bind to CD41, CD61, CD9, and phosphatidylserine in the kit of the present invention can be appropriately set within the range commonly used in this field, depending on the measurement method. For example, when immobilizing the CD41-binding substance, CD61-binding substance, and CD9-binding substance are used, the concentration at the time of use is preferably 10 to 20,000 ng / mL, more preferably 100 to 10,000 ng / mL, and when used for detection, the concentration at the time of use is preferably 10 to 5,000 ng / mL, more preferably 100 to 500 ng / mL. Similarly, when immobilizing the phosphatidylserine-binding substance is used, the concentration at the time of use is preferably 10 to 20,000 ng / mL, more preferably 100 to 10,000 ng / mL, and when used for detection, the concentration at the time of use is preferably 10 to 5,000 ng / mL, more preferably 100 to 500 ng / mL.

[0083] Furthermore, reagents commonly used in this field, such as buffers, reaction accelerators, sugars, proteins, salts, surfactants and other stabilizers, and preservatives, may be present in the presence of substances that bind to CD41, CD61, CD9, and / or phosphatidylserine. The concentrations and pH of these substances should be appropriately selected from the ranges commonly used in this field.

[0084] The kit of the present invention may include, in addition to a substance that binds to CD41 or CD61, and optionally a substance that binds to CD9 and / or phosphatidylserine, reagents necessary for measuring the amount of CD41 or CD61 using these substances. Examples of such reagents include a washing agent, a sample diluent (a reagent for diluting the sample), a reagent for detecting the labeling substance, a reagent for binding the labeling substance to these substances, a reagent for immobilizing these substances on a solid phase, and a reagent for binding avidins or biotins to these substances or the labeling substance. The concentrations, pH, etc., of these reagents can be appropriately selected from the ranges commonly used in this field.

[0085] The kit of the present invention may include standards used to create calibration curves for the amount of CD41 or CD61. Examples of standards include CD41 or CD61, or extracellular vesicles containing CD41 or CD61. The standards may be in solution, frozen, dried, or lyophilized state. Reagents commonly used in this field, such as buffers, reaction accelerators, sugars, proteins, salts, stabilizers such as surfactants, and preservatives, may also be present in the presence of the standards. The concentrations and pH of these reagents may be appropriately selected from ranges commonly used in this field.

[0086] The kit of the present invention may include accompanying documents and instruction manuals. Examples of accompanying documents and instruction manuals include those that describe measuring the amount of CD41 or CD61 in a biological sample derived from a subject, and / or determining whether the subject has a neurodegenerative disease based on the amount of CD41 or CD61. These accompanying documents and instruction manuals may be written in multiple documents or combined into a single document.

[0087] Examples of the kits of the present invention include, specifically, a kit in which either a substance that binds to CD41 or CD61 and a substance that binds to phosphatidylserine are immobilized on a solid phase, and the other of the substance that binds to CD41 or CD61 and the other of the substance that binds to phosphatidylserine are bound to a labeling substance, or a kit that includes the other substance and a component for indirectly binding it to the labeling substance.

[0088] A preferred kit of the present invention is one which includes a substance that binds to CD41 or CD61 and a substance that binds to phosphatidylserine, both of which are immobilized on a solid phase, and one of the following selected from (1) to (3). (1) A substance that binds to CD41 or CD61 and the other substance that binds to phosphatidylserine are bound to a labeling substance; (2) A substance that binds to CD41 or CD61 and the other substance that binds to phosphatidylserine, as well as a secondary affinity substance that specifically binds to the above substance, bound to the labeling substance; (3) A substance that binds to CD41 or CD61 and a substance that binds to phosphatidylserine, the remaining of which is bound to either an avidin or a biotin, and the remaining of which is bound to a labeling substance.

[0089] According to the kit of the present invention, the method for assisting in the diagnosis of neurodegenerative diseases and the method for assisting in the differentiation between Alzheimer's disease and Parkinson's disease according to the present invention can be performed simply, quickly, and accurately.

[0090] <A diagnostic aid for neurodegenerative diseases, and a diagnostic aid for differentiating between Alzheimer's disease and Parkinson's disease> The method for assisting the diagnosis of neurodegenerative diseases according to the present invention can be performed using a diagnostic aid for neurodegenerative diseases. The method for assisting in the differentiation between Alzheimer's disease and Parkinson's disease according to the present invention can be performed using a device for assisting in the differentiation between Alzheimer's disease and Parkinson's disease.

[0091] The devices used to assist in the diagnosis of neurodegenerative diseases and the devices used to assist in differentiating between Alzheimer's disease and Parkinson's disease are collectively referred to as the "devices in this invention."

[0092] The diagnostic aid for neurodegenerative diseases in the present invention is A measuring unit for measuring the amount of CD41 or CD61 in extracellular vesicles in the test sample; and A determination unit that determines whether a patient has a neurodegenerative disease based on the amount of CD41 or CD61 mentioned above; It has.

[0093] The present invention provides an aid in differentiating between Alzheimer's disease and Parkinson's disease. A measuring unit for measuring the amount of CD41 or CD61 in extracellular vesicles and the amount of CD9 in extracellular vesicles in the test sample; and A diagnostic unit that differentiates between Alzheimer's disease and Parkinson's disease using the ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9, as indicators: It has.

[0094] The biological sample, subject, and extracellular vesicles in the apparatus of the present invention are the same as those described above, and the preferred ones are also the same.

[0095] The measuring unit, determination unit, and other components that constitute the apparatus in the present invention may be located within the same apparatus, or they may be separate components. The measuring unit in the apparatus of the present invention is a part that measures the amount of CD41 or CD61 (and optionally the amount of CD9) in extracellular vesicles in a biological sample introduced into the apparatus. The size and configuration of the measuring unit are not particularly limited.

[0096] Examples of measurement devices include microplate readers or imaging devices that use CCDs for imaging in ELISA, imaging devices used in Western blotting or microarray (microchip) methods, mass spectrometers used in mass spectrometry, intermolecular interaction analyzers, flow cytometers used in flow cytometry, and ultraviolet-visible light detectors or fluorescence detectors used in HPLC and capillary electrophoresis.

[0097] The method for measuring the amount of CD41 or CD61 (and optionally the amount of CD9) in extracellular vesicles, and the substances used for the above measurement, are the same as those described above in the ND diagnostic support method and the AD / PD differential diagnosis support method, and the preferred substances and specific examples are also the same.

[0098] The apparatus in the present invention may include a calculation unit. The calculation unit is a part that converts measured values ​​(for example, absorbance, change in absorbance, transmitted light, change in transmitted light, fluorescence intensity, change in fluorescence intensity, luminescence, change in luminescence, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, change in reflectance, refractive index, change in refractive index, etc.) that correlate with the amount or concentration of CD41 or CD6 (and optionally CD9) in extracellular vesicles in the biological sample obtained by the measurement unit into mass, concentration, etc. The measured values ​​and the calculated mass, concentration, etc., may be stored in a memory device such as a hard disk or other storage device provided in the device.

[0099] The determination unit in the apparatus of the present invention is The amount of CD41 or CD61 obtained by the above measurement unit or calculation unit is used as an indicator to determine the site of neurodegenerative disease; or A site used to differentiate between Alzheimer's disease and Parkinson's disease, using as an indicator the ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9, obtained by the above-mentioned measurement unit or calculation unit; That is the case.

[0100] The predetermined reference values ​​used for diagnosing neurodegenerative diseases or differentiating between Alzheimer's disease and Parkinson's disease, and the methods for determining these predetermined reference values, are the same as those described above for the ND diagnostic support method and the AD / PD differential diagnosis support method, and the preferred values ​​and specific examples are also the same.

[0101] The predetermined reference values ​​in the apparatus of the present invention may be stored in the apparatus of the present invention beforehand, or they may be entered from the input port of the apparatus of the present invention during the determination process.

[0102] The apparatus in the present invention may include an output unit. The output unit performs processing such as displaying or outputting the determination result to a display device such as a display or a printing device such as a printer.

[0103] <Biomarkers and biomarker sets> The present invention relates to a diagnostic biomarker for neurodegenerative diseases, comprising an extracellular vesicle having CD41 or an extracellular vesicle having CD61. The present invention further relates to a set of diagnostic biomarkers for neurodegenerative diseases, comprising extracellular vesicles having CD41, or extracellular vesicles having CD61, and extracellular vesicles having CD9. The present invention further relates to a set of biomarkers for differentiating Alzheimer's disease from Parkinson's disease, comprising extracellular vesicles having CD41, or extracellular vesicles having CD61, and extracellular vesicles having CD9.

[0104] In biomarkers and biomarker sets, extracellular vesicles are the same as those described above, and preferred vesicles are also the same. In biomarkers and biomarker sets, extracellular vesicles containing CD41, or extracellular vesicles containing CD61, and extracellular vesicles containing CD9 may have CD41 or CD61 on their membrane surface and may also contain it within the extracellular vesicle.

[0105] The present invention provides a biomarker for the diagnosis of neurodegenerative diseases, which includes an index containing the amount of CD41 or CD61 in extracellular vesicles. This index can be used to determine whether or not a subject has a neurodegenerative disease. According to the biomarker set for the diagnosis of neurodegenerative diseases of the present invention, it is possible to obtain the ratio of the amount of CD41 to the amount of CD9 in extracellular vesicles, or the ratio of the amount of CD61 to the amount of CD9 in extracellular vesicles, and these indicators can also be used as indicators to determine whether or not a subject has a neurodegenerative disease.

[0106] According to the biomarker set for differentiating between Alzheimer's disease and Parkinson's disease of the present invention, it is possible to obtain the ratio of the amount of CD41 to the amount of CD9 in extracellular vesicles, or the ratio of the amount of CD61 to the amount of CD9 in extracellular vesicles, and these indicators can also be used as indicators for differentiating between Alzheimer's disease and Parkinson's disease in subjects.

[0107] <Specific examples of methods to assist in the diagnosis of neurodegenerative diseases> An example of a method to assist in the diagnosis of neurodegenerative diseases is described below. In the following example, extracellular vesicles are measured using a Tim4 protein-anti-CD41 antibody sandwich ELISA method. The EDTA plasma of the subject is centrifuged, for example, at 10,000 g for 30 minutes, and the supernatant is collected. The obtained supernatant is diluted to an appropriate ratio to obtain a sample diluent for measurement. Dispense the sample diluent for measurement into the wells of a plate immobilized with Tim4 protein and allow to react at room temperature for 2 hours with stirring at approximately 500 rpm. After the reaction, discard the reaction solution and wash the wells. Next, dispense 100 μL of the biotin-labeled antibody reaction solution, obtained by diluting biotin-labeled anti-CD41 mouse monoclonal antibody to a final concentration of 250 ng / mL, into the wells. Allow to react at room temperature for 1 hour with stirring at approximately 500 rpm using a microplate shaker. After the reaction, discard the reaction solution and wash the wells with washing solution.

[0108] Next, dispense 100 μL of the reaction solution containing HRP-labeled streptavidin into each well and allow it to react at room temperature for 2 hours while stirring at approximately 500 rpm using a microplate shaker. After the reaction is complete, discard the reaction solution and wash the wells with washing solution.

[0109] Next, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) solution is dispensed into the wells, stirred for approximately 1 minute using a microplate shaker, and then allowed to stand at room temperature (20-25°C) for 30 minutes. After that, 100 μL of reaction stop solution is added to the wells, stirred for approximately 5 seconds using a microplate shaker, and then the absorbance at 450 nm and the absorbance at the secondary wavelength of 620 nm are measured immediately using a 96-well microplate reader. The absorbance value at 450 nm is subtracted from the absorbance value at the secondary wavelength of 620 nm to be defined as the "absorbance value," and the absorbance value of the sample diluent for measurement is subtracted from the absorbance value of the blank to be defined as the "corrected sample absorbance value." Then, a standard curve is created from the absorbance value of the COLO201 cell culture supernatant-derived extracellular vesicle dilution series (calibrator) minus the absorbance value of the blank and the protein concentration of the calibrator. Using a standard curve, the corrected sample absorbance value can be converted to protein concentration, and the resulting converted value can be multiplied by the sample dilution ratio to obtain the "sample measurement value" [ng / mL].

[0110] <Methods for obtaining data to assist in the diagnosis of neurodegenerative diseases, and methods for obtaining data to assist in the differentiation between Alzheimer's disease and Parkinson's disease> According to the present invention, data can be obtained to assist physicians in diagnosing neurodegenerative diseases in subjects. According to the present invention, data can be obtained to assist physicians in differentiating between Alzheimer's disease and Parkinson's disease in subjects. The present invention provides a method for obtaining data to assist in the diagnosis of neurodegenerative diseases, which includes detecting the amount of CD41 or CD61 in extracellular vesicles in a test sample. The present invention provides a method for obtaining data to assist in differentiating between Alzheimer's disease and Parkinson's disease, comprising detecting the amount of CD41 or CD61 in extracellular vesicles and the amount of CD9 in extracellular vesicles in a test sample.

[0111] The data obtained in the method for obtaining data of the present invention may include: (i) an amount obtained by detecting the amount of CD41 or CD61 in extracellular vesicles in a test sample, and optionally the amount of CD9 in extracellular vesicles; (ii) a value obtained by further subjecting the above amount to multivariate analysis such as multiple logistic regression analysis, discriminant analysis, Poisson regression analysis, multiple regression analysis, Cox proportional hazards model, or path analysis; (iii) data (comparison results) showing the magnitude relationship between the amount obtained in (i) or the amount obtained in (ii) above and the aforementioned reference value (cutoff value), etc.; and (iv) data suggesting that the subject may have a neurodegenerative disease or that the subject has a high risk of having a neurodegenerative disease. (i) or (iii) is preferred, and (i) is more preferred.

[0112] The "detection of the amount of CD41 or CD61 in extracellular vesicles, and the detection of the amount of CD9 in extracellular vesicles" in the method for obtaining data of the present invention is as described above in this specification, and the preferred examples and specific examples are also the same.

[0113] According to the present invention, data can be obtained to assist physicians in diagnosing neurodegenerative diseases in subjects. If a physician determines that a subject has Parkinson's disease, an example of a neurodegenerative disease, based on the present invention, the physician may further perform tests such as a medical interview, MIBG myocardial scintigraphy, dopamine transporter scintigraphy, and other tests related to Parkinson's disease biomarkers recommended in Parkinson's disease treatment guidelines, as well as tests using candidate biomarkers for Parkinson's disease. The physician can then make a diagnosis of Parkinson's disease in the subject, taking these results into consideration.

[0114] <Methods for measurement and calculation to assist in the diagnosis of neurodegenerative diseases, and methods for measurement and calculation to assist in the differentiation between Alzheimer's disease and Parkinson's disease> The present invention provides a method for measuring and calculating to assist in the diagnosis of neurodegenerative diseases, which includes detecting the amount of CD41 or CD61 in extracellular vesicles in a test sample. The present invention provides a method for measuring and calculating to assist in differentiating between Alzheimer's disease and Parkinson's disease, comprising detecting the amount of CD41 or CD61 in extracellular vesicles and the amount of CD9 in extracellular vesicles in a test sample.

[0115] The "detection of the amount of CD41 or CD61 in extracellular vesicles and the amount of CD9 in extracellular vesicles" in the measurement and calculation method of the present invention is as described above in this specification, and the preferred examples and specific examples are also the same.

[0116] <Methods to assist in the diagnosis and treatment of neurodegenerative diseases, as well as methods to assist in the differentiation between Alzheimer's disease and Parkinson's disease and methods to treat Parkinson's disease> The present invention provides a method for assisting in the diagnosis of neurodegenerative diseases and for treating neurodegenerative diseases, which includes providing appropriate treatment to a patient who has been determined to have a neurodegenerative disease according to the present invention. The present invention provides a method for assisting in the differentiation between Alzheimer's disease and Parkinson's disease and for treating Parkinson's disease, and includes providing appropriate treatment to patients who have been determined to have Parkinson's disease rather than Alzheimer's disease according to the present invention.

[0117] In one example, if a subject is diagnosed with Parkinson's disease according to the present invention, they may be administered Parkinson's disease medications such as levodopa (L-dopa) (medications or treatments that slow the progression of Parkinson's disease), or surgery may be performed.

[0118] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. In the examples, AD refers to a patient with Alzheimer's disease, and PD refers to a patient with Parkinson's disease. [Examples]

[0119] Example 1 and Comparative Example 1: Evaluation of Parkinson's disease using the amount of CD41 in extracellular vesicles containing PS as an indicator. Extracellular vesicles were measured using a Tim4 protein-anti-CD41 antibody sandwich ELISA method, and AUC and p-values ​​were calculated.

[0120] (1) Preparation of the calibrator Extracellular vesicles were isolated from pooled human EDTA plasma using the MagCapture® Exosome Isolation Kit PS Ver.2 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in accordance with the instructions provided with the kit. The protein concentration in the obtained extracellular vesicle solution was measured using the BCA method (bicinchoninate method) with the Protein Assay BCA Kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0121] Next, the obtained extracellular vesicle solution was diluted using the Reaction Buffer included with the PS Capture Exosome ELISA Kit, Streptavidin HRP (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "Kit A") to concentrations of 0.0313, 0.0625, 0.125, 0.25, 0.50, 1.0, and 2.0 μg / mL, based on the protein concentrations measured by the BCA method, thereby obtaining a series of seven concentrations of pooled human EDTA plasma-derived extracellular vesicle dilutions (hereinafter referred to as "calibrators").

[0122] (2) Pre-treatment of specimens for measurement EDTA plasma samples from 37 healthy individuals, 18 patients with Alzheimer's disease (AD), and 16 patients with Parkinson's disease (PD), purchased from PrecisionMed, were centrifuged at 10,000 g for 30 minutes, and the supernatant was collected. The obtained supernatants were each diluted 200-fold using the Reaction Buffer included with Kit A to prepare the "sample diluents for measurement."

[0123] (3) Measurement by ELISA method The sample dilution prepared in (2) was measured using a Tim4 protein-anti-CD41 antibody sandwich ELISA method, in which Tim4 protein was immobilized on the solid phase and an anti-CD41 antibody was used as the detection antibody. Specifically, a biotin-labeled anti-CD41 mouse monoclonal antibody (MEM-06, Thermo Fisher Scientific) was used. Reagents other than the detection antibody were those included in the above-mentioned Kit A.

[0124] First, the Washing Buffer (10×) included in Kit A was diluted 10-fold with purified water (distilled water), and then 1 / 100th the volume of Exosome Binding Enhancer (100×) included in Kit A was added to the resulting diluted solution. The resulting solution was designated as "Washing Solution (1×)". Next, each well of the "96-well plate with Tim4 protein immobilized on the solid phase" included in Kit A was washed three times with 300-350 μL of Washing Solution (1×).

[0125] Next, 100 μL each of the following were dispensed into each well of the plate: the sample diluent for measurement of healthy control samples prepared in (2) (37 samples, n=2, 74 wells), the sample diluent for measurement of Alzheimer's disease patient samples (18 samples, n=2, 36 wells), the sample diluent for measurement of Parkinson's disease patient samples (16 samples, n=2, 32 wells), the calibrator prepared in (1) (7 points per plate, n=2, 14 wells), and the Reaction Buffer included with Kit A as a blank (n=2 per plate, 2 wells). Then, a plate seal was attached to the plate, and the mixture was reacted at room temperature for 2 hours while being stirred at approximately 500 rpm using a microplate shaker. After the reaction, the reaction solution was discarded, and each well was washed three times with 300-350 μL of washing solution (×1). Next, the biotin-labeled anti-CD41 mouse monoclonal antibody was diluted to a final concentration of 250 ng / mL using the Reaction Buffer provided in Kit A to obtain a biotin-labeled antibody reaction solution. 100 μL of the obtained biotin-labeled antibody reaction solution was dispensed into each well, a plate seal was applied, and the mixture was reacted at room temperature for 1 hour while stirring at approximately 500 rpm using a microplate shaker. After the reaction, the reaction solution was discarded, and each well was washed three times with 300-350 μL of washing solution (×1).

[0126] To the Reaction Buffer included in Kit A, 1 / 100 volume of HRP-conjugated Streptavidin (100×) was added and thoroughly mixed to prepare HRP-labeled streptavidin reaction solution (1×). 100 μL of the obtained HRP-labeled streptavidin reaction solution (1×) was dispensed into each well, a plate seal was applied, and the mixture was reacted at room temperature for 2 hours while stirring at approximately 500 rpm using a microplate shaker. After the reaction was complete, the reaction solution was discarded, and each well was washed five times with 300-350 μL of washing solution (1×).

[0127] Next, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) Solution, included with Kit A (which had been returned to room temperature), was dispensed into each well. After stirring for approximately 1 minute using a microplate shaker, a plate seal was applied, and the mixture was allowed to stand at room temperature (20-25°C) for 30 minutes. Subsequently, 100 μL of Stop Solution, included with Kit A (which had also been returned to room temperature), was added to each well. After stirring for approximately 5 seconds using a microplate shaker, the absorbance at 450 nm and the absorbance at the secondary wavelength of 620 nm were immediately measured using a 96-well microplate reader (Tecan, Safire2). The "absorbance value" was calculated by subtracting the 620 nm absorbance value from the 450 nm absorbance value, and the "corrected sample absorbance value" was calculated by subtracting the blank absorbance value from the absorbance value of the sample diluent used for measurement. Then, a standard curve was created from the absorbance values ​​of the extracellular vesicle dilution series (calibrator) derived from COLO201 cell culture supernatant, minus the absorbance value of the blank, and the protein concentration of the calibrator. Using the standard curve, the corrected sample absorbance values ​​were converted to protein concentrations, and the resulting converted value was multiplied by the sample dilution ratio to obtain the "sample measurement value" [ng / mL].

[0128] (4) Calculation of AUC Based on the sample measurements obtained in (3), statistical analysis software EZR was used to perform a U test to determine the significance of differences between healthy individuals and Parkinson's disease patients, and between Alzheimer's disease patients and Parkinson's disease patients, and the p-value was calculated. In addition, based on the sample measurements obtained in (3), logistic regression analysis was performed using statistical analysis software, and the area under the curve (AUC) was calculated from the obtained receiver operating characteristic curve (ROC curve).

[0129] (5) Results The results obtained are shown in Table 1 below. A box plot created based on the sample measurements is shown in Figure 1. In the figure, the vertical axis represents the sample measurements, the horizontal axis shows Control (results from healthy individuals), AD (results from patients with Alzheimer's disease), and PD (results from patients with Parkinson's disease). The measured values ​​in samples from Parkinson's disease patients were lower than those in healthy individuals. Differentiation between healthy individuals and Parkinson's disease patients was statistically significant, with an AUC of 0.851 and a p-value of less than 0.0001 (Example 1). Differentiation between Alzheimer's disease patients and Parkinson's disease patients was not statistically significant, with an AUC of 0.649 and a p-value of 0.144 (Comparative Example 1). These results indicate that the amount of CD41 in extracellular vesicles can be used to differentiate between healthy individuals and Parkinson's disease patients with high accuracy.

[0130] Comparative Examples 2 and 3: Evaluation of Parkinson's disease using the amount of CD9 in extracellular vesicles with PS as an indicator. (1) Preparation of the calibrator Exosomes, derived from COLO201 cells, purified product (manufactured by Fujifilm Wako Pure Chemical Corporation) were diluted using the Reaction Buffer included in Kit A to concentrations of 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 ng / mL, based on the protein concentrations indicated on the product. This resulted in a series of seven concentrations of extracellular vesicle dilutions derived from COLO201 cell culture supernatant (hereinafter referred to as "calibrators").

[0131] (2) Measurement by ELISA method Using the calibrator prepared in (1), the sample was diluted 100-fold, and extracellular vesicles were measured by Tim4 protein-anti-CD9 antibody sandwich ELISA in the same manner as in Example 1, except that an anti-CD9 antibody was used instead of an anti-CD41 antibody as the detection antibody. Significant difference tests were performed between healthy individuals and Parkinson's disease patients, and between Alzheimer's disease patients and Parkinson's disease patients, and AUC and p-values ​​were calculated. The anti-CD9 antibody used as the detection antibody was anti-CD9, rat monoclonal antibody (77B), biotin-conjugated (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0132] (3) Results The results obtained are shown in Table 1 below. Figure 2 shows a box plot created based on the sample measurements. In the figure, the vertical axis represents the sample measurements, and the horizontal axis shows the results for healthy individuals (Control), the results for patients with Alzheimer's disease (AD), and the results for patients with Parkinson's disease (PD). The differentiation between healthy individuals and Parkinson's disease patients (Comparative Example 2) showed an AUC of 0.753 and a p-value of 0.00308. Furthermore, the differentiation between Alzheimer's disease patients and Parkinson's disease patients (Comparative Example 3) showed an AUC of 0.49 and a p-value of 0.932, with no significant difference observed.

[0133] Examples 2 and 3: Evaluation of Parkinson's disease using the ratio of the amount of CD41 in extracellular vesicles with PS to the amount of CD9 in extracellular vesicles with PS as an indicator. The "sample measurement value" (value (A)) obtained in Example 1 or Comparative Example 1 (Tim4 protein-anti-CD41 antibody sandwich ELISA method) was divided by the "sample measurement value" (value (B)) obtained in Comparative Example 2 or Comparative Example 3 (Tim4 protein-anti-CD9 antibody sandwich ELISA method) to calculate (A) / (B) (hereinafter referred to as "corrected sample measurement value"). Based on the calculated corrected sample measurement value, significance tests were performed between healthy individuals and Parkinson's disease patients, and between Alzheimer's disease patients and Parkinson's disease patients using the same method as in Example 1(4), and the AUC and p-value were calculated.

[0134] The results obtained are shown in Table 1 below. Figure 3 shows a box plot created based on the corrected sample measurements. In the figure, the vertical axis represents the corrected sample measurements ((A) / (B)), the horizontal axis shows Control (results from healthy individuals), AD (results from patients with Alzheimer's disease), and PD (results from patients with Parkinson's disease). After correction, the measured values ​​of the samples were lowest in Parkinson's disease patients, followed by Alzheimer's disease patients, and then healthy controls. Differentiation between healthy controls and Parkinson's disease patients (Example 2) showed a statistically significant difference with an AUC of 0.927 and a p-value of less than 0.0001, and an AUC greater than 0.9. Similarly, differentiation between Alzheimer's disease patients and Parkinson's disease patients (Example 3) showed a statistically significant difference with an AUC of 0.84 and a p-value of 0.000428. These results indicate that the ratio of CD41 to CD9 in extracellular vesicles allows for differentiation between healthy controls and Parkinson's disease patients with very high accuracy. Furthermore, the ratio of CD41 to CD9 in extracellular vesicles allows for differentiation between Alzheimer's disease patients and Parkinson's disease patients with high accuracy.

[0135] Examples 4 and 5: Evaluation of Parkinson's disease using the ratio of the amount of CD9 in extracellular vesicles with PS to the amount of CD41 in extracellular vesicles with PS as an indicator. The "sample measurement value" (value (B)) obtained in Comparative Example 2 or Comparative Example 3 (Tim4 protein-anti-CD9 antibody sandwich ELISA method) was divided by the "sample measurement value" (value (A)) obtained in Example 1 or Comparative Example 1 (Tim4 protein-anti-CD41 antibody sandwich ELISA method) to calculate (B) / (A) (hereinafter referred to as "corrected sample measurement value"). Based on the calculated corrected sample measurement value, significance tests were performed between healthy individuals and Parkinson's disease patients, and between Alzheimer's disease patients and Parkinson's disease patients using the same method as in Example 1(4), and the AUC and p-value were calculated.

[0136] The results obtained are shown in Table 1 below. Figure 4 shows a box plot created based on the corrected sample measurements. In the figure, the vertical axis represents the corrected sample measurements ((B) / (A)), the horizontal axis shows Control for healthy individuals, AD for patients with Alzheimer's disease, and PD for patients with Parkinson's disease. After correction, the measured values ​​of the samples were highest in Parkinson's disease patients, followed by Alzheimer's disease patients, and then healthy controls. Differentiation between healthy controls and Parkinson's disease patients (Example 4) showed a statistically significant difference with an AUC of 0.927 and a p-value of less than 0.0001, and an AUC greater than 0.9. Similarly, differentiation between Alzheimer's disease patients and Parkinson's disease patients (Example 5) showed a statistically significant difference with an AUC of 0.84 and a p-value of 0.000428. These results indicate that the ratio of CD9 to CD41 in extracellular vesicles allows for differentiation between healthy controls and Parkinson's disease patients with very high accuracy. Furthermore, the ratio of CD9 to CD41 in extracellular vesicles allows for differentiation between Alzheimer's disease patients and Parkinson's disease patients with high accuracy.

[0137] Example 6: Evaluation of Parkinson's disease using the amount of CD61 in extracellular vesicles containing PS as an indicator. EDTA plasma from 30 healthy individuals and 16 Parkinson's disease (PD) patients was used as the measurement sample. Instead of biotin-labeled anti-CD41 mouse monoclonal antibody, biotin-labeled anti-CD61 mouse monoclonal antibody (1-55-4, MBL) using the biotin-labeling kit NH2 (Dojin Chemical Laboratories) was used as the detection antibody. Extracellular vesicles were measured by the Tim4 protein-anti-CD61 antibody sandwich ELISA method in the same manner as in Example 1. A significance test was performed between healthy individuals and Parkinson's disease patients using the obtained "sample measurement values," and the AUC and p-value were calculated.

[0138] The results obtained are shown in Table 2 below. Figure 5 shows a box plot created based on the sample measurement values. In the figure, the vertical axis represents the sample measurement values, the horizontal axis shows the results for healthy individuals (Control) and Parkinson's disease patients (PD). The sample measurement values ​​for Parkinson's disease patients were lower than those for healthy individuals. Differentiation between healthy individuals and Parkinson's disease patients was possible with an AUC of 0.788 and a p-value of 0.00108. From these results, it was found that healthy individuals and Parkinson's disease patients can be differentiated with high accuracy based on the amount of CD61 in extracellular vesicles.

[0139] Example 7: Evaluation of Parkinson's disease using the amount of CD41 in extracellular vesicles as an indicator. The same sample used for measurement as in Example 6 was used. Anti-CD41 mouse monoclonal antibody (MEM-06, abcam) was used as the capture sample, and biotin-labeled anti-CD41 mouse monoclonal antibody (PM6 / 248, SIGMA) using the biotin-labeling kit-NH2 (Dojin Chemical Laboratories) was used as the detection antibody. Extracellular vesicles were measured by the anti-CD41 antibody-anti-CD41 antibody sandwich ELISA method in the same manner as in Example 1. A significance test was performed between healthy individuals and Parkinson's disease patients using the obtained "sample measurement values," and the AUC and p-value were calculated.

[0140] The results obtained are shown in Table 2 below. Figure 6 shows a box plot created based on the sample measurement values. In the figure, the vertical axis represents the sample measurement values, the horizontal axis shows the results for healthy individuals (Control) and Parkinson's disease patients (PD). The sample measurement values ​​for Parkinson's disease patients were lower than those for healthy individuals. Differentiation between healthy individuals and Parkinson's disease patients was possible with an AUC of 0.708 and a p-value of 0.0206. From these results, it was found that healthy individuals and Parkinson's disease patients can be differentiated with high accuracy based on the amount of CD41.

[0141] Example 8: Evaluation of Parkinson's disease using the amount of CD61 in extracellular vesicles as an indicator. The same sample used for measurement as in Example 6 was used. Anti-CD61 mouse monoclonal antibody (1-55-4, MBL) was used as the capture antibody, and biotin-labeled anti-CD61 mouse monoclonal antibody (1-55-4, MBL) using the biotin-labeling kit-NH2 (Dojin Chemical Research Institute) was used as the detection antibody. Extracellular vesicles were measured by the anti-CD61 antibody-anti-CD61 antibody sandwich ELISA method in the same manner as in Example 1. A significance test was performed between healthy individuals and Parkinson's disease patients using the obtained "sample measurement values," and the AUC and p-value were calculated.

[0142] The results obtained are shown in Table 2 below. A box plot created based on the sample measurements is shown in Figure 7. In the figure, the vertical axis represents the sample measurements, the horizontal axis shows Control (results from healthy individuals) and PD (results from Parkinson's disease patients). The sample measurements from Parkinson's disease patients were lower than those from healthy individuals. Differentiation between healthy individuals and Parkinson's disease patients was possible with an AUC of 0.762 and a p-value of 0.00307. From these results, it was found that the amount of CD61 in extracellular vesicles can be used to differentiate between healthy individuals and Parkinson's disease patients with high accuracy.

[0143] Example 9: Evaluation of Alzheimer's disease using the amount of CD61 in extracellular vesicles containing PS as an indicator. EDTA plasma from 30 healthy individuals and 19 patients with Alzheimer's disease (AD) was used as measurement samples. Biotin-labeled anti-CD61 mouse monoclonal antibody (MBL) using the biotin-labeling kit NH2 (Dojin Chemical Laboratories) was used as the detection antibody. Extracellular vesicles were measured by the Tim4 protein-anti-CD61 antibody sandwich ELISA method in the same manner as in Example 1. A significance test was performed between healthy individuals and Alzheimer's disease patients using the obtained "sample measurement values," and AUC and p-values ​​were calculated.

[0144] The results obtained are shown in Table 3 below. Figure 8 shows a box plot created based on the sample measurement values. In the figure, the vertical axis represents the sample measurement values, the horizontal axis shows Control (results from healthy individuals) and AD (results from Alzheimer's disease patients). The sample measurement values ​​from Parkinson's disease patients were lower than those from healthy individuals. Differentiation between healthy individuals and Alzheimer's disease patients was achieved with an AUC of 0.774 and a p-value of 0.00105. From these results, it was found that the amount of CD61 in extracellular vesicles can be used to differentiate between healthy individuals and Alzheimer's disease patients with high accuracy.

[0145] [Table 1]

[0146] [Table 2]

[0147] Table 3

Claims

1. A method for assisting the diagnosis of neurodegenerative diseases, comprising detecting the amount of CD41 or CD61 in extracellular vesicles in a test sample, A method to aid in the diagnosis of neurodegenerative diseases, wherein an indicator including the amount of CD41 or CD61 is lower than that of subjects without neurodegenerative diseases, suggesting a neurodegenerative disease.

2. The aforementioned detection further includes detecting the amount of CD9 in extracellular vesicles, The index including the amount of CD41 or the amount of CD61 is the ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9. The method according to claim 1.

3. The method according to claim 1 or 2, wherein the extracellular vesicle is an extracellular vesicle having phosphatidylserine.

4. The method according to claim 1 or 2, wherein the test sample is a blood sample or cerebrospinal fluid.

5. The method according to claim 1 or 2, wherein the amount of CD41-positive extracellular vesicles or the amount of CD61-positive extracellular vesicles is used as the amount of CD41 or the amount of CD61, respectively.

6. The method according to claim 1 or 2, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease.

7. To detect the amount of CD41 or CD61 in extracellular vesicles in the test sample, and A method to assist in differentiating between Alzheimer's disease and Parkinson's disease, comprising detecting the amount of CD9 in extracellular vesicles, A lower ratio of the amount of CD41 to the amount of CD9, or the ratio of the amount of CD61 to the amount of CD9, compared to subjects without Parkinson's disease, suggests Parkinson's disease. A method to help differentiate between Alzheimer's disease and Parkinson's disease.

8. The method according to claim 7, wherein the extracellular vesicle is an extracellular vesicle having phosphatidylserine.

9. The method according to claim 7 or 8, wherein the test sample is a blood sample or cerebrospinal fluid.

10. The method according to claim 7 or 8, wherein the amount of CD41-positive extracellular vesicles, the amount of CD61-positive extracellular vesicles, or the amount of CD9-positive extracellular vesicles are used as the amount of CD41, the amount of CD61, or the amount of CD9, respectively.

11. A diagnostic kit for neurodegenerative diseases containing a substance that binds to CD41 or a substance that binds to CD61.

12. The kit according to claim 11, further comprising a substance that binds to CD9.

13. The kit according to claim 11 or 12, further comprising a substance that binds to phosphatidylserine.

14. A substance that binds to CD41, or a substance that binds to CD61, and A diagnostic kit for differentiating between Alzheimer's disease and Parkinson's disease, containing a substance that binds to CD9.

15. The identification kit according to claim 14, further comprising a substance that binds to phosphatidylserine.

16. A diagnostic biomarker for neurodegenerative diseases, comprising extracellular vesicles containing CD41 or CD61.

17. Extracellular vesicles having CD41, or extracellular vesicles having CD61, and Extracellular vesicles containing CD9 A set of biomarkers for the diagnosis of neurodegenerative diseases, including [specific biomarkers].

18. Extracellular vesicles having CD41, or extracellular vesicles having CD61, and Extracellular vesicles containing CD9 A set of biomarkers for differentiating between Alzheimer's disease and Parkinson's disease, including [specific biomarkers].

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