Methods, biomarkers, and reagent kits to aid in the differentiation of Lewy body disease from Alzheimer's disease

Abstract

The present invention relates to a method for assisting differentiation of Lewy body disease and Alzheimer's disease, a biomarker, and a reagent kit, said method including measuring the quantity of extracellular vesicles that have CD9 or extracellular vesicles that have phosphatidylserine and CD9, and using said quantity of extracellular vesicles that have CD9 or extracellular vesicles that have phosphatidylserine and CD9 as an indicator to determine whether a test subject has Lewy body disease or Alzheimer's disease.

Description

[Technical Field]

[0001] The present invention relates to methods, biomarkers, and reagent kits to aid in the differentiation of Lewy body disease from Alzheimer's disease. [Background technology]

[0002] Alzheimer's disease (AD) is a cause of dementia characterized by memory impairment and one or more cognitive impairments (aphasia, aphasia, agnosia, and executive dysfunction). The brains of Alzheimer's disease patients are characterized by numerous senile plaques formed by the accumulation of amyloid beta (Aβ) protein, and neurofibrillary tangles formed by the accumulation of phosphorylated tau protein, mainly in the cerebral cortex and hippocampus. AD is thought to develop as a result of neuronal cell death, synaptic loss, and decreased acetylcholine levels.

[0003] Parkinson's disease (PD) is a progressive degenerative disorder of the extrapyramidal system characterized by four symptoms: resting tremor, muscle rigidity, akinesia, and impaired postural reflexes. It is thought that the degeneration of nerve cells in the substantia nigra of the brain, combined with a lack of dopamine in the striatum, causes the body to become unable to move smoothly. Parkinson's disease patients frequently suffer from cognitive impairment, and when this becomes severe enough to interfere with daily life, the condition is called Parkinson's disease dementia (PDD).

[0004] Lewy body disease (LBD) is a disease concept that encompasses all pathological conditions characterized by the presence of Lewy bodies. Lewy body diseases include dementia with Lewy bodies (DLB), Parkinson's disease (PD), and Parkinson's disease-type dementia (PDD). The diagnostic criteria for DLB include progressive cognitive decline that interferes with daily activities, as well as the presence of two or more core features: one or more Parkinsonism symptoms (resting tremor, muscle rigidity, akinesia, impaired postural reflexes), cognitive fluctuations with marked changes in attention and clarity, recurring, concrete, and structured visual hallucinations, and rapid eye movement (REM) sleep behavior disorder. In Parkinson's disease, Lewy bodies are found in neurons in the substantia nigra and locus coeruleus of the midbrain, which are part of the brainstem. In dementia with Lewy bodies, Lewy bodies tend to be found throughout the brain, including neurons in the limbic system and cerebral cortex. Because the symptoms of Lewy body dementia (LDB) and Parkinson's disease dementia (PDD) are similar, in clinical practice, Parkinson's disease dementia is diagnosed when parkinsonism is present for more than one year before the onset of dementia, and Lewy body dementia is diagnosed when cognitive symptoms appear before parkinsonism or within one year after the onset of parkinsonism.

[0005] One method for testing for dementia is the Mini-Mental State Examination (MMSE), and subjects are suspected of having dementia if they score 23 or less out of a possible 30 points on the MMSE. A method for diagnosing Alzheimer's disease has been reported in which neuron-derived extracellular vesicles in the blood are concentrated using anti-NCAM antibodies or anti-L1CAM antibodies, and Aβ42, tau, phosphorylated tau, and other components contained in the neuron-derived extracellular vesicles are used as indicators to determine Alzheimer's disease (Patent Document 1, Patent Document 2, Non-Patent Document 1, Non-Patent Document 2). Another method for diagnosing Parkinson's disease has been reported in which neuron-derived extracellular vesicles in the blood are concentrated using anti-NCAM antibodies or anti-L1CAM antibodies, and α-synuclein and other components contained in the extracellular vesicles are used as indicators to determine Parkinson's disease (Patent Document 1, Patent Document 2, Non-Patent Document 1, Non-Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2016-535283 [Patent Document 2] Special Publication No. 2017-525976 [Non-patent literature]

[0007] [Non-Patent Document 1] CN Winston et al. / Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring (2016) 3 :63-72 [Non-patent document 2] Mustapic M et al. Front. Neurosci. (2017) 11:278 Summary of the Invention [Problem to be solved by the invention]

[0008] However, no biomarker is known that can distinguish between Lewy body disease and Alzheimer's disease. In view of the above-mentioned situation, an objective of the present invention is to provide a method, a biomarker, and a reagent kit that assist in distinguishing between Lewy body disease and Alzheimer's disease. [Means for solving the problem]

[0009] The present inventors investigated whether specific extracellular vesicles could be biomarkers to assist in the differentiation of dementia with Lewy bodies from Alzheimer's disease. As a result, the present inventors newly discovered that, among extracellular vesicles, extracellular vesicles having CD9 or extracellular vesicles having phosphatidylserine and CD9 could be biomarkers to assist in the differentiation of dementia with Lewy bodies from Alzheimer's disease, thereby completing the present invention.

[0010] That is, it has been found that the above object can be achieved by the following configuration. [1] A method for assisting in the differentiation of Lewy body disease from Alzheimer's disease, comprising measuring the amount of extracellular vesicles having CD9 or the amount of extracellular vesicles having phosphatidylserine and CD9 in a biological sample derived from a subject suspected of having Lewy body disease or Alzheimer's disease, and determining whether the subject has Lewy body disease or Alzheimer's disease using the amount of extracellular vesicles having CD9 or the amount of extracellular vesicles having phosphatidylserine and CD9 as an indicator. [2] A method for assisting in the differentiation of Lewy body disease from Alzheimer's disease according to [1], wherein the Lewy body disease is Parkinson's disease-type dementia, Lewy body dementia, or / and Parkinson's disease. [3] The method for assisting in the differentiation of Lewy body disease and Alzheimer's disease described in [2], wherein the determination is made as Parkinson's disease dementia or Lewy body dementia when the amount of extracellular vesicles is greater than a first reference value, or / and as Parkinson's disease when the amount of extracellular vesicles is equal to or less than a second reference value. [4] A method for assisting in the differentiation of Lewy body disease from Alzheimer's disease according to any one of claims [1] to [3], wherein the Lewy body disease is Parkinson's disease-type dementia or Lewy body dementia, the measurement of the amount of extracellular vesicles is measuring the amount of extracellular vesicles having CD9, and the determination is made using the amount of extracellular vesicles having CD9 as an indicator. [5] A method for assisting in the differentiation of Lewy body disease from Alzheimer's disease, described in any one of [1] to [3], wherein the Lewy body disease is Parkinson's disease-type dementia or Lewy body dementia, the measurement of the amount of extracellular vesicles is measuring the amount of extracellular vesicles having the phosphatidylserine and CD9, and the determination is made using the amount of extracellular vesicles having the phosphatidylserine and CD9 as an indicator. [6] A method for assisting in the differentiation of Lewy body disease from Alzheimer's disease according to any one of [1] to [3], wherein the Lewy body disease is Parkinson's disease, the measurement of the amount of extracellular vesicles is measuring the amount of extracellular vesicles having CD9, and the determination is made using the amount of extracellular vesicles having CD9 as an indicator. [7] A method for assisting in the differentiation of Lewy body disease from Alzheimer's disease described in [4], in which the above-mentioned judgment is made to judge the disease as Parkinson's disease type dementia or Lewy body dementia if the amount of the above-mentioned extracellular vesicles is greater than a first reference value. [8] A method for assisting in the differentiation between Lewy body disease and Alzheimer's disease according to [4] or [7], wherein the determination is made that the patient has Alzheimer's disease if the amount of extracellular vesicles is equal to or less than a first standard value and greater than a second standard value. [9] A method for assisting in the differentiation of Lewy body disease from Alzheimer's disease described in [5], wherein the determination is made as Parkinson's disease dementia or Lewy body dementia if the amount of extracellular vesicles is greater than a first reference value.

[10] A method for assisting in the differentiation between Lewy body disease and Alzheimer's disease according to [5] or [9], wherein the determination is made that the patient has Alzheimer's disease if the amount of extracellular vesicles is equal to or less than a first standard value and greater than a second standard value.

[11] The method for assisting in the differentiation of Lewy body disease and Alzheimer's disease described in [6], wherein the determination is made as Parkinson's disease if the amount of extracellular vesicles is equal to or less than a second standard value.

[12] A method for assisting in the differentiation between Lewy body disease and Alzheimer's disease according to [6] or

[11] , wherein the determination is made that the patient has Alzheimer's disease if the amount of extracellular vesicles is equal to or less than a first standard value and greater than a second standard value.

[13] A reagent kit for assisting in the differentiation of Lewy body disease from Alzheimer's disease, containing a substance with affinity for CD9.

[14] The reagent kit for assisting in the differentiation between Lewy body disease and Alzheimer's disease according to

[13] , further comprising a substance having affinity for phosphatidylserine.

[15] A reagent kit for assisting in the differentiation of Lewy body disease and Alzheimer's disease according to

[13] or

[14] , wherein the Lewy body disease is Parkinson's disease-type dementia, Lewy body dementia, or / and Parkinson's disease.

[16] Biomarkers to assist in the differentiation of Lewy body disease from Alzheimer's disease, including extracellular vesicles bearing CD9 or extracellular vesicles bearing phosphatidylserine and CD9.

[17] The biomarker for assisting in the differentiation of Lewy body disease and Alzheimer's disease according to

[16] , wherein the Lewy body disease is Parkinson's disease-type dementia, Lewy body dementia, or / and Parkinson's disease.

[0011] Furthermore, the present inventors have also found that extracellular vesicles having CD9, or extracellular vesicles having phosphatidylserine and CD9, can serve as biomarkers to assist in the differentiation of Parkinson's disease from Parkinson's disease-type dementia or dementia with Lewy bodies, and have completed the following inventions. [1A] A method for assisting in the differentiation of Parkinson's disease-type dementia or dementia with Lewy bodies from Parkinson's disease, comprising measuring the amount of CD9-bearing extracellular vesicles or the amount of extracellular vesicles bearing phosphatidylserine and CD9 in a biological sample derived from a subject suspected of having Parkinson's disease-type dementia or dementia with Lewy bodies, or Parkinson's disease, and determining whether the subject has Parkinson's disease-type dementia or dementia with Lewy bodies or Parkinson's disease using the amount of CD9-bearing extracellular vesicles or the amount of phosphatidylserine and CD9 as an indicator. [2A] The method for assisting in the differentiation of Parkinson's disease dementia or dementia with Lewy bodies from Parkinson's disease according to [1A], wherein the determination is made to determine the presence of Parkinson's disease dementia or dementia with Lewy bodies when the amount of extracellular vesicles is greater than a third reference value (the amount of the biomarker of the present invention for differentiating Parkinson's disease dementia or dementia with Lewy bodies from Parkinson's disease, which is set, for example, to be equal to or greater than the second reference value and equal to or less than the first reference value), and / or to determine the presence of Parkinson's disease when the amount of extracellular vesicles is equal to or less than the third reference value. [3A] A reagent kit for assisting in the differentiation of Parkinson's disease from Parkinson's disease-type dementia or dementia with Lewy bodies, which comprises a substance having affinity for CD9. [4A] A reagent kit for assisting in the differentiation of Parkinson's disease type dementia or dementia with Lewy bodies from Parkinson's disease according to [3A], further comprising a substance having affinity for phosphatidylserine. [5A] A biomarker for assisting in the differentiation of Parkinson's disease type dementia or dementia with Lewy bodies from Parkinson's disease, comprising extracellular vesicles having CD9 or extracellular vesicles having phosphatidylserine and CD9. [Effects of the Invention]

[0012] According to the present invention, it is possible to assist in the differentiation between Lewy body disease and Alzheimer's disease. Specifically, it is possible to assist in the differentiation between Parkinson's disease-type dementia, Lewy body dementia, and / or Parkinson's disease and Alzheimer's disease. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 is a box-and-whisker graph showing the evaluation of plasma samples from Alzheimer's disease patients and patients with Parkinson's disease dementia or Lewy body dementia using the amount of CD9-bearing exosomes as an indicator. [Figure 2] Figure 2 is a box-and-whisker graph showing the evaluation of plasma samples from Alzheimer's disease patients and patients with Parkinson's disease dementia or dementia with Lewy bodies using the amount of exosomes containing CD9 and phosphatidylserine as an indicator. DETAILED DESCRIPTION OF THE INVENTION

[0014] In this specification, when indicating the upper and lower limits of a range, unless otherwise specified, A to B indicates that the range is equal to or greater than A and equal to or less than B. Furthermore, in this specification, measurement includes quantitative, semi-quantitative, and qualitative.

[0015] The extracellular vesicles of the present invention are small membrane vesicles derived from cells and composed of a lipid bilayer membrane. In the present invention, the diameter of the extracellular vesicles can be measured, for example, by nanoparticle tracking analysis (NTA) using a nanoparticle analysis system (NanoSight). The diameter of the extracellular vesicles refers to the number-average particle size. Examples of the extracellular vesicles include those having a diameter of 20 nm to 1,000 nm, preferably 50 nm to 800 nm, more preferably 50 nm to 500 nm, and particularly preferably 50 nm to 200 nm. The extracellular vesicles can be classified in various ways based on their origin and the size of the small membrane vesicles, as described, for example, in Nature Reviews Immunology 9, 581-593 (August 2009) and "Obesity Research" Vol. 13 No. 2 2007 Topics, Naoto Aoki et al. Specific examples include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, apoptotic bodies, adiposomes, etc., with exosomes and microvesicles being preferred, and exosomes being more preferred.

[0016] The exosomes are small membrane vesicles derived from cells and composed of a lipid bilayer membrane, and may have a diameter of, for example, 50 to 200 nm, preferably 50 to 150 nm, and more preferably 50 to 100 nm. Exosomes are thought to be derived from late endosomes.

[0017] The microvesicles are small membrane vesicles derived from cells and composed of a lipid bilayer membrane, and may have a diameter of, for example, 100 nm to 1000 nm, preferably 100 nm to 800 nm, and more preferably 100 nm to 500 nm. Microvesicles are believed to be derived from cell membranes.

[0018] The extracellular vesicles may be contained in a biological sample derived from a subject, or may be isolated from a biological sample derived from a subject, and are preferably isolated from a biological sample derived from a subject.

[0019] The subject-derived biological sample may be any sample capable of containing extracellular vesicles, including blood-derived samples such as serum, plasma, whole blood, and buffy coat, as well as body fluid samples such as cerebrospinal fluid, urine, saliva, semen, thoracic exudate, cerebrospinal fluid, tears, cysts, mucus, lymph, ascites, pleural effusion, amniotic fluid, bladder washings, and bronchoalveolar lavage fluid. Blood-derived samples or cerebrospinal fluid are preferred, with serum, plasma, or cerebrospinal fluid being more preferred, serum or plasma being even more preferred, and plasma being particularly preferred. Blood-derived samples are more useful because they impose a lighter burden on the subject. The subject-derived biological sample may be collected directly from the subject, or may have undergone pretreatment such as collection, concentration, purification, isolation, dilution with a buffer solution, or filtration sterilization. These pretreatments may be performed as appropriate according to conventional methods. Hereinafter, the subject-derived biological sample may be referred to as the "biological sample."

[0020] The method for isolating extracellular vesicles from the biological sample may be carried out according to a conventional method and is not particularly limited. Examples of the method for isolating extracellular vesicles from the biological sample include affinity methods (e.g., PS affinity methods), differential centrifugation methods (e.g., ultracentrifugation such as pellet-down methods, sucrose cushion methods, and density gradient centrifugation), immunoprecipitation, chromatography methods (e.g., ion exchange chromatography, gel permeation chromatography), density gradient methods (e.g., sucrose density gradient methods), electrophoresis methods (e.g., organelle electrophoresis), magnetic separation methods (e.g., magnetic activated cell sorting (MACS)), ultrafiltration concentration methods (e.g., nanomembrane ultrafiltration concentration methods), Percoll gradient isolation methods, methods using microfluidic devices, and PEG precipitation methods. Among these, affinity methods are preferred because they can obtain highly purified extracellular vesicles, and differential centrifugation methods are preferred because they theoretically enable unbiased recovery. Affinity methods or ultracentrifugation methods are more preferred, and affinity methods are particularly preferred. Among affinity methods, the PS affinity method, which is affinity purification for phosphatidylserine, is preferred. The affinity method and differential centrifugation may be performed, for example, according to the method described in International Publication No. 2016 / 088689. These isolation methods may be used alone or in combination of two or more. Furthermore, isolation by one isolation method may be repeated two or more times.

[0021] The subject in the present invention is a subject suspected of having Alzheimer's disease or Lewy body disease (e.g., dementia with Lewy bodies, Parkinson's disease dementia, Parkinson's disease), and examples thereof include humans suspected of having developed Alzheimer's disease or Lewy body disease, or humans suspected of being at risk of developing Alzheimer's disease or Lewy body disease.

[0022] <Biomarkers to assist in the differentiation of Lewy body disease and Alzheimer's disease> The biomarker of the present invention for assisting in the differentiation of Lewy body disease from Alzheimer's disease (hereinafter sometimes abbreviated as the biomarker of the present invention) comprises extracellular vesicles having CD9, or extracellular vesicles having phosphatidylserine and CD9. The extracellular vesicles in the biomarker of the present invention are the same as those described above, and preferred examples are also the same.

[0023] <Methods to help differentiate between Lewy body disease and Alzheimer's disease> The method of the present invention for assisting in the differentiation between Lewy body disease and Alzheimer's disease (hereinafter sometimes abbreviated as the differentiation assistance method of the present invention) comprises measuring the amount of extracellular vesicles having CD9 or the amount of extracellular vesicles having phosphatidylserine and CD9 in a biological sample derived from a subject (hereinafter sometimes abbreviated as the measurement step), and determining whether the subject has Lewy body disease or Alzheimer's disease using the amount of extracellular vesicles having CD9 or the amount of extracellular vesicles having phosphatidylserine and CD9 as an indicator (hereinafter sometimes abbreviated as the determination step).

[0024] The biological sample, subject, and extracellular vesicles in the differentiation assistance method of the present invention are the same as those described above, and the preferred embodiments are also the same.

[0025] The "quantity" in the discrimination assistance method of the present invention includes mass and concentration. The "quantity" also includes measured values ​​that correlate with mass or concentration (e.g., absorbance, absorbance change, transmitted light, transmitted light change, fluorescence intensity, fluorescence intensity change, luminescence amount, luminescence change, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change rate, refractive index, refractive index change rate, etc.).

[0026] In the differentiation assisting method of the present invention, the measurement of the amount of CD9-bearing extracellular vesicles and the measurement of the amount of phosphatidylserine and CD9-bearing extracellular vesicles (measurement of the amount of the biomarker of the present invention) are not particularly limited as long as they are methods commonly used in this field. Examples of methods for measuring the amount of the biomarker of the present invention include a measurement method utilizing the affinity between a substance having affinity for the biomarker of the present invention and the biomarker of the present invention, mass spectrometry, and a combination of these. Furthermore, examples of substances having affinity for the biomarker of the present invention include substances having affinity for CD9 or phosphatidylserine contained in the biomarker of the present invention, and substances having affinity for phosphatidylserine, and substances that specifically bind to the biomarker of the present invention are preferred.

[0027] The substance having affinity for CD9 may be any substance that binds to CD9, and is preferably a substance that specifically binds to CD9. Examples of the substance having affinity for CD9 include antibodies that bind to CD9 (hereinafter sometimes abbreviated as anti-CD9 antibodies), proteins that bind to CD9 such as lectins that bind to sugar chains on CD9, and nucleic acids that bind to CD9 (aptamers), with proteins that bind to CD9 being preferred and anti-CD9 antibodies being more preferred.

[0028] The substance having affinity for phosphatidylserine may be any substance that binds to phosphatidylserine, and is preferably a substance that specifically binds to phosphatidylserine. Examples of the substance having affinity for phosphatidylserine include proteins that bind to phosphatidylserine and nucleic acids (aptamers) 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 (hereinafter sometimes abbreviated as anti-PS antibodies) and phosphatidylserine-affinity proteins, and phosphatidylserine-affinity proteins are preferred. Examples of anti-PS antibodies include anti-phosphatidylserine antibody 1H6 (Merck, Inc.). Examples of phosphatidylserine affinity proteins include Tim proteins such as Tim1 (T-cell immunoglobulin-mucin domain-containing molecule 1, T-cell immunoglobulin-mucin-domain 1), Tim2 (T-cell immunoglobulin-mucin domain-containing molecule 2, T-cell immunoglobulin-mucin-domain 2), Tim3 (T-cell immunoglobulin-mucin domain-containing molecule 3, T-cell immunoglobulin-mucin-domain 3), and Tim4 (T-cell immunoglobulin-mucin domain-containing molecule 4, T-cell immunoglobulin-mucin-domain 4); Annexin V; and MFG-E8. Tim proteins and anti-PS antibodies are preferred, with Tim proteins being more preferred. Tim proteins are also preferred, with Tim1 and Tim4 being more preferred. The above-mentioned substances having affinity for phosphatidylserine may be used alone or in combination, with the use of only one type being preferred.

[0029] The above-mentioned substance having affinity for CD9 and the above-mentioned substance having affinity for phosphatidylserine may be commercially available or may be prepared by standard methods. The above-mentioned anti-CD9 antibody and the above-mentioned anti-PS antibody may be either a polyclonal antibody or a monoclonal antibody, and these may be used alone or in combination. The above-mentioned anti-CD9 antibody and the above-mentioned anti-PS antibody may be not only an intact immunoglobulin molecule, but also fragment antibodies such as Fab, F(ab'), and F(ab') that are capable of binding to antigens, or synthetic antibodies such as single-chain Fv, diabodies, triabodies, and tetrabodies. These antibodies may be prepared, for example, according to the method described in "Immunoassay Methods" (edited by the Biochemical Measurement Research Group, Kodansha, 2014).

[0030] The substance having affinity for CD9 and the substance having affinity for phosphatidylserine may be labeled with a labeling substance, such as enzymes such as peroxidase, microperoxidase, and alkaline phosphatase; 99m Tc, 131 I, 125 I, 14 C. 3 H, 32 P, 35Examples of suitable labeling agents include radioisotopes such as S; 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 act as spin labeling agents, such as compounds containing an oxyl group, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl; and nanoparticles such as gold colloids and quantum dots. Enzymes and fluorescent substances are preferred. Measurement of these labeling agents can be performed according to known methods appropriate for the labeling agent. Furthermore, a secondary affinity substance (e.g., a secondary antibody) that binds to the substance having affinity for CD9 or the substance having affinity for phosphatidylserine (hereinafter referred to as the primary affinity substance) may also be used. The secondary affinity substance is preferably labeled with a labeling substance, and a secondary antibody labeled with a labeling substance is more preferred. The labeling substance is the same as that described above, and the preferred methods are also the same. The labeling method for the primary affinity substance and the secondary affinity substance is not particularly limited. The primary affinity substance and the secondary affinity substance may be directly labeled, or may be indirectly labeled via a ligand and receptor, such as avidin and biotin. Examples of indirect labeling methods include a method in which either avidin or biotin is bound to a substance having affinity for CD9 or a substance having affinity for phosphatidylserine (primary affinity substance), or a method in which the remaining one of avidin and biotin is bound to a labeling substance, thereby utilizing the affinity between avidin and biotin. Examples of biotins include biotin, iminobiotin, desthiobiotin, biocytin, biotin sulfoxide, etc., with biotin being preferred. Examples of avidins include avidin, tamapidine, tamapidine 2, streptavidin, etc., with streptavidin being preferred.The method of binding either a ligand or a receptor (e.g., avidins and biotins) to the substance having affinity for CD9 or the substance having affinity for phosphatidylserine (primary affinity substance), and the method of binding the other ligand or receptor (e.g., avidins and biotins) to a labeled substance may be carried out according to conventional methods. The method of indirectly labeling a secondary affinity substance is the same as the method of indirectly labeling a primary affinity substance, and specific examples and preferred examples are also the same.

[0031] The substance having affinity for CD9 and the substance having affinity for phosphatidylserine may be immobilized on a solid phase, and it is preferable that the substance having affinity for phosphatidylserine is immobilized on a solid phase.

[0032] Examples of the solid phase include synthetic polymers such as latex, polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber; and inorganic materials such as porous glass, ground glass, ceramics, alumina, silica gel, activated carbon, and metal oxides. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples include microtiter plates (ELISA plates), beads, tubes (microtubes), particles, dedicated trays with multiple tubes molded into one piece, disk-shaped pieces, and test tubes. The method for immobilizing the substance having affinity for CD9 and the substance having affinity for phosphatidylserine on the solid phase is not particularly limited, as long as it is a method commonly used in this field, and may be performed according to a conventional method.

[0033] The combination of the substance having affinity for CD9 and the substance having affinity for phosphatidylserine is not particularly limited, but is preferably a combination of the protein that binds to CD9 and the protein that binds to phosphatidylserine, and more preferably a combination of the anti-CD9 antibody and the anti-PS antibody or the phosphatidylserine affinity protein. Examples of the combination of the anti-CD9 antibody and the anti-PS antibody or the phosphatidylserine affinity protein include a combination of an anti-CD9 antibody and a Tim protein or an anti-PS antibody, preferably a combination of an anti-CD9 antibody and a Tim protein, more preferably a combination of an anti-CD9 antibody and Tim1 or Tim4, and particularly preferably a combination of an anti-CD9 antibody and Tim4.

[0034] Examples of the measurement method utilizing the affinity for the biomarker of the present invention include the sandwich method (e.g., sandwich ELISA method, AlphaLISA TM Law (Exoscreen TM immunoassay methods using antibodies that bind to the biomarkers of the present invention, such as immunoassays (fluorescence resonance energy transfer (FRET) method, bioluminescence resonance energy transfer (BRET) method, etc.), competitive methods, agglutination methods (immunoturbidimetry, immunoturbidimetry), immunochromatography, capillary electrophoresis, Western blot method, and surface plasmon resonance (SPR) method, are also included; sandwich methods (e.g., sandwich ELISA method, AlphaLISA method, etc.) TM Law (Exoscreen TMThe measurement principle may be either a homogeneous method or a heterogeneous method. Immunological measurement methods classified by labeling are not particularly limited, and examples include enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescent enzyme immunoassay (FEIA), fluorescent immunoassay (FIA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), and electrochemiluminescent immunoassay (ECLIA), with enzyme-linked immunosorbent assay (ELISA) and chemiluminescent enzyme immunoassay (CLEIA) being preferred. Furthermore, when a substance having affinity for the biomarker of the present invention other than an antibody that binds to the biomarker of the present invention is used, the immunoassay may be performed in accordance with the above-mentioned immunoassay, for example, using a substance having affinity for the tetraspanin other than an antibody that binds to the tetraspanin, or a substance having affinity for the phosphatidylserine other than the anti-PS antibody, and preferred methods include those similar to the above-mentioned immunoassay. The above-mentioned immunoassay and methods similar to the above-mentioned immunoassay are preferred as methods for measuring the biomarker of the present invention.

[0035] Specifically, the amount of the biomarker of the present invention may be measured in accordance with the method described in International Publication No. 2016 / 088689, the entire disclosure of which is incorporated herein by reference.

[0036] Specifically, a preferred method for measuring the amount of a biomarker of the present invention is, for example, a method (sandwich method) comprising the steps of: (1) contacting a biomarker of the present invention in a biological sample with a first affinity substance for the biomarker of the present invention (hereinafter sometimes abbreviated as the first affinity substance) and a second affinity substance for the biomarker of the present invention (hereinafter sometimes abbreviated as the second affinity substance) to form a complex containing the biomarker of the present invention in the biological sample, the first affinity substance, and the second affinity substance (hereinafter sometimes abbreviated as the "complex formation step"); and (2) measuring the amount of the complex (hereinafter sometimes abbreviated as the "complex amount measurement step").

[0037] The complex formation process preferably includes a first step of contacting a biological sample with a first affinity substance to form a first complex consisting of a biomarker of the present invention in the biological sample and the first affinity substance, and a second step of contacting the first complex with a second affinity substance to form a second complex consisting of the first complex and the second affinity substance.

[0038] When measuring extracellular vesicles having CD9 as a biomarker of the present invention, the first affinity substance and the second affinity substance are substances having affinity for the above-mentioned CD9, and specific and preferred examples are the same as those described above. Furthermore, the combination of the first affinity substance and the second affinity substance is not particularly limited, and a combination in which both the first affinity substance and the second affinity substance are anti-CD9 antibodies is preferred. Furthermore, the first affinity substance and the second affinity substance may be the same or different (for example, the first affinity substance and the second affinity substance do not compete with each other).

[0039] When measuring extracellular vesicles having phosphatidylserine and CD9 as biomarkers of the present invention, the first affinity substance is a substance having affinity for the phosphatidylserine, and the second affinity substance is a substance having affinity for the CD9, and specific examples and preferred examples are the same as those described above. The combination of the first affinity substance and the second affinity substance is the same as the "combination of a substance having affinity for CD9 and a substance having affinity for phosphatidylserine" described above, and specific examples and preferred examples are the same as those described above.

[0040] In the complex formation step, the order of contacting the first affinity substance, the second affinity substance, and the biological sample is not particularly limited, but it is preferable to contact the first affinity substance with the biological sample, and then contact the second affinity substance. Specifically, when measuring extracellular vesicles having phosphatidylserine and CD9 as the biomarkers of the present invention, it is preferable to contact the biological sample with a substance having affinity for phosphatidylserine as the first affinity substance, and then contact the biological sample with a substance having affinity for CD9 as the second affinity substance. Furthermore, when measuring extracellular vesicles having CD9 as the biomarker of the present invention, it is preferable to contact the biological sample with a substance having affinity for CD9 as the first affinity substance, and then contact the biological sample with a substance having affinity for CD9 as the second affinity substance.

[0041] More specifically, in the complex formation step, for example, when measuring extracellular vesicles having CD9 as a biomarker of the present invention, a biological sample is contacted with an anti-CD9 antibody immobilized on a solid phase to form a first complex between the anti-CD9 antibody and the extracellular vesicles having phosphatidylserine and CD9 in the biological sample, and the first complex is contacted with the anti-CD9 antibody to form a second complex between the first complex and the anti-CD9 antibody. Furthermore, when measuring extracellular vesicles having phosphatidylserine and CD9 as a biomarker of the present invention, a biological sample is contacted with an anti-PS antibody or Tim protein (preferably Tim1 or Tim4, more preferably Tim4) immobilized on a solid phase to form a first complex between the anti-PS antibody or Tim protein and the extracellular vesicles having phosphatidylserine and CD9 in the biological sample, and the first complex is contacted with the anti-CD9 antibody to form a second complex between the first complex and the anti-CD9 antibody.

[0042] After forming the complex, it is preferable to carry out a washing operation (B / F separation) at least before the step of measuring the amount of the complex. Specifically, for example, in the above-mentioned method, the washing operation may be carried out after forming the first complex and / or after forming the second complex, and it is preferable to carry out the washing operation (B / F separation) after forming the first complex, and then carry out the washing operation (B / F separation) after forming the second complex.

[0043] The complex amount measurement step is a step of measuring the amount of a complex (second complex) obtained in the complex formation step, which contains a first affinity substance, a second affinity substance, and a biomarker of the present invention. Any method capable of measuring the amount of the complex may be used. For example, the first affinity substance or the second affinity substance in the second complex, which is directly or indirectly labeled with a labeling substance, may be measured. Specifically, the complex amount measurement step preferably measures the second complex obtained in the complex formation step by, for example, (1) a method using a second affinity substance directly or indirectly labeled with a labeling substance, or (2) a method using a secondary affinity substance (e.g., a secondary antibody directly or indirectly labeled with a labeling substance) that binds to the second affinity substance (primary affinity substance) and is further labeled directly or indirectly with a labeling substance. In the complex amount measurement step, a washing procedure (B / F separation) is preferably performed before detecting the labeled substance.

[0044] More specifically, the complex amount measuring step preferably measures the second complex obtained in the complex formation step by, for example, a method using (1) an anti-CD9 antibody directly or indirectly labeled with a labeling substance, or (2) a secondary antibody that binds to the anti-CD9 antibody (primary affinity substance) and is further labeled directly or indirectly with a labeling substance. Furthermore, in the complex amount measuring step, a washing procedure (B / F separation) is preferably performed before detecting the labeling substance.

[0045] The amount of the biomarker of the present invention can be measured, as an embodiment of the sandwich method, by a proximity-based measurement method (e.g., AlphaLISA) that utilizes a proximity signal generated by the acceptor and / or donor when the acceptor and donor are brought into close proximity. TM The proximity-based measurement method may be performed by, for example, a method in which the first affinity substance and the second affinity substance are immobilized on different first and second beads (acceptor or donor), respectively. One of the first and second beads is a donor, and a photosensitizer contained in the donor releases singlet oxygen upon excitation. The other of the first and second beads is an acceptor. A complex is formed between the first bead bound to the first affinity substance, the biomarker of the present invention, and the second bead bound to the second affinity substance, and the complex is irradiated with excitation light, thereby releasing singlet oxygen from the photosensitizer of the donor. Only when the complex is formed and the first and second beads are in close proximity, singlet oxygen reaches the acceptor, causing the acceptor to initiate a chemiluminescent reaction, and the resulting light is detected, allowing the amount of the biomarker of the present invention to be measured. The amount of the biomarker of the present invention can also be measured using AlphaLISA. TM Improved Exoscreen TM This may also be done using the method (Yoshioka et al., NatCommun, 2014).

[0046] The amount of biological sample, the amount (concentration) of proteins in the biological sample, the amount (concentration) and particle number of the biomarker of the present invention in the biological sample, the amount (concentration) of the substance having affinity for the biomarker of the present invention to be reacted with these, the labeling substance, the labeling method, etc. may be appropriately determined depending on the type of biological sample, the required measurement sensitivity, the measurement method and measurement device to be used, etc. The amount (concentration) of the biomarker of the present invention in the biological sample may also be calculated by preparing a calibration curve using a standard. Examples of the standard include extracellular vesicles containing phosphatidylserine and CD9, extracellular vesicles containing CD9, etc.

[0047] The method of the present invention for assisting in the differentiation between Lewy body disease and Alzheimer's disease (the differentiation assistance method of the present invention) comprises determining whether a subject has Lewy body disease or Alzheimer's disease using the amount of a biomarker of the present invention as an index. That is, the differentiation assistance method of the present invention comprises providing the amount of a biomarker of the present invention as an index for determining whether a subject has Lewy body disease or Alzheimer's disease. Examples of the determination include determining whether a subject suspected of having Lewy body disease or Alzheimer's disease is likely to have Lewy body disease or Alzheimer's disease (determining whether a subject is likely to have Lewy body disease, determining whether a subject is likely to have Alzheimer's disease), or determining whether a subject is likely to have Lewy body disease or Alzheimer's disease (determining whether a subject is likely to have Lewy body disease, determining whether a subject is likely to have Alzheimer's disease). Examples of the determination include determining whether a subject suspected of having Lewy body disease or Alzheimer's disease has a high risk of developing Lewy body disease or Alzheimer's disease (determining whether a subject has a high risk of developing Lewy body disease, determining whether a subject has a high risk of developing Alzheimer's disease), or determining whether a subject has a risk of developing Lewy body disease or Alzheimer's disease (determining whether a subject has a risk of developing Lewy body disease, determining whether a subject has a risk of developing Alzheimer's disease). Examples of the determination include determining whether a subject suspected of having Lewy body disease or Alzheimer's disease is likely to have Lewy body disease or Alzheimer's disease, or determining whether a subject has Lewy body disease or Alzheimer's disease. Furthermore, by determining whether a subject has Lewy body disease or Alzheimer's disease, changes in the likelihood of the subject having the disease or the risk of developing the disease in the subject may be monitored over time.

[0048] More specifically, the diagnosis assistance method of the present invention comprises determining whether a subject has dementia with Lewy bodies, Parkinson's disease, Alzheimer's disease, or Parkinson's disease using the amount of the biomarker of the present invention as an index. That is, the diagnosis assistance method of the present invention comprises providing the amount of the biomarker of the present invention as an index for determining whether a subject has dementia with Lewy bodies, Parkinson's disease, Alzheimer's disease, or Parkinson's disease. Examples of the above determination include determination of the possibility of a subject having dementia with Lewy bodies, dementia of Parkinson's disease, Alzheimer's disease, or Parkinson's disease, such as determination of whether or not the subject is likely to have dementia with Lewy bodies, dementia of Parkinson's disease, Alzheimer's disease, or Parkinson's disease (determination of whether or not the subject is likely to have dementia with Lewy bodies or dementia of Parkinson's disease, determination of whether or not the subject is likely to have Alzheimer's disease, determination of whether or not the subject is likely to have Parkinson's disease), or determination of the possibility of a subject having dementia with Lewy bodies, dementia of Parkinson's disease, Alzheimer's disease, or Parkinson's disease (determination of whether or not the subject is likely to have dementia with Lewy bodies or dementia of Parkinson's disease, determination of whether or not the subject is likely to have Alzheimer's disease, determination of whether or not the subject is likely to have Parkinson's disease).The above-mentioned determination may include determination of whether or not a subject suspected of being at risk of developing dementia with Lewy bodies or Parkinson's disease, Alzheimer's disease, or Parkinson's disease has a high risk of developing dementia with Lewy bodies or Parkinson's disease, Alzheimer's disease, or Parkinson's disease (determination of whether or not a subject has a high risk of developing dementia with Lewy bodies or Parkinson's disease, determination of whether or not a subject has a high risk of developing Alzheimer's disease, determination of whether or not a subject has a high risk of developing Parkinson's disease), determination of the presence or absence of a risk of developing dementia with Lewy bodies or Parkinson's disease, Alzheimer's disease, or Parkinson's disease (determination of whether or not a subject has a high risk of developing dementia with Lewy bodies or Parkinson's disease, Alzheimer's disease, or Parkinson's disease). Examples of such determinations include determinations for assessing the risk of onset, such as determination of whether or not a subject is at risk of developing dementia with Lewy bodies or Parkinson's disease-type dementia, determination of whether or not a subject is at risk of developing Alzheimer's disease, and determination of whether or not a subject is at risk of developing Parkinson's disease. Preferred are determinations of whether or not a subject suspected of having dementia with Lewy bodies or Parkinson's disease-type dementia, Alzheimer's disease, or Parkinson's disease is likely to have dementia with Lewy bodies or Parkinson's disease-type dementia, Alzheimer's disease, or Parkinson's disease, and determination of whether or not a subject is likely to have dementia with Lewy bodies or Parkinson's disease-type dementia, Alzheimer's disease, or Parkinson's disease.

[0049] In the differentiation assistance method of the present invention, the determination of Lewy body disease and Alzheimer's disease is made, for example, by comparing the amount of the biomarker of the present invention in a biological sample derived from a subject, obtained by measuring the amount of the biomarker of the present invention, with a predetermined reference value (cutoff value). As the reference, for example, a first reference value for determining Parkinson's disease dementia or dementia with Lewy bodies from Alzheimer's disease, and / or a second reference value for determining Alzheimer's disease from Parkinson's disease, may be set. The first reference value is a value (amount of the biomarker of the present invention) greater than the second reference value. By using the first reference value, it can be determined that a subject has a high or possible risk of suffering from Parkinson's disease dementia or dementia with Lewy bodies (or simply Lewy body disease) when the amount of the biomarker of the present invention is greater than the first reference value. By using the second reference value, when the amount of the biomarker of the present invention is equal to or less than the second reference value, it can be determined that the subject is highly likely or likely to have Parkinson's disease (or simply Lewy body disease), or is at high risk or at risk of developing Parkinson's disease. Furthermore, by using the first reference value and the second reference value, it can be determined that the subject is highly likely or likely to have Alzheimer's disease, or is at high risk or at risk of developing Alzheimer's disease, when the amount of the biomarker of the present invention is equal to or less than the first reference value and greater than the second reference value.

[0050] In the differentiation assistance method of the present invention, the determination of Lewy body disease and Alzheimer's disease may be performed using only the first reference value to determine whether a subject is suffering from Parkinson's disease dementia or Lewy body dementia, or Alzheimer's disease, in a subject suspected of having Parkinson's disease dementia or Lewy body dementia, or Alzheimer's disease. Specifically, if the amount of the biomarker of the present invention is greater than the first reference value, the subject is determined to have a high or possible risk of Parkinson's disease dementia or Lewy body dementia, or to have a high or possible risk of developing the disease. If the amount of the biomarker of the present invention is equal to or less than the first reference value, the subject is determined to have a high or possible risk of Alzheimer's disease, or to have a high or possible risk of developing the disease. Furthermore, it is preferable to determine that if the amount of the biomarker of the present invention is equal to or less than the first reference value and greater than the second reference value, the subject is determined to have a high or possible risk of Alzheimer's disease, or to have a high or possible risk of developing the disease. In the differentiation assistance method of the present invention, the determination of Lewy body disease and Alzheimer's disease may be performed solely using the second reference value to determine whether a subject is suffering from Alzheimer's disease or Parkinson's disease. Specifically, when the amount of the biomarker of the present invention is equal to or less than the second reference value, the subject is determined to have a high or possible risk of Parkinson's disease, or to have a high or possible risk of developing Parkinson's disease. When the amount of the biomarker of the present invention is equal to or less than the first reference value, the subject is determined to have a high or possible risk of Alzheimer's disease, or to have a high or possible risk of developing Alzheimer's disease. Furthermore, when the amount of the biomarker of the present invention is equal to or less than the first reference value and greater than the second reference value, the subject is preferably determined to have a high or possible risk of Alzheimer's disease, or to have a high or possible risk of developing Alzheimer's disease.

[0051] The first reference value is the amount of the biomarker of the present invention, which is preset for distinguishing between Parkinson's disease dementia or dementia with Lewy bodies and Alzheimer's disease. The method for determining the first reference value is not particularly limited. For example, the first reference value can be determined by measuring the amount of the biomarker of the present invention contained in biological samples obtained from a patient with or at risk of Parkinson's disease dementia or dementia with Lewy bodies and a patient with or at risk of Alzheimer's disease, and then using the obtained amounts of the biomarker to perform statistical analysis such as receiver operating characteristic analysis (ROC analysis). The biological samples obtained from a patient with or at risk of Parkinson's disease dementia or dementia with Lewy bodies and a patient with or at risk of Alzheimer's disease are preferably similar biological samples. The second reference value is preset for distinguishing between Alzheimer's disease and Parkinson's disease. It can be determined by a method similar to that for the first reference value, except that biological samples obtained from a patient with or at risk of Parkinson's disease and a patient with or at risk of Alzheimer's disease are used. Furthermore, when setting the reference value, it is preferable to take into consideration sensitivity, specificity, positive predictive value, negative predictive value, etc. The predetermined reference value can be set, for example, so that the sensitivity is 60% or more, preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more, and can be set, for example, so that the specificity is 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more.

[0052] <Reagent kit for assisting in the differentiation of Alzheimer's disease and Parkinson's disease> The reagent kit for assisting in the differentiation between Alzheimer's disease and Parkinson's disease of the present invention (hereinafter referred to as the differentiation assisting reagent kit of the present invention) contains a substance having affinity for CD9. The differentiation assisting reagent kit of the present invention may further contain a substance having affinity for phosphatidylserine.

[0053] The substance having affinity for CD9 and the substance having affinity for phosphatidylserine in the differentiation-assisting reagent kit of the present invention are the same as those in the differentiation-assisting method of the present invention, and the preferred examples are also the same. The substance having affinity for CD9 and the substance having affinity for phosphatidylserine may be in a solution state, a frozen state, a dried state, or a lyophilized state. The substance having affinity for CD9 and the substance having affinity for phosphatidylserine may be immobilized on a solid phase or labeled with a labeling substance. The solid phase, the method of immobilization on the solid phase, the labeling substance, and the labeling method are the same as those in the differentiation-assisting method of the present invention, and the preferred examples are also the same.

[0054] The differentiation-assisting reagent kit of the present invention may further comprise a secondary affinity substance (e.g., a secondary antibody) that binds to the substance having affinity for CD9 (the primary affinity substance), and / or a secondary affinity substance (e.g., a secondary antibody) that binds to the substance having affinity for phosphatidylserine (the primary affinity substance), and the secondary affinity substance is preferably labeled with a labeling substance. Furthermore, the affinity substances (the primary affinity substance and the secondary affinity substance) in the differentiation-assisting reagent kit of the present invention may be labeled directly or indirectly with a labeling substance. The affinity substances (the primary affinity substance and the secondary affinity substance), labeling substance, and labeling method in the differentiation-assisting reagent kit of the present invention are the same as those in the differentiation-assisting method of the present invention, and specific examples and preferred examples are also the same.

[0055] The concentrations (amounts) of the substance having affinity for CD9 and the substance having affinity for phosphatidylserine (primary affinity substance) in the differentiation-assisting reagent kit of the present invention may be appropriately selected within the ranges commonly used in this field depending on the assay method. When immobilizing the substance having affinity for CD9 and / or the substance having affinity for phosphatidylserine (primary affinity substance), the concentration upon use is, for example, 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, for solid phase immobilization. When used for detection, the concentration is, for example, 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL. Reagents commonly used in this field, such as buffers, reaction promoters, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc., may also be present together with the substance having affinity for CD9 and / or the substance having affinity for phosphatidylserine. The concentrations and pH of these may also be appropriately selected within the ranges commonly used in this field.

[0056] The differentiation-assisting reagent kit of the present invention may contain, in addition to the substance having affinity for CD9, reagents necessary for measuring the biomarker of the present invention. Examples of such reagents include detergents, sample diluents, reagents for detecting labeled substances, reagents for directly or indirectly binding labeled substances to the affinity substances, reagents for immobilizing the affinity substances on solid phases, and reagents for binding avidins or biotins to the affinity substances or labeled substances. The concentrations, pH, and other parameters of these reagents may be selected appropriately from ranges commonly used in this field.

[0057] The differentiation aid reagent kit of the present invention may contain a standard used to prepare a calibration curve for extracellular vesicles bearing CD9, or a standard used to prepare a calibration curve for extracellular vesicles bearing CD9 and phosphatidylserine. Examples of such standards include CD9-bearing extracellular vesicles and phosphatidylserine-bearing extracellular vesicles. The standard may be in a solution state, a frozen state, a dried state, or a lyophilized state. Furthermore, reagents commonly used in this field, such as buffers, reaction promoters, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc., may be present together with the standard. The concentrations and pH of these may also be selected appropriately from ranges commonly used in this field.

[0058] The differentiation aid reagent kit of the present invention may include a package insert or instruction manual. Examples of the package insert or instruction manual include a package insert or instruction manual that describes measuring the amount of the biomarker of the present invention in a biological sample derived from a subject and / or assisting in the differentiation between Alzheimer's disease and Parkinson's disease using the amount of the biomarker of the present invention as an index (the differentiation aid method of the present invention). These package inserts or instruction manuals may be written in multiple documents or may be written together in a single document.

[0059] Specific examples of the differentiation aid reagent kit of the present invention include the following. <1> (A) a kit containing the above-mentioned substance having affinity for CD9; <1-1> A kit comprising (A-1) a substance having affinity for the CD9 immobilized on a solid phase, and (B-1) a labeled substance having affinity for the CD9; <1-2> A kit comprising (A-1) a substance having affinity for the CD9 immobilized on a solid phase, (B) a substance having affinity for the CD9, and (C) a labeled substance having affinity for "(B) the substance having affinity for the CD9" (e.g., an antibody (secondary antibody) against the (B) substance having affinity for the CD9); <2> (D) a kit comprising the substance having affinity for phosphatidylserine and (A) the substance having affinity for CD9; <2-1> A kit comprising (D-1) a substance having affinity for the phosphatidylserine immobilized on a solid phase and (A-2) a substance having affinity for the labeled CD9; <2-2> (D-1) a kit comprising a substance having affinity for the phosphatidylserine immobilized on a solid phase, (A) a substance having affinity for CD9, and (E) a labeled substance having affinity for "(A) the substance having affinity for CD9" (e.g., an antibody (secondary antibody) against the (A) substance having affinity for CD9); <2-3> A kit comprising (D-2) a substance having affinity for the labeled phosphatidylserine and (A-1) a substance having affinity for the CD9 immobilized on a solid phase; <2-4> A kit comprising (D) a substance having affinity for the above-mentioned phosphatidylserine, (F) a labeled substance having affinity for the "(D) substance having affinity for the above-mentioned phosphatidylserine" (for example, an antibody (secondary antibody) against the above-mentioned (D) substance having affinity for the above-mentioned phosphatidylserine), and (A-1) a substance having affinity for the above-mentioned CD9 immobilized on a solid phase. <1> ,<1-1>,<1-2>, <2> The labeled affinity substance may be configured as a kit in which either a ligand such as an avidin or a biotin or a receptor is bound to an unlabeled affinity substance, and the other ligand such as an avidin or a biotin or a receptor is bound to a labeled substance.

[0060] More specifically, the following are more preferred examples of the differentiation aid reagent kit of the present invention. [1] (A) A kit containing an anti-CD9 antibody; [1-1] A kit comprising (A-1) an anti-CD9 antibody immobilized on a solid phase and (B-1) a labeled anti-CD9 antibody; [1-2] A kit including (A-1) an anti-CD9 antibody immobilized on a solid phase, (B) an anti-CD9 antibody, and (C) a labeled antibody (secondary antibody) against "(B) anti-CD9 antibody"; [2] (D) a kit comprising an anti-PS antibody or a phosphatidylserine affinity protein (preferably a Tim protein, more preferably Tim1 or Tim4, and more preferably Tim4) and (A) an anti-CD9 antibody; [2-1] (D-1) a kit comprising an anti-PS antibody or a phosphatidylserine affinity protein (preferably a Tim protein, more preferably Tim1 or Tim4, and more preferably Tim4) immobilized on a solid phase, and (A-2) a labeled anti-CD9 antibody; [2-2] A preferred kit includes (D-1) a solid-phase-immobilized anti-PS antibody or phosphatidylserine affinity protein (preferably a Tim protein, preferably Tim1 or Tim4, with Tim4 being more preferred), (A) an anti-CD9 antibody, and (E) a labeled anti-CD9 antibody. The labeled antibody or phosphatidylserine affinity protein may be configured as an unlabeled antibody phosphatidylserine affinity protein bound to either a ligand such as avidin or biotin or a receptor, or as an unlabeled antibody phosphatidylserine affinity protein bound to the other ligand or receptor such as avidin or biotin, and a labeling substance. Furthermore, each antibody and phosphatidylserine affinity protein may be present in a solution (reagent) at a concentration of, for example, 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL.

[0061] According to the present invention, data (determination results) can be obtained to assist in the differentiation of Lewy body disease and Alzheimer's disease in a subject. When the present invention determines that a subject is likely or likely to have Alzheimer's disease, or that the subject is at high risk or at risk of developing Alzheimer's disease, further tests, such as a medical interview, a diagnostic method using an imaging device such as amyloid PET diagnosis, an MMSE test, tests for Alzheimer's disease biomarkers recommended in dementia disease treatment guidelines, such as a decrease in Aβ42 in cerebrospinal fluid or an increase in tau or phosphorylated tau, or tests using candidate substances for Alzheimer's disease biomarkers, can be performed. Taking these results into consideration, a diagnosis of Alzheimer's disease in the subject can be made. Furthermore, drugs or therapeutic agents that slow the progression of Alzheimer's disease, such as cholinesterase inhibitors, may be administered, or surgery may be performed. Furthermore, when the present invention determines that a subject is likely or likely to have Parkinson's disease, or that the subject is at high risk or at risk of developing Parkinson's disease, further tests for Parkinson's disease recommended in the Parkinson's disease treatment guidelines, such as a medical interview, MIBG myocardial scintigraphy, and dopamine transporter scintigraphy, or tests using candidate biomarkers for Parkinson's disease, can be performed, and the results of these tests can be taken into consideration to diagnose the subject's Parkinson's disease. Furthermore, drugs or therapeutic agents that slow the progression of Parkinson's disease, such as levodopa (L-dopa), can be administered, or surgery can be performed. Furthermore, if the subject is determined by the present invention to have a high or possible risk of having Parkinson's disease dementia or dementia with Lewy bodies, or to have a high or possible risk of developing Parkinson's disease dementia or dementia with Lewy bodies, there is a risk of hypersensitivity to the drug, so the dosage of anti-dementia drugs such as cholinesterase inhibitors or anti-Parkinson's disease drugs such as levodopa (L-dopa) may be adjusted; for example, the dosage may be reduced compared to that for Parkinson's disease patients, or administration may not be necessary. [Industrial Applicability]

[0062] The method, biomarker, and reagent kit of the present invention for assisting in the differentiation between Lewy body disease and Alzheimer's disease are useful in the field of clinical testing because they enable the determination of Lewy body disease and Alzheimer's disease in a subject. [Example]

[0063] EXAMPLES The present invention will be specifically described below based on examples and comparative examples, but the present invention is not limited to these examples in any way.

[0064] Example 1. Evaluation of Alzheimer's disease specimens and Parkinson's disease-type dementia or Lewy body dementia specimens using the amount of CD9-bearing exosomes as an index Exosomes were measured using an anti-CD9 antibody-anti-CD9 antibody sandwich ELISA, and the AUC and p-value were calculated. (1) Preparation of calibrators Exosomes were isolated from COLO201 cell culture supernatant using the MagCapture® Exosome Isolation Kit PS (Fujifilm Wako Pure Chemical Industries, Ltd., hereafter referred to as "Kit A") according to the instructions attached to Kit A. The exosomes were then eluted using the elution solution provided with Kit A. The protein concentration in the resulting exosome solution was measured by the bicinchoninic acid (BCA) method using a Protein Assay BCA Kit (Fujifilm Wako Pure Chemical Industries, Ltd.). Next, the obtained exosome solution was diluted using the Reaction Buffer included in the PS Capture Exosome ELISA Kit, Streptavidin HRP (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "Kit B") to 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0, and 20.0 ng / mL based on the protein concentration measured by the BCA method, to obtain a dilution series of exosomes derived from COLO201 cell culture supernatant consisting of eight concentrations (hereinafter referred to as "calibrators"). (2) Pretreatment of the measurement sample EDTA plasma samples from 10 Alzheimer's disease (AD) patients and 5 Parkinson's disease / Dementia with Lewy Bodies patients (PDD / DLB, MMSE score 23 or less) purchased from PrecisionMed were centrifuged at 10,000 g for 20 minutes, and the supernatants were collected. The resulting supernatants were diluted 100-fold with the Reaction Buffer included in Kit A to prepare the "sample dilution solution for measurement." (3) Measurement by ELISA The diluted sample solution prepared in (2) was measured using an anti-CD9 antibody-anti-CD9 antibody sandwich ELISA, in which the anti-CD9 antibody was immobilized on a solid phase and used as the detection antibody. The reagents included in Kit B were used, except for the detection antibody and solid-phase immobilization plate. (i) Preparation of biotin-labeled anti-CD9 mouse monoclonal antibody: Anti-CD9 mouse monoclonal antibody (1K, Fujifilm Wako Pure Chemical Industries, Ltd.) was reduced with 1.6 mM DTT and then reacted with biotin-PEAC5-maleimide (Dojindo Laboratories, Ltd.) at 37°C for 1.5 hours to prepare a biotin-labeled anti-CD9 mouse monoclonal antibody. (ii) Preparation of anti-CD9 antibody-immobilized plate The washing buffer (10x) included with Kit B was diluted 10-fold with purified (distilled) water, and then 1 / 100th of the Exosome Binding Enhancer (100x) included with Kit B was added to the resulting dilution. The resulting solution was designated "washing solution (1x)." Anti-CD9 mouse monoclonal antibody (lK) (Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted to a concentration of 10 μg / mL with 50 mM MOPS (pH 7.5), and 100 μL was added to the wells of a 96-well microplate (Nunc Corporation) and incubated overnight in a refrigerator. After washing the wells three times with TBST (Tris-Buffered Saline, pH 7.4), 300 μL of 10 mg / mL Block Ace in TBS (Tris-Buffered Saline, pH 7.4) was added and incubated overnight in a refrigerator to obtain an anti-CD9 antibody-immobilized plate. (iii) Measurement of CD9-bearing exosomes Each well of the anti-CD9 antibody-immobilized plate was washed three times with 300–350 μL of washing solution (1×). Next, 100 μL of the diluted sample solution for measurement from Alzheimer's disease patients (10 samples, n = 2, 20 wells), Parkinson's disease dementia or dementia with Lewy bodies patients (5 samples, n = 2, 10 wells), the calibrator prepared in (2) (8 samples, n = 2, 16 wells), and the Reaction Buffer included in Kit B (n = 2, 2 wells) as a blank were dispensed into each well. A plate seal was then affixed to the plate, and the reaction was allowed to proceed for 2 hours at room temperature 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×). Biotin-labeled anti-CD9 mouse monoclonal antibody was diluted to a final concentration of 250 ng / mL using the Reaction Buffer included with Kit B to obtain a biotin-labeled antibody reaction solution. 100 μL of the resulting biotin-labeled antibody reaction solution was dispensed into each well, a plate seal was attached, and the reaction was allowed to proceed at room temperature for 1 hour while stirring at approximately 500 rpm on 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). HRP-conjugated streptavidin (100×) was added to 1 / 100 of the Reaction Buffer included with Kit B and mixed thoroughly to prepare the HRP-labeled streptavidin reaction solution (1×). 100 μL of the resulting HRP-labeled streptavidin reaction solution (1×) was dispensed into each well, a plate seal was attached, and the reaction was allowed to proceed at room temperature for 2 hours while stirring at approximately 500 rpm on a microplate shaker. After the reaction was completed, the reaction solution was discarded, and each well was washed five times with 300-350 μL of washing solution (1×). 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) Solution included in the B kit was returned to room temperature and dispensed into each well. The plate was then stirred for approximately 1 minute using a microplate shaker, and a plate seal was attached. The plate was then left to react at room temperature (20-25°C) for 30 minutes.After cooling to room temperature, 100 μL of StopSolution (included in Kit B) was added to each well. After shaking for approximately 5 seconds on a microplate shaker, the absorbance at 450 nm and at 620 nm was immediately measured using a 96-well microplate reader (Tecan, Safire2). The absorbance at 450 nm minus the absorbance at 620 nm was calculated as the "absorbance value," and the corrected sample absorbance was calculated by subtracting the blank absorbance from the absorbance of the sample dilution solution. A standard curve was then constructed using the absorbance values ​​of the COLO201 cell culture supernatant-derived exosome dilution series (calibrator) minus the blank absorbance and the calibrator protein concentration. Using the standard curve, the corrected sample absorbance values ​​were converted to protein concentrations, and the resulting converted values ​​were multiplied by the sample dilution factor to determine the "sample measurement value" (ng / mL). (4) Calculation of AUC Based on the sample measurements obtained in (3), a Wilcoxon / Kruskal-Wallis test (rank sum) was used to test for significance between Alzheimer's disease patients and Parkinson's disease dementia or Lewy body dementia patients using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA), and a p-value was calculated. Furthermore, based on the sample measurements obtained in (3), a logistic regression analysis was performed using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA), and the area under the curve (AUC) was calculated from the resulting receiver operating characteristic curve (ROC curve). The results are shown in Table 1 and Figure 1 below. In the figure, the vertical axis represents the sample measurements, the horizontal axis represents the results for Alzheimer's disease patients, and the horizontal axis represents the results for Parkinson's disease dementia or Lewy body dementia patients.

[0065] Example 2: Evaluation of Alzheimer's disease specimens and Parkinson's disease-type dementia or Lewy body dementia specimens using the amount of exosomes containing PS (phosphatidylserine) and CD9 as indicators Exosomes were measured by Tim4-anti-CD9 antibody sandwich ELISA in the same manner as in Example 1, except that the Tim4-immobilized plate included in the PSCapture Exosome ELISA Kit, Streptavidin HRP (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., kit B above) was used instead of the anti-CD9 antibody-immobilized plate. A significant difference test was performed between the measured values ​​of Alzheimer's disease (AD) specimens and those of Parkinson's disease-type dementia or dementia with Lewy bodies (PDD / DLB), and the AUC and p-value were calculated. The results are shown in Table 1 below and Figure 2.

[0066] Comparative Example 1: Evaluation of Alzheimer's disease specimens and Parkinson's disease-type dementia or Lewy body dementia specimens using the amount of CD63-bearing exosomes as an index Except for using anti-CD63 antibody (3-13) (Fujifilm Wako Pure Chemical Industries, Ltd.) instead of anti-CD9 antibody as the antibody immobilized on the antibody-immobilized plate, and using the biotin-labeled anti-CD63 antibody included in the PS Capture Exosome ELISA Kit, Streptavidin HRP (Fujifilm Wako Pure Chemical Industries, Ltd., the above-mentioned "Kit B") as the detection antibody, exosomes were measured by anti-CD63 antibody-anti-CD63 antibody sandwich ELISA in the same manner as in Example 1, and a significant difference test was performed between the measured values ​​of Alzheimer's disease specimens and those of Parkinson's disease dementia or dementia with Lewy bodies specimens, and the P value was calculated. The results are shown in Table 1 below.

[0067] Comparative Example 2: Evaluation of Alzheimer's disease specimens and Parkinson's disease-type dementia or Lewy body dementia specimens using the amount of CD81-bearing exosomes as an index Except for using an anti-CD81 mouse monoclonal antibody (17B1) (Fujito Film Wako Pure Chemical Industries, Ltd.) instead of an anti-CD9 antibody as the antibody immobilized on the antibody-immobilized plate, and using the above-mentioned anti-CD81 antibody biotin-labeled in the same manner as in Example 1 as the detection antibody, exosomes were measured by the anti-CD81 antibody-anti-CD81 antibody sandwich ELISA method in the same manner as in Example 1, and a significant difference test was performed between the measured values ​​of Alzheimer's disease specimens and those of Parkinson's disease dementia or dementia with Lewy bodies specimens, and the p-value was calculated. The results are shown in Table 1 below.

[0068] Comparative Example 3: Evaluation of Alzheimer's disease specimens and Parkinson's disease-type dementia or Lewy body dementia specimens using the amount of exosomes containing PS and CD63 as indicators Exosomes were measured using a Tim4-anti-CD63 antibody sandwich ELISA in the same manner as in Example 2, except that an anti-CD63 antibody was used instead of an anti-CD9 antibody as the detection antibody. A significant difference test was performed between the measured values ​​of Alzheimer's disease specimens and those of Parkinson's disease dementia or Lewy body dementia specimens, and a p-value was calculated. The anti-CD63 antibody used as the detection antibody was biotin-labeled with the same τ as in Comparative Example 1. The results are shown in Table 1 below.

[0069] Comparative Example 4: Evaluation of Alzheimer's disease specimens and Parkinson's disease-type dementia or Lewy body dementia specimens using the amount of exosomes containing PS and CD81 as indicators Exosomes were measured by Tim4-anti-CD81 antibody sandwich ELISA in the same manner as in Example 2, except that an anti-CD81 antibody was used instead of an anti-CD9 antibody as the detection antibody. A significant difference test was performed between the measured values ​​of Alzheimer's disease specimens and those of Parkinson's disease dementia or Lewy body dementia specimens, and p-values ​​were calculated. The same biotin-labeled anti-CD81 antibody was used as the detection antibody in Comparative Example 2. The results are shown in Table 1 below.

[0070] [Table 1]

[0071] As shown in Table 1, when plasma samples from patients with Alzheimer's disease and patients with Parkinson's disease or dementia with Lewy bodies were used, the AUC was 0.949 when the amount of CD9-bearing exosomes was used as an indicator to assess Alzheimer's disease, Parkinson's disease, or Lewy body dementia. The AUC was 0.90 when the amount of exosomes bearing phosphatidylserine and CD9 was used as an indicator. On the other hand, there was no statistically significant difference when the amount of exosomes bearing CD63, CD81, phosphatidylserine and CD63, or phosphatidylserine and CD81 were used as indicators. These results demonstrate that the amount of CD9-bearing exosomes and the amount of exosomes bearing phosphatidylserine and CD9 can be used as indicators to determine Alzheimer's disease, Parkinson's disease, or Lewy body dementia. In particular, the amount of CD9-bearing exosomes was found to provide a highly accurate diagnosis.

[0072] Example 3. Evaluation of Alzheimer's disease specimens and Parkinson's disease specimens using the amount of CD9-bearing exosomes as an index Exosomes were measured by the same method as in Example 1 using an anti-CD9 antibody-anti-CD9 antibody sandwich ELISA, except that "EDTA plasma from 18 Alzheimer's disease patients purchased from PrecisionMed" was used as Alzheimer's disease patient samples, and "EDTA plasma from 5 Parkinson's disease patients purchased from PrecisionMed" (MMSE score 27 or higher) was used instead of "Parkinson's disease dementia or Lewy body dementia samples." A significant difference test was performed between the measured values ​​of the Alzheimer's disease samples and the Parkinson's disease samples, and the AUC and P values ​​were calculated. The results are shown in Table 2 below.

[0073] Comparative Example 5: Evaluation of Alzheimer's disease specimens and Parkinson's disease specimens using the amount of CD63-bearing exosomes as an index Exosomes were measured using the same method as in Comparative Example 1 using an anti-CD63 antibody-anti-CD63 antibody sandwich ELISA, except that "EDTA plasma from 18 Alzheimer's disease patients purchased from PrecisionMed" was used as Alzheimer's disease patient samples, and "EDTA plasma from 5 Parkinson's disease patients purchased from PrecisionMed" (MMSE score 27 or higher) was used instead of "Parkinson's disease dementia or Lewy body dementia samples." A significant difference test was performed between the measured values ​​of the Alzheimer's disease samples and the Parkinson's disease sample samples, and a p-value was calculated. The results are shown in Table 2 below.

[0074] Comparative Example 6: Evaluation of Alzheimer's disease specimens and Parkinson's disease specimens using the amount of CD81-bearing exosomes as an index Exosomes were measured using the same method as in Comparative Example 2 using an anti-CD81 antibody-anti-CD81 antibody sandwich ELISA, with the exception that "EDTA plasma from 18 Alzheimer's disease patients purchased from PrecisionMed" was used as Alzheimer's disease patient samples, and "EDTA plasma from 5 Parkinson's disease patients purchased from PrecisionMed" (MMSE score 27 or higher) was used instead of "Parkinson's disease dementia or Lewy body dementia samples." A significant difference test was performed between the measured values ​​of the Alzheimer's disease samples and the Parkinson's disease sample samples, and a p-value was calculated. The results are shown in Table 2 below.

[0075] [Table 2]

[0076] As shown in Table 2, when plasma samples from Alzheimer's disease patients and Parkinson's disease patients were used, and the amount of CD9-bearing exosomes was used as an indicator to evaluate Alzheimer's disease and Parkinson's disease, the AUC was 0.819. On the other hand, when the amount of CD63-bearing exosomes or the amount of CD81-bearing exosomes was used as an indicator, there was no statistically significant difference. These results demonstrate that Alzheimer's disease and Parkinson's disease can be diagnosed using the amount of CD9-bearing exosomes as an indicator.

[0077] Example 4. Evaluation of specimens from Parkinson's disease dementia or dementia with Lewy bodies (MMSE score of 23 or less) and Parkinson's disease (MMSE score of 27 or more) using the amount of CD9-bearing exosomes as an index. Five EDTA plasma samples from Parkinson's disease dementia or dementia with Lewy bodies patients (PDD / DLB, MMSE score of 23 or less) were used as "Parkinson's disease dementia or dementia with Lewy bodies specimens." Instead of Alzheimer's disease specimens, eight EDTA plasma samples from Parkinson's disease patients (MMSE score of 27 or more) purchased from PrecisionMed were used. Exosomes were measured by an anti-CD9 antibody-anti-CD9 antibody sandwich ELISA in the same manner as in Example 1, and a significant difference test was performed between the measured values ​​of the Parkinson's disease dementia or dementia with Lewy bodies specimens and those of the Parkinson's disease specimens. AUC and p-values ​​were calculated. The results are shown in Table 3 below.

[0078] Example 5. Evaluation of specimens from Parkinson's disease dementia or Lewy body dementia (MMSE score of 23 or less) and Parkinson's disease (MMSE score of 27 or more) using the amount of exosomes containing PS and CD9 as indicators Exosomes were measured using the Tim4-anti-CD9 antibody sandwich ELISA method in the same manner as in Example 2, except that "EDTA plasma from five Parkinson's disease dementia or Lewy body dementia patients (PDD / DLB, MMSE score of 23 or less)" was used as the "Parkinson's disease dementia or Lewy body dementia samples," and "EDTA plasma from eight Parkinson's disease patients (MMSE score of 27 or more) purchased from PrecisionMed" was used instead of Alzheimer's disease patient samples. A significant difference test was performed between the measured values ​​of the Parkinson's disease dementia or Lewy body dementia samples and the measured values ​​of the Parkinson's disease samples, and the AUC and p-value were calculated. The results are shown in Table 3 below.

[0079] [Table 3]

[0080] As shown in Table 3, when plasma from patients with Parkinson's disease dementia or dementia with Lewy bodies (MMSE of 23 or less) and patients with Parkinson's disease (MMSE of 27 or more) was used as samples, and the amount of CD9-bearing exosomes and PS and CD9-bearing exosomes was used as an indicator to evaluate Parkinson's disease dementia or dementia with Lewy bodies (MMSE of 23 or less) and Parkinson's disease (MMSE of 27 or more), the AUC was 1.000. These results show that Parkinson's disease dementia or dementia with Lewy bodies (MMSE of 23 or less) and Parkinson's disease (MMSE of 27 or more) can be determined by using the amount of CD9-bearing exosomes or the amount of PS and CD9-bearing exosomes as an indicator.

Claims

1. A method for obtaining data to assist in distinguishing between Lewy body disease and Alzheimer's disease in a subject, comprising measuring the amount of CD9-bearing extracellular vesicles or the amount of extracellular vesicles bearing phosphatidylserine and CD9 in a blood-derived sample from the subject suspected of having Lewy body disease or Alzheimer's disease, using the amount of CD9-bearing extracellular vesicles or the amount of phosphatidylserine and CD9 as an indicator.

2. 2. The method of claim 1, wherein the Lewy body disease is Parkinson's disease dementia, Lewy body dementia, or / and Parkinson's disease.

3. The method of claim 2, wherein the differentiation is made from Parkinson's disease dementia or Lewy body dementia when the amount of extracellular vesicles is greater than a first reference value, or / and from Parkinson's disease when the amount of extracellular vesicles is equal to or less than a second reference value.

4. the Lewy body disease is Parkinson's disease dementia or Lewy body dementia, and measuring the amount of extracellular vesicles is measuring the amount of CD9-bearing extracellular vesicles; The method of claim 1, wherein the differentiation is performed using the amount of extracellular vesicles bearing CD9 as an indicator.

5. the Lewy body disease is Parkinson's disease dementia or Lewy body dementia, measuring the amount of extracellular vesicles is measuring the amount of extracellular vesicles having phosphatidylserine and CD9; The method of claim 1, wherein the differentiation is performed using the amount of extracellular vesicles containing phosphatidylserine and CD9 as an indicator.

6. the Lewy body disease is Parkinson's disease, Measuring the amount of extracellular vesicles is measuring the amount of extracellular vesicles bearing CD9; The differentiation is performed using the amount of extracellular vesicles having CD9 as an indicator. The method of claim 1.

7. A reagent kit for assisting in the differentiation of Lewy body disease and Alzheimer's disease, comprising a substance having affinity for CD9 on extracellular vesicles in a blood-derived sample.

8. 8. The reagent kit for assisting in the differentiation between Lewy body disease and Alzheimer's disease according to claim 7, wherein the Lewy body disease is Parkinson's disease-type dementia, Lewy body dementia, or / and Parkinson's disease.

9. A biomarker for assisting in the differentiation of Lewy body disease and Alzheimer's disease, comprising extracellular vesicles having CD9 in a blood-derived sample, or extracellular vesicles having phosphatidylserine and CD9 in a blood-derived sample.

10. The biomarker for assisting in the differentiation between Lewy body disease and Alzheimer's disease according to claim 9, wherein the Lewy body disease is Parkinson's disease-type dementia, Lewy body dementia, or / and Parkinson's disease.

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