Methods, biomarkers, kits, and devices to aid in the diagnosis of parkinson's disease

Abstract

The present application provides a method, a biomarker, a kit, and a device for assisting diagnosis of Parkinson's disease. The present application relates to a method, a biomarker, a kit, and a device for assisting diagnosis of Parkinson's disease, the method comprising the steps of: measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin or the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having tetraspanin; and determining whether a subject is Parkinson's disease using the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin or the amount of extracellular vesicles having the phosphatidylserine and tetraspanin to the amount of extracellular vesicles having the tetraspanin as an index.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for aiding diagnosis of Parkinson's disease, a biomarker, a kit, and a device. BACKGROUND

[0002] Parkinson's disease (PD) refers to a neurodegenerative disease in which nerve cells in the substantia nigra in the brain degenerate and striated dopamine becomes insufficient to move the body flexibly. The prevalence of Parkinson's disease is as high as 100 to 150 per 100,000 people, and the prevalence increases with age, so the number of patients increases dramatically with aging.

[0003] There is currently no radical treatment for Parkinson's disease, but it is known that by taking a dopamine precursor substance treatment drug such as levodopa (L-Dopa) or a dopamine agonist to supplement the reduced dopamine action in the brain, the motor symptoms can be improved. Therefore, a biomarker that can objectively determine the condition of Parkinson's disease is required.

[0004] As a method for diagnosing Parkinson's disease, a method of concentrating a neural-derived extracellular vesicle in blood using an anti-NCAM antibody or an anti-L1CAM antibody, and determining Parkinson's disease using alpha synuclein or the like contained in the extracellular vesicle as an index has been reported (Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2).

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: Japanese Patent Application 2016-550673

[0008] Patent Document 2: Japanese Patent Application 2017-520760

[0009] Non-Patent Documents

[0010] Non-Patent Document 1: C. N. Winston et al. / Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring (2016) 3: 63-72

[0011] Non-Patent Document 2: Mustapic M et al. Front. Neurosci. (2017) 11: 278 SUMMARY

[0012] Technical Problem to be Solved by the Invention

[0013] However, the method for diagnosing Parkinson's disease using the nerve-derived extracellular vesicles in blood as an index requires an operation of affinity concentration of the nerve-derived extracellular vesicles from the test subject, and thus is complicated. Also, the test subject cannot be directly measured, and thus it is difficult to apply to multiple test subject measurements because of errors between content measurements.

[0014] In view of the foregoing, an object of the present application is to provide a method, a biomarker, a kit, and a device for assisting in the diagnosis of Parkinson's disease.

[0015] Means for solving the technical problem

[0016] The present inventors have studied whether or not specific extracellular vesicles can be used as a biomarker for assisting in the diagnosis of Parkinson's disease.

[0017] As a result, the present inventors have newly found that specific extracellular vesicles having phosphatidylserine and tetraspanin in extracellular vesicles are a biomarker for assisting in the diagnosis of Parkinson's disease, and thus have completed the present application.

[0018] The present application relates to a method, a biomarker, a kit, and a device for assisting in the diagnosis of Parkinson's disease.

[0019] [1] A method for assisting in the diagnosis of Parkinson's disease, comprising the steps of:

[0020] measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanin or the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having tetraspanin in a biological sample derived from a test subject; and

[0021] determining whether or not the test subject has Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanin or the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin to the amount of extracellular vesicles having tetraspanin as an index.

[0022] [2] The method for assisting in the diagnosis of Parkinson's disease according to [1], wherein the determination of Parkinson's disease is such that when the amount of extracellular vesicles having phosphatidylserine and tetraspanin or the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin to the amount of extracellular vesicles having tetraspanin is below a reference value, it is determined that the test subject has Parkinson's disease.

[0023] [3] The method for assisting in the diagnosis of Parkinson's disease according to [1] or [2], wherein,

[0024] The measuring of the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin is measuring the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin using a substance having an affinity for the tetraspanin and a substance having an affinity for the phosphatidylserine,

[0025] The determination of the Parkinson's disease is determining whether the test subject is the Parkinson's disease using the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin as an index,

[0026] The tetraspanin is selected from CD9, CD63, and CD81.

[0027] [4] The method of assisting the diagnosis of the Parkinson's disease according to any one of [1] to [3], wherein

[0028] The measuring of the amount of the extracellular vesicle is measuring the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin,

[0029] The measuring of the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin is measuring the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin using a substance having an affinity for the tetraspanin and a substance having an affinity for the phosphatidylserine,

[0030] The determination of the Parkinson's disease is determining whether the test subject is the Parkinson's disease using the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin as an index.

[0031] [5] The method of assisting the diagnosis of the Parkinson's disease according to [4], wherein the substance having an affinity for the phosphatidylserine is a T cell immunoglobulin·mucin-containing protein.

[0032] [6] The method of assisting the diagnosis of the Parkinson's disease according to any one of [1] to [5], wherein

[0033] The measuring of the amount of the extracellular vesicle is measuring the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin,

[0034] The determination of the Parkinson's disease is determining whether the test subject is the Parkinson's disease using the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin as an index.

[0035] The biological sample is a blood sample or cerebrospinal fluid.

[0036] [7] The method of assisting the diagnosis of the Parkinson's disease according to [1] or [2], wherein

[0037] The measuring of the amount of the extracellular vesicle is measuring the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin and the amount of the extracellular vesicle having the tetraspanin,

[0038] The determination of the Parkinson's disease refers to determining whether a test subject is a Parkinson's disease using the ratio of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount of the extracellular vesicle having the tetraspanin as an index,

[0039] The tetraspanin is CD9 or CD63.

[0040] [8] The method for assisting the diagnosis of Parkinson's disease according to any one of [1], [2], and [7], wherein

[0041] The determination of the amount of the extracellular vesicle refers to determining the amount of the extracellular vesicle having phosphatidylserine and tetraspanin and the amount of the extracellular vesicle having tetraspanin,

[0042] The determination of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin refers to determining the amount of the extracellular vesicle having phosphatidylserine and tetraspanin using a substance having affinity for tetraspanin and a substance having affinity for phosphatidylserine,

[0043] The determination of the amount of the extracellular vesicle having the tetraspanin refers to determining the amount of the extracellular vesicle having the tetraspanin using a substance having affinity for the tetraspanin,

[0044] The determination of the Parkinson's disease refers to determining whether a test subject is a Parkinson's disease using the ratio of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount of the extracellular vesicle having the tetraspanin as an index.

[0045] [9] The method for assisting the diagnosis of Parkinson's disease according to [8], wherein the substance having affinity for phosphatidylserine is a T cell immunoglobulin·mucin-containing protein.

[0046]

[10] The method for assisting the diagnosis of Parkinson's disease according to any one of [1] to [2] and [7] to [9], wherein

[0047] The determination of the amount of the extracellular vesicle refers to determining the amount of the extracellular vesicle having phosphatidylserine and tetraspanin and the amount of the extracellular vesicle having tetraspanin,

[0048] The determination of the Parkinson's disease refers to determining whether a test subject is a Parkinson's disease using the ratio of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount of the extracellular vesicle having the tetraspanin as an index,

[0049] The biological sample is a blood sample or cerebrospinal fluid.

[0050]

[11] The method for assisting the diagnosis of Parkinson's disease according to any one of [1] to [2] and [7] to

[10] , wherein the determination of Parkinson's disease is determination of which disease, a Parkinson's disease with a dementia symptom or a Parkinson's disease without a dementia symptom, the test subject has, with the proportion of the amount of the extracellular vesicle with phosphatidylserine and tetraspanin-4 to the amount of the extracellular vesicle with the tetraspanin-4 as an index.

[0051]

[12] A kit for assisting the diagnosis of Parkinson's disease, comprising a substance having affinity for tetraspanin-4 and a substance having affinity for phosphatidylserine.

[0052]

[13] A biomarker for assisting the diagnosis of Parkinson's disease, comprising an extracellular vesicle with phosphatidylserine and tetraspanin-4.

[0053]

[14] An apparatus for assisting the diagnosis of Parkinson's disease, comprising:

[0054] a measurement unit that measures the amount of the extracellular vesicle with phosphatidylserine and tetraspanin-4 in a biological sample derived from a test subject; and

[0055] a determination unit that determines the test subject to be Parkinson's disease with the amount of the extracellular vesicle with the phosphatidylserine and tetraspanin-4 as an index.

[0056]

[15] An apparatus for assisting the diagnosis of Parkinson's disease, comprising:

[0057] a measurement unit that measures the amount of the extracellular vesicle with phosphatidylserine and tetraspanin-4 and the amount of the extracellular vesicle with tetraspanin-4 in a biological sample derived from a test subject;

[0058] a calculation unit that calculates the proportion of the amount of the extracellular vesicle with the phosphatidylserine and tetraspanin-4 to the amount of the extracellular vesicle with the tetraspanin-4; and

[0059] a determination unit that determines the test subject to be Parkinson's disease with the proportion of the amount of the extracellular vesicle with the phosphatidylserine and tetraspanin-4 to the amount of the extracellular vesicle with the tetraspanin-4 as an index.

[0060]

[16] A biomarker group for assisting the diagnosis of Parkinson's disease, comprising an extracellular vesicle with phosphatidylserine and tetraspanin-4 and an extracellular vesicle with tetraspanin-4.

[0061] EFFECT OF INVENTION

[0062] According to the present application, it is possible to simply assist in the diagnosis of Parkinson's disease. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 A box plot of a plasma specimen derived from a Parkinson's disease patient having no dementia symptoms and a plasma specimen derived from a healthy person was evaluated with the amount of exosomes having phosphatidylserine and CD9 as an index.

[0064] Figure 2 A box plot of a plasma specimen derived from a Parkinson's disease patient having no dementia symptoms, a plasma specimen derived from a Parkinson's disease patient having dementia symptoms, and a plasma specimen derived from a healthy person was evaluated with the proportion of the amount of exosomes having phosphatidylserine and CD9 to the amount of extracellular vesicles having CD9 as an index.

[0065] Figure 3 A box plot of a plasma specimen derived from a Parkinson's disease patient having no dementia symptoms, a plasma specimen derived from a Parkinson's disease patient having dementia symptoms, and a plasma specimen derived from a healthy person was evaluated with the proportion of the amount of exosomes having phosphatidylserine and CD9 to the amount of extracellular vesicles having CD9 as an index.

[0066] Figure 4 A box plot of a plasma specimen derived from a Parkinson's disease patient and a plasma specimen derived from a healthy person was evaluated with the amount of exosomes having phosphatidylserine and CD9 as an index.

[0067] Figure 5 A box plot of a plasma specimen derived from a Parkinson's disease patient and a plasma specimen derived from a healthy person was evaluated with the proportion of the amount of exosomes having phosphatidylserine and CD9 to the amount of extracellular vesicles having CD9 as an index. DETAILED DESCRIPTION

[0068] In the present specification, when the upper limit and the lower limit of the range are shown, A ~ B indicates A or more and B or less, unless otherwise specified.

[0069] The extracellular vesicle is a small membrane vesicle composed of a lipid bilayer derived from a cell. The extracellular vesicle can be exemplified by, for example, an extracellular vesicle having 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. As the extracellular vesicle, for example, as described in Nature Reviews Immunology 9, 581-593 (August 2009), "Fat research" Vol. 13 No. 2 2007 main body Naoki Aoki et al., extracellular vesicles classified into a plurality of types according to the origin of their production or the size of the small membrane vesicle, and the like can be exemplified. Specifically, exosome, microvesicle, ectosome, membrane particle, exosome-like vesicle, apoptotic body, and fat body, and the like can be exemplified, and exosome and microvesicle are preferable, and exosome is more preferable.

[0070] The exosome is a small membrane vesicle composed of a lipid bilayer derived from a cell, and can be exemplified by, for example, an exosome having a diameter of 50 nm to 200 nm, preferably 50 nm to 150 nm, and more preferably 50 nm to 100 nm. In addition, it is considered that the exosome is derived from a late endosome.

[0071] The microvesicle is a small membrane vesicle composed of a lipid bilayer derived from a cell, and can be exemplified by, for example, a microvesicle having a diameter of 100 nm to 1000 nm, preferably 100 nm to 800 nm, and more preferably 100 nm to 500 nm. In addition, it is considered that the microvesicle is derived from a cell membrane.

[0072] The extracellular vesicle can be contained in a biological sample derived from a test subject, and can be isolated from a biological sample derived from a test subject, and is preferably isolated from a biological sample derived from a test subject.

[0073] As the biological sample derived from the test subject, any sample can be used as long as it can contain an extracellular vesicle, and can be exemplified by, for example, a sample derived from blood such as serum, plasma, whole blood, and buffy coat; a body fluid sample such as cerebrospinal fluid, urine, saliva, semen, pleural effusion, tears, sputum, mucus, lymph, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid, and bronchoalveolar lavage fluid, and preferably a sample derived from blood and cerebrospinal fluid, and more preferably serum, plasma, and cerebrospinal fluid, and further preferably serum and plasma, and particularly preferably plasma. Furthermore, since the burden on the test subject is light, the sample derived from blood is more effective.

[0074] The biological sample derived from the subject to be tested may be directly collected from the subject to be tested, or may be a biological sample that has been pretreated by recovery, concentration, purification, separation, dilution based on a buffer, and filtration sterilization, or the like. These pretreatments are appropriately performed according to conventional methods. Hereinafter, the biological sample derived from the subject to be tested is sometimes simply referred to as "biological sample".

[0075] As the method for isolating extracellular vesicles from the biological sample, a method according to a conventional method can be used, and there is no particular limitation. As the method for isolating extracellular vesicles from the biological sample, for example, an affinity method (e.g., a PS affinity method), a density gradient centrifugation method (e.g., a pellet down method, a sucrose gradient method, a density gradient centrifugation method, or the like), an immunoprecipitation method, a chromatography method (e.g., an ion exchange chromatography method, a gel permeation chromatography method), a density gradient method (e.g., a sucrose density gradient method), an electrophoresis method (e.g., an organelle electrophoresis method), a magnetic separation method (e.g., a magnetic activated cell sorting (MACS) method), an ultrafiltration concentration method (e.g., a nanomembrane ultrafiltration concentration method), a Percoll gradient separation method, a method using a microfluidic device, a PEG precipitation method, or the like can be used, and from the viewpoint of obtaining extracellular vesicles with high purity, an affinity method is preferred, or from the viewpoint of being able to perform recovery without theoretical bias, a density gradient centrifugation method is preferred, and more preferably an affinity method or a density gradient centrifugation method, and particularly preferably an affinity method. Among the affinity methods, a phosphatidylserine affinity purification method, i.e., a PS affinity method is preferred. The affinity method and the density gradient centrifugation method may, for example, be performed according to the method described in WO2016 / 088689.

[0076] These separation methods can be used alone or in combination with two or more. Also, the separation based on one separation method can be repeated two or more times.

[0077] As the subject to be tested, there is no particular limitation, and for example, a person diagnosed as having Parkinson's disease according to a diagnostic criterion, a person diagnosed as having a risk of developing symptoms of Parkinson's disease according to a diagnostic criterion, a person who has not received a diagnosis of Parkinson's disease, a person not diagnosed as having Parkinson's disease according to a diagnostic criterion, or a person not diagnosed as having a risk of developing symptoms of Parkinson's disease according to a diagnostic criterion can be mentioned, and preferably, a person diagnosed as having Parkinson's disease according to a diagnostic criterion, a person diagnosed as having a risk of developing symptoms of Parkinson's disease according to a diagnostic criterion, and a person who has not received a diagnosis of Parkinson's disease are preferred.

[0078] As the diagnostic criterion, for example, a diagnostic criterion used when Parkinson's disease is diagnosed based on the results of an examination related to a biomarker of Parkinson's disease or the like recommended in the Parkinson's Disease Treatment Guidelines, an examination related to a candidate substance for a biomarker of Parkinson's disease, or the like can be mentioned.

[0079] The present application relates to a method for aiding diagnosis of Parkinson's disease, a biomarker, a kit, and a device.

[0080] In the present application, there are included a case where a biomarker comprising an extracellular vesicle having phosphatidylserine and tetraspanin is used to determine whether a test subject is Parkinson's disease (hereinafter, sometimes abbreviated as "1st Invention") with the amount of the extracellular vesicle having the phosphatidylserine and tetraspanin as an index; and a case where a biomarker comprising an extracellular vesicle having phosphatidylserine and tetraspanin and an extracellular vesicle having tetraspanin are used in combination to determine whether a test subject is Parkinson's disease (hereinafter, sometimes abbreviated as "2nd Invention") with the ratio of the amount of the extracellular vesicle having the phosphatidylserine and tetraspanin to the amount of the extracellular vesicle having the tetraspanin as an index.

[0081] 1. 1st Invention

[0082] < Biomarker for aiding diagnosis of Parkinson's disease >

[0083] The biomarker for aiding diagnosis of Parkinson's disease (hereinafter, sometimes abbreviated as "PD marker") in the 1st Invention comprises an extracellular vesicle having phosphatidylserine and tetraspanin.

[0084] The extracellular vesicle in the PD marker is the same as the aforementioned content, and preferably the same as the aforementioned content.

[0085] The extracellular vesicle having phosphatidylserine and tetraspanin in the PD marker has at least one tetraspanin selected from the group consisting of CD9, CD63, CD81, and CD151, and phosphatidylserine on the membrane surface, preferably has at least one tetraspanin selected from the group consisting of CD9, CD63, and CD81, and phosphatidylserine, more preferably has at least one tetraspanin selected from the group consisting of CD9 and CD81, and phosphatidylserine, and particularly preferably has CD9 and phosphatidylserine.

[0086] < Method for aiding diagnosis of Parkinson's disease >

[0087] The method for aiding diagnosis of Parkinson's disease (hereinafter, sometimes abbreviated as "PD diagnosis aiding method") in the 1st Invention comprises the steps of measuring the amount of an extracellular vesicle having phosphatidylserine and tetraspanin in a biological sample; and determining whether a test subject is Parkinson's disease with the amount of the extracellular vesicle having phosphatidylserine and tetraspanin in the biological sample as an index.

[0088] The biological sample, the test subject, and the extracellular vesicle in the PD diagnosis aiding method are the same as the aforementioned content, and preferably the same as the aforementioned content.

[0089] The extracellular vesicles having phosphatidylserine and tetraspanins in the PD diagnosis support method are preferably the same as the aforementioned, and more preferably the same as the aforementioned in the PD marker.

[0090] As the "amount" in the PD diagnosis support method, a mass or a concentration can be given. Also, the "amount" includes a measured value (e.g., absorbance, absorbance change amount, transmitted light, transmitted light change amount, fluorescence intensity, fluorescence intensity change amount, luminescence amount, luminescence change amount, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change amount, refractive index, refractive index change amount, etc.) that has a correlation with a mass or a concentration.

[0091] In the measurement of the amount of the extracellular vesicles having phosphatidylserine and tetraspanins in the PD diagnosis support method, the amount of the extracellular vesicles having only one kind of tetraspanin and phosphatidylserine (e.g., extracellular vesicles having CD9 and phosphatidylserine) can be set as the measurement target, the amount of the extracellular vesicles having two or more kinds of tetraspanins and phosphatidylserine (e.g., extracellular vesicles having CD9 and phosphatidylserine and extracellular vesicles having CD81 and phosphatidylserine) can be set as the measurement target, or preferably the amount of the extracellular vesicles having only one kind of tetraspanin and phosphatidylserine is set as the measurement target.

[0092] The measurement of the amount of the extracellular vesicles having phosphatidylserine and tetraspanins in the PD diagnosis support method is not particularly limited as long as it is a method generally performed in the field. The measurement can be performed, for example, using an immunological measurement method using a substance having affinity for a tetraspanin and a substance having affinity for phosphatidylserine, a mass analysis method, a method combining them, or the like, and preferably an immunological measurement method using a substance having affinity for a tetraspanin and a substance having affinity for phosphatidylserine. In addition, the immunological measurement method includes not only a method utilizing an immune reaction (antigen-antibody reaction), but also a method utilizing a binding force between two molecules other than an antigen-antibody reaction, such as a method utilizing the binding of a lectin to a protein (a method in accordance with an immunological measurement method).

[0093] As the substance having affinity for the tetraspanin, any substance that specifically binds to the tetraspanin can be used, such as an antibody that specifically binds to the tetraspanin, a lectin that specifically binds to a sugar chain possessed by the tetraspanin, and the like, and preferably a protein that specifically binds to the tetraspanin, and more preferably an antibody that specifically binds to the tetraspanin. As the antibody that specifically binds to the tetraspanin, for example, an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, an anti-CD151 antibody, and the like can be used, and preferably an anti-CD9 antibody, an anti-CD63 antibody, and an anti-CD81 antibody, and more preferably an anti-CD9 antibody and an anti-CD81 antibody, and particularly preferably an anti-CD9 antibody. The substance having affinity for the tetraspanin can be used alone or in combination of two or more, and is preferably used alone.

[0094] As the substance having affinity for the phosphatidylserine, any substance that specifically binds to the phosphatidylserine can be used, such as a protein that specifically binds to the phosphatidylserine and the like, and preferably a protein that specifically binds to the phosphatidylserine. As the protein that specifically binds to the phosphatidylserine, for example, an antibody that specifically binds to the phosphatidylserine or a phosphatidylserine affinity protein can be used, and preferably a phosphatidylserine affinity protein. As the antibody that specifically binds to the phosphatidylserine, for example, an anti-phosphatidylserine antibody 1H6 (Merck KGaA) and the like can be used. As the phosphatidylserine affinity protein, for example, a Tim protein such as Timl (T-cell immunoglobulin-mucin-domain 1), Tim2 (T-cell immunoglobulin-mucin-domain 2), Tim3 (T-cell immunoglobulin-mucin-domain 3), Tim4 (T-cell immunoglobulin-mucin-domain 4), and the like; an anti-phosphatidylserine antibody; Annexin V; MFG-E8; and the like can be used, and preferably a Tim protein and an anti-phosphatidylserine antibody, and more preferably a Tim protein. As the Tim protein, preferably Timl and Tim4, and more preferably Tim4.

[0095] The substance having affinity for the phosphatidylserine can be used singly or in two or more kinds, and is preferably used singly.

[0096] The substance having affinity for the tetraspanin and the substance having affinity for the phosphatidylserine can be commercially available or can be appropriately prepared by a conventional method. Further, the antibody specifically binding to the tetraspanin or the antibody specifically binding to the phosphatidylserine can be either of a polyclonal antibody and a monoclonal antibody, and can be used singly or can be used in appropriate combination. Further, the antibody specifically binding to the tetraspanin or the antibody specifically binding to the phosphatidylserine can be used not only as an intact immunoglobulin but also as a fragment thereof having a binding ability to an antigen, such as a fragment antibody of Fab, F(ab')2, F(ab') and the like, a synthetic antibody of single chain Fv, double chain antibody, triple chain antibody, quadruple chain antibody and the like. Further, when these antibodies are prepared, a method described in, for example, “Immunoassay” (edited by Biochemical Assay Research Society, KODANSHA LTD., 2014) and the like can be employed.

[0097] The substance having affinity for the tetraspanin and / or the substance having affinity for the phosphatidylserine can be labeled with a labeling substance. As the labeling substance, for example, enzymes such as peroxidase, microperoxidase, alkaline phosphatase and the like; 99m Tc, 131 I, 125 I, 14 C, 3 H, 32 P, 35 S and the like radioisotopes; fluorescein, fluorescein isothiocyanate (FITC), 4-methylumbelliferone, HiLyte, Alexa, CyDye or rhodamine, or a derivative thereof or the like fluorescent substance; luciferin, luminol, ruthenium complex or the like luminescent substance; phenol, naphthol or anthracene, or a derivative thereof or the like substance having an absorption in the ultraviolet region; 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl or the like compound having an oxyl group as a spin labeling agent; gold colloid, quantum dot or the like nanoparticle, and the like, and is preferably enzymes and fluorescent substances. The method for labeling the substance having affinity for the tetraspanin and / or the substance having affinity for the phosphatidylserine with the labeling substance is not particularly limited, and can be performed according to a labeling method known per se. The measurement of these labeling substances can be performed according to a measurement method known per se corresponding to the labeling substance.

[0098] The substance having affinity for the tetraspanin and the substance having affinity for the phosphatidylserine can further use a secondary affinity substance (e.g., secondary antibody) that specifically binds to the primary affinity substance. The secondary affinity substance can be labeled with a labeling substance, preferably a labeled secondary affinity substance, more preferably a secondary antibody labeled with a labeling substance. The labeling substance or labeling method is the same as described above, preferably the same as described above.

[0099] Further, the labeling based on the labeling substance can use the binding of avidin and biotin using a substance that binds one of the avidin and biotin to the substance having affinity for the tetraspanin and / or the substance having affinity for the phosphatidylserine and a substance that binds the remaining one of the avidin and biotin to the labeling substance. As the biotin, biotin, iminobiotin, desthiobiotin, biotin sulfoxide, and the like can be given, and biotin is preferred. As the avidin, avidin, Tamavidin, Tamavidin 2, streptavidin, and the like can be given, and streptavidin is preferred. The method of binding one of the avidin and biotin to the substance having affinity for the tetraspanin and / or the substance having affinity for the phosphatidylserine and the method of binding the remaining one of the avidin and biotin to the labeling substance can be performed according to a conventional method.

[0100] The substance having affinity for the tetraspanin and / or the substance having affinity for the phosphatidylserine can be immobilized to a solid phase, and preferably the substance having affinity for the phosphatidylserine is immobilized to a solid phase.

[0101] As the solid phase, for example, synthetic high molecular compounds such as latex, polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polymethacrylic acid glycidyl ester, polyvinyl chloride, polyethylene, polychlorocarbonic acid ester, silicone resin, silicone rubber, and the like; inorganic substances such as porous glass, ground glass, ceramic, alumina, silica gel, activated carbon, metal oxide, and the like can be given, and two or more of them can be used in combination.

[0102] The shape of the solid phase is not particularly limited, and for example, microplate (ELISA plate), beads, hose (microtube), particles, a dedicated tray in which a plurality of hoses are integrally formed, a disc-shaped sheet, a test tube, and the like can be given.

[0103] As a method for immobilizing a substance having affinity for tetraspanin and / or a substance having affinity for phosphatidylserine to the solid phase, any method commonly used in the field can be used, and the method is not particularly limited. The method can be performed according to a conventional method.

[0104] In the PD diagnosis assistance method, the combination of the substance having affinity for tetraspanin and the substance having affinity for phosphatidylserine is not particularly limited, but is preferably a combination of a protein specifically binding to the tetraspanin and a protein specifically binding to the phosphatidylserine, more preferably a combination of an antibody specifically binding to the tetraspanin and an antibody or phosphatidylserine affinity protein specifically binding to the phosphatidylserine.

[0105] As the combination of the antibody specifically binding to the tetraspanin and the antibody or phosphatidylserine affinity protein specifically binding to the phosphatidylserine, for example, a combination of an antibody specifically binding to the tetraspanin and an antibody specifically binding to Tim protein or phosphatidylserine can be given, and is preferably a combination of an anti-CD9 antibody, an anti-CD63 antibody, or an anti-CD81 antibody and Tim protein, more preferably a combination of an anti-CD9 antibody or an anti-CD81 antibody and Tim protein, further preferably a combination of an anti-CD9 antibody or an anti-CD81 antibody and Timl or Tim4, particularly preferably a combination of an anti-CD9 antibody or an anti-CD81 antibody and Tim4, most preferably a combination of an anti-CD9 antibody and Tim4.

[0106] Specifically, the measurement of the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin can be exemplified by enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence enzyme immunoassay (FEIA), fluorescence immunoassay (FIA), chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), immunocomplex transfer method, immunochromatography (ICA), Luminescent Oxygen Channeling Immunoassay (LOCI), Liquid-phase Binding Assay-ElectroKinetic Analyte Transport Assay (LBA-EATA), capillary electrophoresis method such as lectin electrophoresis, immunoblotting, immunoscattering turbidimetry (NIA) such as latex immunoscattering turbidimetry, immunotransmission turbidimetry (TIA) such as latex immunotransmission turbidimetry, immunoprecipitation method such as microparticle counting immunoprecipitation assay (PCIA), surface plasmon resonance (SPR), AlphaLISA, measurement of the amount using fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) to detect the presence of the target molecule, and other well-known immunological measurement methods or mass analysis methods, preferably enzyme-linked immunosorbent assay (ELISA), chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), and capillary electrophoresis method, more preferably enzyme-linked immunosorbent assay (ELISA), chemiluminescence enzyme immunoassay (CLEIA), and LBA-EATA, and particularly preferably enzyme-linked immunosorbent assay (ELISA).

[0107] The measurement principle of the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin is not particularly limited, and can be exemplified by sandwich method and competitive method, preferably sandwich method. Also, as the measurement principle, homogeneous method and heterogeneous method can be used, preferably heterogeneous method.

[0108] As the method in accordance with the immunological measurement method, a method in which a substance having affinity to the tetraspanin or a substance having affinity to the phosphatidylserine other than an antibody specifically binding to the tetraspanin or an antibody specifically binding to the phosphatidylserine is used to perform the immunological measurement method can be exemplified, and the preferable method can also be exemplified by the same method.

[0109] Specifically, the measurement of the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin can be performed in accordance with the method described in WO2016 / 088689, and all descriptions in this publication are incorporated into the present specification.

[0110] Specifically, the measurement of the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin in the PD diagnosis support method can be, for example, a method including the following steps: (1) a step of bringing the extracellular vesicle having the phosphatidylserine and the tetraspanin in a biological sample, a substance having affinity for the tetraspanin, and a substance having affinity for the phosphatidylserine into contact to form a complex body containing the extracellular vesicle having the phosphatidylserine and the tetraspanin in the biological sample, the substance having affinity for the tetraspanin, and the substance having affinity for the phosphatidylserine (hereinafter, sometimes referred to as "complex body formation step"); and (2) a step of measuring the amount of the complex body (hereinafter, sometimes referred to as "complex body amount measurement step").

[0111] Specific examples, preferred examples, and the like of the substance having affinity for the tetraspanin, the substance having affinity for the phosphatidylserine, and combinations thereof are the same as described above.

[0112] The order of bringing the substance having affinity for the tetraspanin, the substance having affinity for the phosphatidylserine, and the biological sample into contact in the complex body formation step is not particularly limited, but it is preferable to bring the biological sample into contact with the substance having affinity for the phosphatidylserine and then bring the substance having affinity for the tetraspanin into contact.

[0113] That is, the complex body formation step preferably includes: a first order of bringing the biological sample into contact with the substance having affinity for the phosphatidylserine to form a first complex body composed of the extracellular vesicle in the biological sample and the substance having affinity for the phosphatidylserine; and a second order of bringing the first complex body into contact with the substance having affinity for the tetraspanin to form a second complex body composed of the first complex body and the substance having affinity for the tetraspanin.

[0114] Specifically, the complex body formation step, for example, brings an antibody or a Tim protein (preferably Tim1 or Tim4, and more preferably Tim4) that is immobilized to a solid phase and specifically binds to the phosphatidylserine into contact with the biological sample to form a first complex body of the antibody or the Tim protein that specifically binds to the phosphatidylserine and the extracellular vesicle having the phosphatidylserine and the tetraspanin in the biological sample, and brings the first complex body into contact with an antibody that specifically binds to the tetraspanin to form a second complex body of the antibody that specifically binds to the tetraspanin and the first complex body.

[0115] Preferably, the washing operation (B / F separation) is performed after the formation of the complex, at least before the complex measurement process.

[0116] Specifically, for example, the washing operation can be performed after the formation of the first complex and / or after the formation of the second complex in the above-described method. Preferably, the washing operation (B / F separation) is performed after the formation of the first complex, and further, the washing operation (B / F separation) is performed after the formation of the second complex.

[0117] The complex measurement process is a process of measuring the amount of the complex containing the extracellular vesicle having phosphatidylserine and tetraspanin, the substance having affinity for tetraspanin, and the substance having affinity for phosphatidylserine, which is obtained in the complex formation process. The method can be any method as long as the amount of the complex can be measured.

[0118] More specifically, the complex measurement process can detect the labeled substance in the complex containing the antibody specifically binding to phosphatidylserine or Tim protein (preferably, Timl or Tim4, more preferably, Tim4) immobilized to a solid phase, the extracellular vesicle having phosphatidylserine and tetraspanin, the antibody specifically binding to tetraspanin, and the labeled substance (preferably, enzyme and fluorescent substance) in the biological sample, which is obtained in the above-described complex formation process, using (1) the antibody specifically binding to tetraspanin labeled with a labeled substance, or using (2) the labeled secondary antibody specifically binding to the antibody specifically binding to tetraspanin labeled with a labeled substance, or using (3) the antibody specifically binding to tetraspanin to which one of avidin and biotin is bound and the labeled substance to which the remaining one of avidin and biotin is bound, and the like.

[0119] In the complex measurement process, the washing operation (B / F separation) is preferably performed before the detection of the labeled substance.

[0120] More specifically, in the determination of the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnosis support method, for example, an antibody or Tim protein (preferably, Timl or Tim4, more preferably, Tim4) that specifically binds to phosphatidylserine immobilized on a solid phase plate is brought into contact with a biological sample, and a first complex of the antibody or Tim protein that specifically binds to phosphatidylserine and extracellular vesicles having phosphatidylserine and tetraspanin in the biological sample is formed. After B / F separation as necessary, (1) the first complex is brought into contact with an antibody that specifically binds to tetraspanin labeled with a labeling substance, and a second complex of the first complex and the antibody that specifically binds to tetraspanin labeled with a labeling substance is formed, (2) the first complex is brought into contact with an antibody that specifically binds to tetraspanin, and a second complex of the first complex and the antibody that specifically binds to tetraspanin is formed, and after B / F separation as necessary, the second complex is brought into contact with a labeled secondary antibody that specifically binds to the "antibody that specifically binds to the tetraspanin" labeled with a labeling substance, and a third complex of the second complex and the labeled secondary antibody is formed, or (3) the first complex is brought into contact with an antibody that specifically binds to tetraspanin to which one of an avidin and a biotin is bound, and a second complex of the first complex and the antibody that specifically binds to tetraspanin to which one of an avidin and a biotin is bound is formed, and after B / F separation as necessary, a third complex of the second complex and a labeling substance (preferably, an enzyme and a fluorescent substance) to which the remaining one of an avidin and a biotin is bound is formed, and after B / F separation, the labeling substance of the obtained second complex or third complex is detected.

[0121] The amount of the biological sample or the amount (concentration) of the protein in the biological sample, the amount (concentration) or the number of particles of the extracellular vesicles having phosphatidylserine and tetraspanin in the biological sample, the amount (concentration) of the substance having affinity to tetraspanin and the substance having affinity to phosphatidylserine, the labeling substance or the labeling method, and the like are appropriately set according to the kind of the biological sample, the required determination sensitivity, the used determination method or determination device, and the like.

[0122] Further, the amount (concentration) of the extracellular vesicles having phosphatidylserine and tetraspanin in the biological sample and the like can be calculated by preparing a calibration curve using a standard substance. As the standard substance, for example, extracellular vesicles having phosphatidylserine and tetraspanin and the like can be mentioned.

[0123] The determination of Parkinson's disease in the PD diagnosis support method includes determination of Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanin as an index.

[0124] As the determination of the Parkinson's disease, for example, there can be mentioned determination of whether or not the possibility of the Parkinson's disease of the test subject is high, determination of whether or not there is a possibility of the Parkinson's disease of the test subject, determination of whether or not the risk of the test subject to develop symptoms of the Parkinson's disease is high, and determination of whether or not there is a risk of the test subject to develop symptoms of the Parkinson's disease, and preferably determination of whether or not the possibility of the Parkinson's disease of the test subject is high and determination of whether or not there is a possibility of the Parkinson's disease of the test subject.

[0125] The determination of the Parkinson's disease in the PD diagnosis assistance method is performed, for example, by comparing the amount of the extracellular vesicle having phosphatidylserine and tetraspanin in the biological test sample derived from the test subject obtained by the aforementioned measurement of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin with a predetermined reference value (cut-off value).

[0126] Specifically, when the amount of the extracellular vesicle having phosphatidylserine and tetraspanin is equal to or less than the predetermined reference value, it can be determined that the possibility of the Parkinson's disease of the test subject is high or there is a possibility of the Parkinson's disease of the test subject or the risk of the test subject to develop symptoms of the Parkinson's disease is high or there is a risk of the test subject to develop symptoms of the Parkinson's disease.

[0127] Also, when the amount of the extracellular vesicle having phosphatidylserine and tetraspanin is greater than the predetermined reference value, it can be determined that the possibility of the Parkinson's disease of the test subject is low or there is no possibility of the Parkinson's disease of the test subject or the risk of the test subject to develop symptoms of the Parkinson's disease is low or there is no risk of the test subject to develop symptoms of the Parkinson's disease.

[0128] The predetermined reference value (cut-off value) is the amount of the extracellular vesicle having phosphatidylserine and tetraspanin in a biological test sample that is predetermined in order to distinguish between a Parkinson's disease patient and a healthy person.

[0129] The method for determining the pre-established reference value is not particularly limited, and for example, the amount of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample obtained from a patient with Parkinson's disease and a healthy person is measured, the obtained amount of extracellular vesicles having phosphatidylserine and tetraspanin is used, and a reference value is set by statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis). In the setting of the pre-established reference value, sensitivity, specificity, positive predictive value, and negative predictive value, and the like are preferably taken into consideration. The pre-established 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 further 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 further preferably 90% or more.

[0130] < Parkinson's disease diagnosis aid kit >

[0131] The Parkinson's disease diagnosis aid kit in the first invention (hereinafter, sometimes abbreviated as "PD diagnosis aid kit") contains a substance having affinity for tetraspanin and a substance having affinity for phosphatidylserine.

[0132] The substance having affinity for tetraspanin and the substance having affinity for phosphatidylserine in the PD diagnosis aid kit are each the same as the aforementioned content in the PD diagnosis aid method, and the preferred content is also the same.

[0133] The substance having affinity for tetraspanin and the substance having affinity for phosphatidylserine can each be in a solution state, a frozen state, or a dried state, and can also be in a freeze-dried state. Furthermore, the substance having affinity for tetraspanin and the substance having affinity for phosphatidylserine can be contained in the kit as one reagent, or can be contained in the kit as separate reagents.

[0134] The combination of the substance having affinity for tetraspanin and the substance having affinity for phosphatidylserine in the PD diagnosis aid kit can be the same as the aforementioned combination in the PD diagnosis aid method, and the preferred combination is also the same.

[0135] The substance having affinity for tetraspanin and / or the substance having affinity for phosphatidylserine in the PD diagnosis aid kit can be immobilized to a solid phase, and can also be labeled with a labeling substance. As the solid phase or the method for immobilizing to a solid phase, the labeling substance or the labeling method, the aforementioned content is the same as the aforementioned content in the PD diagnosis aid method, and the preferred content is also the same.

[0136] The kit for aiding in the diagnosis of PD can also include a secondary affinity substance (e.g., a secondary antibody) that specifically binds to the substance having affinity for the four-penetrating membrane protein and / or the substance having affinity for the phosphatidylserine. The secondary affinity substance in the kit for aiding in the diagnosis of PD is the same as, and preferably the same as, that described above in the method for aiding in the diagnosis of PD. Also, the secondary affinity substance in the kit for aiding in the diagnosis of PD can be labeled with a labeling substance or labeling method that is the same as, and preferably the same as, that described above in the method for aiding in the diagnosis of PD.

[0137] The substance having affinity for the four-penetrating membrane protein and / or the substance having affinity for the phosphatidylserine in the kit for aiding in the diagnosis of PD can be combined with one of an avidin and a biotin, which are the same as, and preferably the same as, those described above in the method for aiding in the diagnosis of PD.

[0138] The kit for aiding in the diagnosis of PD can also include a labeling substance or labeling method combined with one of an avidin and a biotin, which are the same as, and preferably the same as, those described above in the method for aiding in the diagnosis of PD.

[0139] The concentration (amount) of the substance having affinity for the four-penetrating membrane protein and the substance having affinity for the phosphatidylserine in the kit for aiding in the diagnosis of PD can be appropriately set within a range generally used in the field according to the measuring method. The substance having affinity for the four-penetrating membrane protein, for example, generally contains an amount of 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, when immobilized to a solid phase, and an amount of 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL, when used for detection. Also, the substance having affinity for the phosphatidylserine, for example, generally contains an amount of 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, when immobilized to a solid phase, and an amount of 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL, when used for detection.

[0140] Also, reagents generally used in the field, such as a buffer, a reaction accelerator, a sugar, a protein, a salt, a surfactant, and the like, can be co-presented with the substance having affinity for the four-penetrating membrane protein and / or the substance having affinity for the phosphatidylserine. Their concentration and pH can also be appropriately selected from a range generally used in the field.

[0141] Moreover, the PD diagnosis aid kit can have, in addition to the substance having affinity for tetraspanins and the substance having affinity for phosphatidylserine, reagents necessary for measuring the amount of extracellular vesicles having tetraspanins and phosphatidylserine using these affinity substances. As such reagents, for example, there can be mentioned washing reagents, sample diluents (reagents for diluting a sample), reagents for detecting a labeling substance, reagents for binding a labeling substance to these affinity substances, reagents for immobilizing these affinity substances to a solid phase, reagents for binding avidin or biotin to these affinity substances or labeling substances, and the like. The concentrations and pH of these reagents can be appropriately selected from the range generally used in the field.

[0142] The PD diagnosis aid kit can contain a standard used for making a calibration curve with respect to extracellular vesicles having phosphatidylserine and tetraspanins. As the standard, for example, there can be mentioned extracellular vesicles having phosphatidylserine and tetraspanins. The standard can be in a solution state, a frozen state, or a dried state, or a freeze-dried state. Moreover, reagents generally used in the field, such as a buffer, a reaction accelerator, a saccharide, a protein, a salt, a surfactant, and the like, a stabilizer, a preservative, and the like, can be coexistent with the standard. The concentrations and pH of these reagents can be appropriately selected from the range generally used in the field.

[0143] The PD diagnosis aid kit can contain an accessory or an instruction manual. As the accessory or the instruction manual, for example, there can be mentioned an accessory or an instruction manual in which contents of measuring the amount of extracellular vesicles having phosphatidylserine and tetraspanins in a biological sample derived from a test subject and / or contents of determining that the test subject is Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanins as an index are described, and the like. These accessories or instruction manuals can be described in multiple parts, or can be described in one part.

[0144] Specifically, the PD diagnosis aid kit can be, for example, a kit containing a substance in which either of a substance having affinity for tetraspanins and a substance having affinity for phosphatidylserine is immobilized to a solid phase, a substance in which the remaining one of the substance having affinity for tetraspanins and the substance having affinity for phosphatidylserine is bound to a labeling substance, or the remaining one of the affinity substances and a component for indirectly binding the labeling substance.

[0145] The PD diagnosis aid kit can be, as a preferable kit, a kit containing a substance in which either of a substance having affinity for tetraspanins and a substance having affinity for phosphatidylserine is immobilized to a solid phase, and any one selected from (1) to (3) below.

[0146] (1) a substance to which the remaining one of a substance having affinity for tetraspanin and a substance having affinity for phosphatidylserine is bound, (2) a substance to which the remaining one of a substance having affinity for tetraspanin and a substance having affinity for phosphatidylserine and a secondary affinity substance specifically bound to the affinity substance are bound, and (3) a substance to which the remaining one of a substance having affinity for tetraspanin and a substance having affinity for phosphatidylserine is bound and one of avidin and biotin, and the remaining one of avidin and biotin is bound to a labeling substance

[0147] As a preferable embodiment of the kit for aiding diagnosis of PD, a kit comprising an antibody or Tim protein (more preferably Tim4 or Timl, and particularly preferably Tim4) specifically bound to phosphatidylserine is immobilized to a solid phase plate and any one selected from the group consisting of (1) to (3) below.

[0148] (1) a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing a substance to which the remaining one of an antibody (more preferably an anti-CD9 antibody or an anti-CD81 antibody, and particularly preferably an anti-CD9 antibody) or Tim protein specifically bound to phosphatidylserine is bound; (2) a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing the remaining one of an antibody or Tim protein specifically bound to phosphatidylserine, and a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing a substance to which a secondary affinity substance specifically bound to the affinity substance is bound; and (3) a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing an antibody (preferably an anti-CD9 antibody or an anti-CD81 antibody, and more preferably an anti-CD9 antibody) specifically bound to tetraspanin to which one of avidin and biotin is bound, and a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing a labeling substance to which the remaining one of avidin and biotin is bound.

[0149] < Parkinson's Disease Diagnosis Aid Device >

[0150] The device for assisting in diagnosis of Parkinson's disease in the first invention (hereinafter, sometimes abbreviated as "PD diagnosis assistance device") has: a measurement unit that measures the amount of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample derived from a test subject; and a determination unit that determines whether the test subject is Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanin as an index.

[0151] The biological sample, the test subject, and the extracellular vesicle in the PD diagnosis assistance device are the same as the aforementioned, and preferably the same as the aforementioned.

[0152] The measurement unit and the determination unit of the PD diagnosis assistance device can be provided in the same device, or can be separate.

[0153] The measurement unit in the PD diagnosis assistance device is a unit that measures the amount of extracellular vesicles having phosphatidylserine and tetraspanin in a biological sample introduced into the device. The size or structure of the measurement unit is not particularly limited. As the measurement unit, for example, a microplate reader used in ELISA, an imaging device that captures an image using a CCD, an imaging device used in immunoblotting or a method using a microarray (microchip), a mass spectrometer used in mass spectrometry, a flow cytometer used in a molecular interaction analysis device or a flow cytometer, a UV-Vis detector or a fluorescence detector used in HPLC or capillary electrophoresis, and the like can be mentioned.

[0154] The extracellular vesicles having phosphatidylserine and tetraspanin, the amount, and the measurement object of the measurement method of the amount of extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnosis assistance device are the same as the aforementioned, and preferably the same as the aforementioned.

[0155] The extracellular vesicles having phosphatidylserine and tetraspanin, and the substance used in the measurement of the extracellular vesicles having phosphatidylserine and tetraspanin in the PD diagnosis assistance device are the same as the aforementioned, and preferably the same as the aforementioned or the specific examples.

[0156] The PD diagnosis assistance device can further include a calculation unit. The calculation unit in the PD diagnosis assistance device is a unit that converts a measured value (for example, absorbance, absorbance change amount, transmitted light, transmitted light change amount, fluorescence intensity, fluorescence intensity change amount, luminescence amount, luminescence change amount, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change amount, refractive index, refractive index change amount, and the like) obtained by the measurement unit, which has a correlation with the mass or concentration of the extracellular vesicles having phosphatidylserine and tetraspanin in the biological sample, into mass or concentration, and the like.

[0157] In addition, the measured value, the mass or concentration converted by the arithmetic unit, or the like can be stored in a memory or a storage device provided in a hard disk or the like.

[0158] The determination section in the PD diagnosis assistance device is a section that determines Parkinson's disease using the amount of extracellular vesicles having phosphatidylserine and tetraspanin-4 obtained by the measurement section or the arithmetic section as an index.

[0159] The determination of Parkinson's disease in the PD diagnosis assistance device or the preset reference value used in the determination or the determination method of the preset reference value is the same as the aforementioned content in the PD diagnosis assistance method, and the preferable content or specific examples are also the same.

[0160] The preset reference value in the PD diagnosis assistance device can be stored in the PD diagnosis assistance device in advance or can be input from an input section of the PD diagnosis assistance device at the time of determination.

[0161] The PD diagnosis assistance device can further include an output section. In the output section, processing of displaying the determination result on a display device such as a display or outputting the determination result to a printing device such as a printer is performed.

[0162] 2. The second invention

[0163] <Biological marker group for diagnosis assistance of Parkinson's disease>

[0164] The biological marker group for diagnosis assistance of Parkinson's disease in the second invention (hereinafter, sometimes abbreviated as "PD marker group") is a combination of biological markers including extracellular vesicles having phosphatidylserine and tetraspanin-4 and extracellular vesicles having tetraspanin-4.

[0165] According to the PD marker group, for example, the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin-4 to the amount of extracellular vesicles having the tetraspanin-4 (hereinafter, sometimes abbreviated as "PD marker (II)") can be obtained, and the ratio can be used as an index for determining whether a test subject has Parkinson's disease.

[0166] Further, according to the PD marker (II), an index for distinguishing whether a test subject has Parkinson's disease with dementia symptoms or Parkinson's disease without dementia symptoms can be used.

[0167] The extracellular vesicles in the PD marker group are the same as the extracellular vesicles described above, and the preferable content is also the same.

[0168] The extracellular vesicle having phosphatidylserine and tetraspanin in the PD marker group has at least one tetraspanin among CD9, CD63, CD81, CD151, and the like, and phosphatidylserine, preferably at least one tetraspanin selected from CD9, CD63, and CD81, and phosphatidylserine, more preferably at least one tetraspanin selected from CD9 and CD63, and phosphatidylserine, and particularly preferably CD9 and phosphatidylserine on the membrane surface.

[0169] The extracellular vesicle having tetraspanin in the PD marker group has at least one tetraspanin among CD9, CD63, CD81, CD151, and the like, preferably at least one tetraspanin selected from CD9, CD63, and CD81, more preferably at least one tetraspanin selected from CD9 and CD63, and particularly preferably CD9 on the membrane surface.

[0170] The tetraspanin in the extracellular vesicle having phosphatidylserine and tetraspanin in the PD marker group can be the same as the tetraspanin in the extracellular vesicle having the tetraspanin, or can be different, and is preferably the same.

[0171] <Method for assisting in diagnosis of Parkinson's disease (II)>

[0172] The method for assisting in diagnosis of Parkinson's disease (hereinafter, sometimes abbreviated as "PD diagnosis assistance method (II)") in the second invention includes the steps of measuring the amount of extracellular vesicle having phosphatidylserine and tetraspanin in a biological sample and the amount of extracellular vesicle having tetraspanin in the biological sample, and calculating the ratio of the amount of extracellular vesicle having phosphatidylserine and tetraspanin to the amount of extracellular vesicle having tetraspanin (PD marker (II)), and determining whether the subject is Parkinson's disease using the ratio of the amount of extracellular vesicle having phosphatidylserine and tetraspanin to the amount of extracellular vesicle having tetraspanin as an index.

[0173] The biological sample, the subject, and the extracellular vesicle in the PD diagnosis assistance method (II) are the same as those described above, and preferably the same.

[0174] The extracellular vesicle having phosphatidylserine and tetraspanin in the PD diagnosis assistance method (II) is the same as that described above in the PD marker group, and preferably the same.

[0175] The amount in the PD diagnosis support method (II), the measurement target of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin, the measurement of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin, or the substance used in the measurement can be the same as the aforementioned content in the PD diagnosis support method, except that the antibody specifically binding to tetraspanin and the combination of the antibody specifically binding to tetraspanin and the antibody or phosphatidylserine affinity protein specifically binding to phosphatidylserine are preferred, and the preferred content or specific example is also the same as the aforementioned content in the PD diagnosis support method.

[0176] As the antibody specifically binding to tetraspanin in the PD diagnosis support method (II), for example, an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and an anti-CD151 antibody, and the like can be given, and an anti-CD9 antibody, an anti-CD63 antibody, and an anti-CD81 antibody are preferred, and an anti-CD9 antibody and an anti-CD63 antibody are more preferred, and an anti-CD9 antibody is particularly preferred.

[0177] The substance having affinity to the tetraspanin can be used only one kind, or two or more kinds can be used, and only one kind is preferred.

[0178] As the combination of the antibody specifically binding to tetraspanin and the antibody or phosphatidylserine affinity protein specifically binding to phosphatidylserine in the PD diagnosis support method (II), for example, a combination of the antibody specifically binding to tetraspanin and the antibody specifically binding to Tim protein or phosphatidylserine can be given, and a combination of an anti-CD9 antibody, an anti-CD63 antibody, or an anti-CD81 antibody and Tim protein is preferred, and a combination of an anti-CD9 antibody or an anti-CD63 antibody and Tim protein is more preferred, and a combination of an anti-CD9 antibody or an anti-CD63 antibody and Tim1 or Tim4 is further preferred, and a combination of an anti-CD9 antibody or an anti-CD63 antibody and Tim4 is particularly preferred, and a combination of an anti-CD9 antibody and Tim4 is most preferred.

[0179] The extracellular vesicle having tetraspanin in the PD diagnosis support method (II) is the same as the aforementioned content in the PD marker group, and the preferred content is also the same.

[0180] The tetraspanin in the extracellular vesicle having the phosphatidylserine and tetraspanin in the PD diagnosis support method (II) can be the same as the tetraspanin in the extracellular vesicle having the tetraspanin, or can be different, and the tetraspanin is preferably the same.

[0181] The measurement of the amount of extracellular vesicles having the tetraspanin in the PD diagnosis support method (II) can be performed on extracellular vesicles having one kind of tetraspanin (e.g., only extracellular vesicles having CD9) or on extracellular vesicles having two or more kinds of tetraspanin (e.g., extracellular vesicles having CD9 and extracellular vesicles having CD63), and preferably, the measurement is performed on extracellular vesicles having one kind of tetraspanin.

[0182] The measurement of the amount of extracellular vesicles having the tetraspanin is not particularly limited as long as it is a method generally used in the field, and for example, an immunological measurement method using a substance having affinity for the tetraspanin, a mass analysis method, and a method combining them can be mentioned, and preferably, an immunological measurement method using a substance having affinity for the tetraspanin is used. In addition, the immunological measurement method includes not only a method using an immune reaction (antigen-antibody reaction), but also a method using a binding force between two molecules other than the antigen-antibody reaction, such as the binding of a lectin to a protein (a method in accordance with the immunological measurement method).

[0183] As the substance having affinity for the tetraspanin in the measurement of the amount of extracellular vesicles having the tetraspanin, the same content as the aforementioned content in the PD diagnosis support method can be mentioned, and preferably, a protein specifically binding to the tetraspanin, and more preferably, an antibody specifically binding to the tetraspanin is used. As the antibody specifically binding to the tetraspanin, for example, an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and an anti-CD151 antibody can be mentioned, and preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody is used. The substance having affinity for the tetraspanin can be used alone or two or more kinds thereof, and preferably, only one kind is used.

[0184] The substance having affinity for the tetraspanin can be a commercially available product or can be appropriately prepared by a conventional method. Also, the antibody specifically binding to the tetraspanin can be any one of a polyclonal antibody and a monoclonal antibody, and can be used alone or can be used in appropriate combination. Also, the antibody specifically binding to the tetraspanin can use not only an intact immunoglobulin but also a fragment thereof having a binding ability to an antigen, such as a fragment antibody of Fab, F(ab')2, F(ab'), a synthetic antibody such as a single chain Fv, a double chain antibody, a triple chain antibody, a quadruple chain antibody, and the like. Also, when these antibodies are prepared, a method described in, for example, "Immunoassay" (edited by the Biochemical Assay Research Society, KODANSHA LTD., 2014) or the like can be used.

[0185] The substance having affinity for the tetraspanin in the PD diagnosis aid method (II) can be labeled with a labeling substance, and the labeling substance or the labeling method is the same as in the PD diagnosis aid method, and the preferable contents or specific examples are also the same.

[0186] Also, as in the PD diagnosis aid method, the substance having affinity for the tetraspanin can be provided as a primary affinity substance, and a secondary affinity substance (for example, a secondary antibody) specifically binding to the primary affinity substance can be further used. The secondary affinity substance can be labeled with a labeling substance, and the labeling substance or the labeling method is the same as the foregoing in the PD diagnosis aid method, and the preferable contents are also the same.

[0187] Also, the labeling based on the labeling substance can use a substance that binds one of an avidin and a biotin to the substance having affinity for the tetraspanin and a substance that binds the remaining one of the avidin and the biotin to the labeling substance, and can use the binding of the avidin and the biotin. The method of binding the avidin or the biotin to the avidin or the biotin and the substance having affinity for the tetraspanin is the same as the foregoing in the PD diagnosis aid method, and the preferable contents or specific examples are also the same.

[0188] The substance having affinity for the tetraspanin in the PD diagnosis aid method (II) can be immobilized to a solid phase, and the solid phase or the method of immobilizing the substance having affinity for the tetraspanin to a solid phase is the same as the foregoing in the PD diagnosis aid method, and the preferable contents or specific examples are also the same.

[0189] The substance having affinity for tetraspanin used in the measurement of the extracellular vesicle having the phosphatidylserine and the tetraspanin and the substance having affinity for tetraspanin used in the measurement of the extracellular vesicle having the tetraspanin in the PD diagnosis aid method (II) can use a substance having affinity for at least one same tetraspanin (for example, a substance having affinity for at least CD9 is used in both measurements), can use a substance having affinity for different kinds of tetraspanin (for example, a substance having affinity for CD9 is used in one measurement and a substance having affinity for CD63 is used in the remaining one measurement), preferably uses a substance having affinity for at least one same tetraspanin, more preferably uses only a substance having affinity for the same tetraspanin (for example, a substance having affinity for only CD9 is used in both measurements), and particularly preferably uses only one substance having affinity for the same tetraspanin (for example, only an anti-CD9 antibody is used in both measurements).

[0190] The measurement of the amount of the extracellular vesicle having the tetraspanin in the PD diagnosis aid method (II), the measurement principle is the same as the measurement of the amount of the extracellular vesicle having the phosphatidylserine and the tetraspanin in the PD diagnosis aid method, and preferably the content is also the same.

[0191] Specifically, the measurement of the amount of the extracellular vesicle having the tetraspanin in the PD diagnosis aid method (II) can cite, for example, a method including the following steps as a preferable method: (1) a step (complex formation step) of bringing the extracellular vesicle having the tetraspanin in the biological test sample into contact with a substance having affinity for the tetraspanin to form a complex containing the extracellular vesicle having the tetraspanin in the biological test sample and the substance having affinity for the tetraspanin; and (2) a step (complex amount measurement step) of measuring the amount of the complex.

[0192] The complex formation step in the measurement of the amount of the extracellular vesicle having the tetraspanin in the PD diagnosis aid method (II) preferably includes: a first sequence of bringing the biological test sample into contact with a substance having affinity for the tetraspanin to form a first complex composed of the extracellular vesicle in the biological test sample and the substance having affinity for the tetraspanin; and a second sequence of bringing the first complex into contact with a substance having affinity for the tetraspanin to form a second complex composed of the first complex and the substance having affinity for the tetraspanin.

[0193] The substance having affinity for the tetraspanin used in the formation of the first complex and the substance having affinity for the tetraspanin used in the formation of the second complex are preferably the same.

[0194] Specifically, the complex formation step in the measurement of the amount of the extracellular vesicle having tetraspanin in the PD diagnosis support method (II) forms, for example, a first complex of the antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) specific to tetraspanin and the extracellular vesicle having phosphatidylserine and tetraspanin in the biological sample by bringing the antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) immobilized on a solid phase into contact with the biological sample, and forms a second complex of the first complex and the antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) specific to tetraspanin by bringing the antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) specific to tetraspanin into contact with the first complex.

[0195] Preferably, the washing operation (B / F separation) is performed after the complex formation and before the complex amount measurement.

[0196] Specifically, for example, the washing operation can be performed after the formation of the first complex and / or after the formation of the second complex in the above-described method, and preferably, the washing operation (B / F separation) is performed after the formation of the first complex and further, the washing operation (B / F separation) is performed after the formation of the second complex.

[0197] The complex amount measurement step in the measurement of the amount of the extracellular vesicle having tetraspanin in the PD diagnosis support method (II) is a step of measuring the amount of the complex obtained in the complex formation step, which contains the extracellular vesicle having tetraspanin and the substance having affinity to tetraspanin, and can be any method as long as the amount of the complex can be measured.

[0198] More specifically, the complex amount measurement step detects the labeled substance in the complex obtained in the above-described complex formation step, which contains the antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) specific to tetraspanin immobilized on a solid phase, the extracellular vesicle having tetraspanin in the biological sample, the antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) specific to tetraspanin, and the labeled substance (preferably, an enzyme and a fluorescent substance) by using, for example, (1) the antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) specific to tetraspanin labeled with a labeled substance, or (2) a labeled secondary antibody labeled with a labeled substance and specific to the "antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) specific to tetraspanin", or (3) the antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) specific to tetraspanin to which one of an avidin and a biotin is bound and the labeled substance to which the remaining one of the avidin and the biotin is bound.

[0199] In the complex measurement process, it is preferable to perform a washing operation (B / F separation) before detecting the labeling substance.

[0200] More specifically, the measurement of the amount of the extracellular vesicle having the tetraspanin in the PD diagnosis support method (II) is performed, for example, by bringing an antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) that specifically binds to the tetraspanin immobilized on a solid phase plate into contact with the biological sample, thereby forming a first complex of the antibody that specifically binds to the tetraspanin and the extracellular vesicle having the tetraspanin in the biological sample, and, as needed, after B / F separation, (1) bringing the first complex into contact with an antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) that specifically binds to the tetraspanin labeled with a labeling substance, thereby forming a second complex of the first complex and the antibody that specifically binds to the tetraspanin labeled with the labeling substance, (2) bringing the first complex into contact with an antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) that specifically binds to the tetraspanin, thereby forming a second complex of the first complex and the antibody that specifically binds to the tetraspanin, and, as needed, after B / F separation, bringing the second complex into contact with a labeled secondary antibody that specifically binds to the "antibody that specifically binds to the tetraspanin" labeled with a labeling substance, thereby forming a third complex of the second complex and the labeled secondary antibody, or (3) bringing the first complex into contact with an antibody (preferably, an anti-CD9 antibody and an anti-CD63 antibody, and more preferably, an anti-CD9 antibody) that specifically binds to the tetraspanin bound to one of an anti-biotin protein and a biotin, thereby forming a second complex of the first complex and the antibody that specifically binds to the tetraspanin bound to one of an anti-biotin protein and a biotin, and, as needed, after B / F separation, forming a third complex of the second complex and a labeling substance (preferably, an enzyme and a fluorescent substance) bound to the remaining one of an anti-biotin protein and a biotin, and, after B / F separation, detecting the labeling substance (preferably, an enzyme and a fluorescent substance) of the obtained second complex or third complex.

[0201] The amount of the biological sample or the amount (concentration) of the protein in the biological sample, the amount (concentration) or the number of particles of the extracellular vesicle having the tetraspanin in the biological sample, the amount (concentration) of the substance having affinity to the tetraspanin, the labeling substance, or the labeling method is appropriately set according to the type of the biological sample, the required measurement sensitivity, the used measurement method or measurement device, and the like.

[0202] Further, the amount (concentration) of the extracellular vesicle having tetraspanin in the biological sample can be calculated by preparing a calibration curve using a standard sample. As the standard sample, for example, an extracellular vesicle having tetraspanin can be mentioned.

[0203] The ratio of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount of the extracellular vesicle having the tetraspanin can be calculated by dividing the amount (A) of the extracellular vesicle having phosphatidylserine and tetraspanin by the amount (B) of the extracellular vesicle having the tetraspanin ((A) / (B)).

[0204] The determination of Parkinson's disease in the PD diagnosis support method (II) includes determining Parkinson's disease with the ratio of the amount (B) of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount (A) of the extracellular vesicle having the tetraspanin ((A) / (B)) as an index.

[0205] As the determination of Parkinson's disease, for example, determination of whether the test subject has a high possibility of Parkinson's disease, determination of whether the test subject has a possibility of Parkinson's disease, determination of whether the test subject has a high risk of symptoms of Parkinson's disease, and determination of whether the test subject has a risk of symptoms of Parkinson's disease can be mentioned, and determination of whether the test subject has a high possibility of Parkinson's disease and determination of whether the test subject has a possibility of Parkinson's disease are preferable.

[0206] Further, the determination of Parkinson's disease in the PD diagnosis support method (II) can include determining (distinguishing) whether the test subject has dementia symptoms of Parkinson's disease or does not have dementia symptoms of Parkinson's disease with the ratio of the amount (B) of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount (A) of the extracellular vesicle having the tetraspanin ((A) / (B)) as an index.

[0207] In the present specification, dementia symptoms mean a state in which cognitive function of the brain is reduced due to acquired organic disorders. Specifically, for example, in the Mini-Mental State Examination (MMSE), when the score is 27 or less out of 30, it is determined that there are dementia symptoms, and for example, it can be considered that there is a possibility of developing symptoms of Alzheimer's dementia or mild cognitive impairment. In the present specification, Parkinson's disease with dementia symptoms is a state in which Parkinson's disease is developed or there is a possibility of developing symptoms of Parkinson's disease and symptoms of dementia symptoms are developed or there is a possibility of developing symptoms of dementia symptoms, for example, a state in which Parkinson's disease is developed or there is a possibility of developing symptoms of Parkinson's disease and the MMSE is 27 or less.

[0208] In this specification, Parkinson's disease without dementia symptoms is a condition of having Parkinson's disease or having the possibility of developing Parkinson's disease symptoms without the presence of dementia symptoms or the possibility of developing dementia symptoms. For example, having Parkinson's disease or having the possibility of developing Parkinson's disease symptoms and having an MMSE score greater than 27.

[0209] The determination of Parkinson's disease in the PD diagnostic auxiliary method (II) is performed, for example, as follows: the ratio of the amount of extracellular vesicles with phosphatidylserine and tetrapermeable protein (B) obtained by measuring the amount of extracellular vesicles with tetrapermeable protein (B) to the amount of extracellular vesicles with said tetrapermeable protein (A) ((A) / (B)) is compared with a pre-set baseline value (cutoff value).

[0210] Specifically, when the ratio ((A) / (B)) of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities (A)) is below a predetermined baseline value, it can be determined that the test subject is highly likely to have Parkinson's disease or that there is a high risk of the test subject developing symptoms of Parkinson's disease.

[0211] Furthermore, when the ratio ((A) / (B)) of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities (A)) is greater than a predetermined baseline value, it can be determined that the subject has a low probability of having Parkinson's disease or no probability of having Parkinson's disease, or that the subject has a low risk of developing symptoms of Parkinson's disease or no risk of developing symptoms of Parkinson's disease.

[0212] The pre-defined baseline (cutoff value) in the PD diagnostic aid method (II) is a pre-defined ratio ((A) / (B)) of the amount of extracellular vesicles with phosphatidylserine and tetrapermeabilities (B) to the amount of extracellular vesicles with tetrapermeabilities (A) in the biological sample, which is used to distinguish between patients with Parkinson's disease and healthy individuals.

[0213] The method for determining the pre-set reference value in the PD diagnosis support method (II) is not particularly limited, for example, the amount (B) of extracellular vesicles having phosphatidylserine and tetraspanin-4 is measured from biological samples obtained from patients with Parkinson's disease and healthy persons, the ratio ((A) / (B)) of the amount (B) of extracellular vesicles having phosphatidylserine and tetraspanin-4 to the amount (A) of extracellular vesicles having tetraspanin-4 is calculated for each, the obtained ratio ((A) / (B)) of the amount (B) of extracellular vesicles having phosphatidylserine and tetraspanin-4 to the amount (A) of extracellular vesicles having tetraspanin-4 is used, and a reference value is set by statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis). In the setting of the pre-set reference value, sensitivity, specificity, positive predictive value, negative predictive value, and the like are preferably taken into consideration. The pre-set reference value can be set, for example, to a sensitivity of 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more, and to a specificity of 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more.

[0214] The determination of Parkinson's disease in the PD diagnosis support method (II) can be performed by using the ratio ((A) / (B)) of the amount (B) of extracellular vesicles having phosphatidylserine and tetraspanin-4 to the amount (A) of extracellular vesicles having tetraspanin-4 as an index, and determining (distinguishing) whether the test subject is a healthy person, a Parkinson's disease patient with dementia symptoms, or a Parkinson's disease patient without dementia symptoms, according to a plurality of pre-set reference values {cut-off values, such as a cut-off value for distinguishing a healthy person from a Parkinson's disease patient with dementia symptoms (hereinafter sometimes referred to as "healthy person / Parkinson's disease patient with dementia symptoms reference value"), and a cut-off value for distinguishing a Parkinson's disease patient with dementia symptoms from a Parkinson's disease patient without dementia symptoms (hereinafter sometimes referred to as "Parkinson's disease patient with dementia symptoms / Parkinson's disease patient without dementia symptoms reference value")}. The determination of Parkinson's disease in the PD diagnosis support method (II) can also be performed by using the ratio ((A) / (B)) of the amount (B) of extracellular vesicles having phosphatidylserine and tetraspanin-4 to the amount (A) of extracellular vesicles having tetraspanin-4 as an index, and determining (distinguishing) whether the test subject is a Parkinson's disease patient with dementia symptoms or a Parkinson's disease patient without dementia symptoms, according to the "Parkinson's disease patient with dementia symptoms / Parkinson's disease patient without dementia symptoms reference value".

[0215] In the PD diagnosis support method (II), when it is determined only whether the test subject is a Parkinson's disease patient with dementia symptoms or a Parkinson's disease patient without dementia symptoms, the test subject is preferably a person diagnosed as having Parkinson's disease according to a diagnosis reference, and a person diagnosed as having a risk of developing symptoms of Parkinson's disease according to a diagnosis reference. The diagnosis reference is the same as described above.

[0216] The determination of whether the subject in the PD diagnosis support method (II) has dementia-symptomatic Parkinson's disease or non-dementia-symptomatic Parkinson's disease is performed, for example, by comparing the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin (B) to the amount of extracellular vesicles having the tetraspanin (A) ((A) / (B)) obtained by the aforementioned measurement of the amount of extracellular vesicles having the tetraspanin and the aforementioned measurement of the amount of extracellular vesicles having phosphatidylserine and tetraspanin to the pre-established "dementia-symptomatic Parkinson's disease / non-dementia-symptomatic Parkinson's disease reference value".

[0217] Specifically, when the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin (B) to the amount of extracellular vesicles having the tetraspanin (A) ((A) / (B)) is "dementia-symptomatic Parkinson's disease / non-dementia-symptomatic Parkinson's disease reference value" or less, it can be determined that the subject has a high possibility of having non-dementia-symptomatic Parkinson's disease or that the subject has a high risk of developing symptoms of non-dementia-symptomatic Parkinson's disease or that there is a risk of the subject developing symptoms of non-dementia-symptomatic Parkinson's disease.

[0218] Also, when the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin (B) to the amount of extracellular vesicles having the tetraspanin (A) ((A) / (B)) is greater than "dementia-symptomatic Parkinson's disease / non-dementia-symptomatic Parkinson's disease reference value", it can be determined that the subject has a high possibility of having dementia-symptomatic Parkinson's disease or that the subject has a high risk of developing symptoms of dementia-symptomatic Parkinson's disease or that there is a risk of the subject developing symptoms of dementia-symptomatic Parkinson's disease.

[0219] The method for determining the "Parkinson's disease with dementia symptoms / Parkinson's disease without dementia symptoms reference value" is not particularly limited, for example, the amount (B) of extracellular vesicles with phosphatidylserine and tetraspanin-4 from a biological sample obtained from a patient with Parkinson's disease with dementia symptoms and a patient with Parkinson's disease without dementia symptoms is measured relative to the amount (A) of extracellular vesicles with tetraspanin-4, the ratio ((A) / (B)) of the amount (B) of extracellular vesicles with phosphatidylserine and tetraspanin-4 to the amount (A) of extracellular vesicles with tetraspanin-4 is calculated for each, the ratio ((A) / (B)) of the amount (B) of extracellular vesicles with phosphatidylserine and tetraspanin-4 to the amount (A) of extracellular vesicles with tetraspanin-4 obtained is used, and by statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis: subject performance characteristics), a reference value can be set. In the setting of the reference value set in advance, sensitivity, specificity, positive predictive value, negative predictive value, etc. are preferably taken into account. The reference value set in advance can be set, for example, to a sensitivity of 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more, for example, to a specificity of 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more.

[0220] In the determination of whether the subject in the PD diagnosis support method (II) is a healthy person, a Parkinson's disease with dementia symptoms, or a Parkinson's disease without dementia symptoms, for example, when the ratio of the amount (B) of the extracellular vesicle with phosphatidylserine and tetraspanin to the amount (A) of the extracellular vesicle with tetraspanin ((A) / (B)) is "Parkinson's disease with dementia symptoms / Parkinson's disease without dementia symptoms reference value" or less, it can be determined that the subject has a high possibility of having a Parkinson's disease without dementia symptoms or that the subject has a high risk of developing symptoms of a Parkinson's disease without dementia symptoms or that there is a risk that the subject will develop symptoms of a Parkinson's disease without dementia symptoms. When the ratio ((A) / (B)) is greater than "Parkinson's disease with dementia symptoms / Parkinson's disease without dementia symptoms reference value" and is "healthy person / Parkinson's disease with dementia symptoms reference value" or less, it can be determined that the subject has a high possibility of having a Parkinson's disease with dementia symptoms or that the subject has a high risk of developing symptoms of a Parkinson's disease with dementia symptoms or that there is a risk that the subject will develop symptoms of a Parkinson's disease with dementia symptoms. When the ratio ((A) / (B)) is greater than "healthy person / Parkinson's disease with dementia symptoms reference value", it can be determined that the subject has a low possibility of having a Parkinson's disease or that there is no possibility of having a Parkinson's disease or that the subject has a low risk of developing symptoms of a Parkinson's disease or that there is no risk that the subject will develop symptoms of a Parkinson's disease.

[0221] The method for determining the "healthy person / Parkinson's disease with dementia symptoms reference value" is not particularly limited, and for example, the amount (B) of the extracellular vesicle with phosphatidylserine and tetraspanin to the amount (A) of the extracellular vesicle with tetraspanin ((A) / (B)) is calculated from the amount (B) of the extracellular vesicle with phosphatidylserine and tetraspanin to the amount (A) of the extracellular vesicle with tetraspanin in biological samples obtained from healthy persons and Parkinson's disease patients with dementia symptoms, respectively, the obtained ratio of the amount (B) of the extracellular vesicle with phosphatidylserine and tetraspanin to the amount (A) of the extracellular vesicle with tetraspanin ((A) / (B)) is used, and a reference value is set by statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis). In the setting of the reference value set in advance, sensitivity, specificity, positive predictive value, negative predictive value, and the like are preferably taken into consideration. The reference value set in advance can be set, for example, to a sensitivity of 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more, and can be set, for example, to a specificity of 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more.

[0222] <Kit for aiding diagnosis of Parkinson's disease (II)>

[0223] The kit for aiding diagnosis of Parkinson's disease in the second invention (hereinafter, sometimes abbreviated as "PD diagnosis aid kit (II)") comprises a substance having affinity to tetraspanin and a substance having affinity to phosphatidylserine.

[0224] The substance having affinity to tetraspanin and the substance having affinity to phosphatidylserine in the PD diagnosis aid kit (II) are respectively the same as the aforementioned contents in the PD diagnosis aid method (II), and the preferable contents are also the same. The substance having affinity to tetraspanin and the substance having affinity to phosphatidylserine can be in a solution state, or in a frozen state or a dried state, or in a freeze-dried state. Furthermore, the substance having affinity to tetraspanin and the substance having affinity to phosphatidylserine can be contained in the kit as one reagent, or can be contained in the kit as separate reagents.

[0225] The combination of the substance having affinity to tetraspanin and the substance having affinity to phosphatidylserine in the PD diagnosis aid kit (II) can be the same as the aforementioned combination in the PD diagnosis aid method (II), and the preferable combination is also the same.

[0226] The substance having affinity to tetraspanin and / or the substance having affinity to phosphatidylserine in the PD diagnosis aid kit (II) can be immobilized to a solid phase, and can also be labeled with a labeling substance. The solid phase or the method of immobilization to a solid phase, the labeling substance or the method of labeling are the same as the aforementioned contents in the PD diagnosis aid method, and the preferable contents are also the same.

[0227] The PD diagnosis aid kit (II) can also comprise a secondary affinity substance (e.g., a secondary antibody) that specifically binds to the substance having affinity to the tetraspanin and / or the substance having affinity to the phosphatidylserine. The secondary affinity substance in the PD diagnosis aid kit (II) is the same as the aforementioned contents in the PD diagnosis aid method, and the preferable contents are also the same. Furthermore, the secondary affinity substance in the PD diagnosis aid kit (II) can be labeled with a labeling substance, and the labeling substance or the method of labeling are the same as the aforementioned contents in the PD diagnosis aid method, and the preferable contents are also the same.

[0228] The substance having affinity for tetraspanin and / or the substance having affinity for phosphatidylserine in the kit for aiding diagnosis of PD (II) can also be combined with one of the avertin and the biotin, and as the avertin and the biotin, the same contents as those described above in the method for aiding diagnosis of PD are preferable.

[0229] The kit for aiding diagnosis of PD (II) can contain a labeling substance combined with one of the avertin and the biotin, and as the labeling substance or the labeling method, the same contents as those described above in the method for aiding diagnosis of PD are preferable.

[0230] The concentrations (amounts) of the substance having affinity for tetraspanin and the substance having affinity for phosphatidylserine in the kit for aiding diagnosis of PD (II) can be appropriately set within the range generally used in the field according to the measuring method. The substance having affinity for tetraspanin, for example, as the concentration at the time of use, when immobilized to a solid phase, generally contains an amount of 10 to 20,000 ng / mL, preferably an amount of 100 to 10,000 ng / mL, and when used for detection, generally contains an amount of 10 to 5,000 ng / mL, preferably an amount of 100 to 500 ng / mL. Also, the substance having affinity for phosphatidylserine, for example, as the concentration at the time of use, when immobilized to a solid phase, generally contains an amount of 10 to 20,000 ng / mL, preferably an amount of 100 to 10,000 ng / mL, and when used for detection, generally contains an amount of 10 to 5,000 ng / mL, preferably an amount of 100 to 500 ng / mL.

[0231] Also, the substance having affinity for tetraspanin and the substance having affinity for phosphatidylserine in the kit for aiding diagnosis of PD (II) can coexist with the reagents generally used in the field, and as the reagents, the same contents as those of the kit for aiding diagnosis of PD are preferable.

[0232] Also, the kit for aiding diagnosis of PD (II) can have, in addition to the substance having affinity for tetraspanin and the substance having affinity for phosphatidylserine, reagents required for measuring the amount of extracellular vesicles having tetraspanin and phosphatidylserine and / or the amount of extracellular vesicles having phosphatidylserine using these affinity substances. As such reagents, the same contents as those of the kit for aiding diagnosis of PD are preferable.

[0233] The kit for aiding diagnosis of PD (II) can contain a standard substance used for making a calibration curve with respect to extracellular vesicles having phosphatidylserine and tetraspanin, and as the standard substance, the same contents as those of the kit for aiding diagnosis of PD are preferable.

[0234] Also, the PD diagnosis aid kit (II) can contain a standard sample used for making a calibration curve with respect to the extracellular vesicle having tetraspanin, and as the standard sample, an extracellular vesicle having tetraspanin can be cited. The standard sample can be in a solution state, or in a frozen state or a dried state, or in a freeze-dried state. Also, a reagent such as a buffer, a reaction promoter, a saccharide, a protein, a salt, a surfactant, and the like, a stabilizer, a preservative, and the like, which are generally used in the field, can be coexistent with the standard sample. Their concentrations and pH can be appropriately selected from the range generally used in the field.

[0235] An accessory or an instruction manual can be contained in the PD diagnosis aid kit (II). As the accessory or the instruction manual, for example, an accessory or an instruction manual, and the like, in which contents of measuring the amount of the extracellular vesicle having tetraspanin and the amount of the extracellular vesicle having phosphatidylserine and tetraspanin in a biological test sample derived from a test subject, and / or contents of determining that the test subject is Parkinson's disease with the amount of the extracellular vesicle having phosphatidylserine and tetraspanin as an index relative to the amount of the extracellular vesicle having tetraspanin (for example, the PD diagnosis aid method (II)) are described, can be cited. These accessories or instruction manuals can be described in multiple parts, or can be described in one part.

[0236] Specifically, the PD diagnosis aid kit (II) can cite, for example, a kit containing the following (A) and (B).

[0237] (A) a substance to which either of a substance having affinity for tetraspanin and a substance having affinity for phosphatidylserine is immobilized to a solid phase, a substance to which the remaining one of the substance having affinity for tetraspanin and the substance having affinity for phosphatidylserine is bound to a labeling substance, or the remaining one of the affinity substances and a component that indirectly binds the same to a labeling substance

[0238] (B) a substance to which a substance having affinity for tetraspanin is immobilized to a solid phase, a substance to which the affinity substance is bound to a labeling substance, or the affinity substance and a component that indirectly binds the same to a labeling substance

[0239] The PD diagnosis aid kit (II) can cite, for example, preferably a kit containing the following (C) and (D).

[0240] (C) a substance to which either of a substance having affinity for tetraspanin and a substance having affinity for phosphatidylserine is immobilized to a solid phase, and any one selected from the following (1) to (3)

[0241] (1) a substance to which the remaining one of a substance having affinity for tetraspanin and a substance having affinity for phosphatidylserine is bound, (2) a substance to which the remaining one of a substance having affinity for tetraspanin and a substance having affinity for phosphatidylserine and a secondary affinity substance specifically binding to the affinity substance are bound, and (3) a substance to which one of avidin and biotin is bound and the remaining one of avidin and biotin is bound to a labeling substance

[0242] (D) a substance to which a substance having affinity for tetraspanin is immobilized to a solid phase and any one selected from the group consisting of (4) to (6) below

[0243] (4) a substance to which a substance having affinity for tetraspanin is bound, (5) a substance to which a substance having affinity for tetraspanin and a secondary affinity substance specifically binding to a substance having affinity for tetraspanin are bound, and (6) any one selected from the group consisting of a substance to which one of a substance having affinity for tetraspanin and avidin and biotin is bound and the remaining one of avidin and biotin is bound to a labeling substance

[0244] As a preferable example of the kit for assisting in the diagnosis of PD (II), for example, the following kits can be given.

[0245] For example, a kit containing a solid phase to which an antibody or a Tim protein (more preferably, Tim4 or Timl, and particularly preferably, Tim4) specifically binding to phosphatidylserine is immobilized, a solid phase to which a tetraspanin antibody (more preferably, an anti-CD9 antibody or an anti-CD63 antibody, and particularly preferably, an anti-CD9 antibody) is immobilized, and any one selected from the group consisting of (1) to (3) below can be given.

[0246] (1) a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing a substance to which a tetraspanin antibody (more preferably an anti-CD9 antibody or an anti-CD63 antibody, and particularly preferably an anti-CD9 antibody) and a labeling substance are bound; (2) a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing a tetraspanin antibody (more preferably an anti-CD9 antibody or an anti-CD63 antibody, and particularly preferably an anti-CD9 antibody), and a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing a secondary affinity substance specifically binding to the tetraspanin antibody and a labeling substance; (3) a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing an antibody (preferably an anti-CD9 antibody or an anti-CD63 antibody, and more preferably an anti-CD9 antibody) specifically binding to a tetraspanin to which one of an avidin and a biotin is bound, and a solution (usually 10 to 5000 ng / mL, preferably 100 to 500 ng / mL) containing a labeling substance to which the remaining one of the avidin and the biotin is bound.

[0247] <Device for assisting diagnosis of Parkinson's disease (II)>

[0248] The device for assisting diagnosis of Parkinson's disease (hereinafter, sometimes abbreviated as "PD diagnosis assistance device (II)") in the second invention has: a measurement unit that measures the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having tetraspanin in a biological sample derived from a test subject; a calculation unit that calculates the proportion of the amount of extracellular vesicles having phosphatidylserine and tetraspanin to the amount of extracellular vesicles having tetraspanin; and a determination unit that determines whether the test subject has Parkinson's disease, using the proportion of the amount of extracellular vesicles having phosphatidylserine and tetraspanin to the amount of extracellular vesicles having tetraspanin in the biological sample as an index.

[0249] The measurement unit, the calculation unit, the determination unit, and the like that constitute the PD diagnosis assistance device (II) can be arranged in the same device, or can be separate bodies.

[0250] The measurement unit in the PD diagnosis assistance device (II) is a site that measures the amount of extracellular vesicles having phosphatidylserine and tetraspanin and the amount of extracellular vesicles having tetraspanin in a biological sample introduced into the device. The size or structure of the measurement unit is not particularly limited.

[0251] As the measuring section, for example, an enzyme marker used in ELISA method, an imaging device using CCD for photographing, an imaging device used in immunoblotting method or a method using microarray (microchip), a mass analysis device used in mass analysis method, a flow cytometry device used in intermolecular interaction analysis device and flow cytometry device, an ultraviolet-visible light detector or a fluorescence detector used in HPLC method or capillary electrophoresis method, and the like can be mentioned.

[0252] The test subject, the biological sample, and the extracellular vesicle in the PD diagnosis assistance device (II) are the same as, and preferably the same as, the aforementioned.

[0253] The extracellular vesicle having phosphatidylserine and tetraspanin, the amount of the extracellular vesicle having phosphatidylserine and tetraspanin, and the measurement target of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin in the PD diagnosis assistance device (II) are the same as, and preferably the same as, the aforementioned in the PD diagnosis assistance method.

[0254] The measurement of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin in the PD diagnosis assistance device (II) or the substance used in the measurement is the same as, and preferably the same as, the aforementioned in the PD diagnosis assistance method, and the specific example is also the same.

[0255] Further, the extracellular vesicle having tetraspanin, the measurement target of the amount of the extracellular vesicle having tetraspanin in the PD diagnosis assistance device (II) are the same as, and preferably the same as, the aforementioned in the PD diagnosis assistance method (II).

[0256] The measurement of the amount of the extracellular vesicle having tetraspanin in the PD diagnosis assistance device (II) or the substance used in the measurement is the same as, and preferably the same as, the aforementioned in the PD diagnosis assistance method (II), and the specific example is also the same.

[0257] The operation section in the PD diagnosis assistance device (II) is a section that calculates the ratio of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount of the extracellular vesicle having tetraspanin, based on the amount of the extracellular vesicle having tetraspanin and the amount of the extracellular vesicle having phosphatidylserine and tetraspanin obtained by the measuring section. The size or structure of the measuring section is not particularly limited.

[0258] The operation section in the PD diagnosis assistance device (II) can calculate the ratio of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount of the extracellular vesicle having the tetraspanin, after converting the measured value (e.g., absorbance, absorbance change amount, transmitted light, transmitted light change amount, fluorescence intensity, fluorescence intensity change amount, luminescence amount, luminescence change amount, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change amount, refractive index, refractive index change amount, etc.) obtained by the measurement section in the PD diagnosis assistance device, which has a correlation with the mass or concentration of the extracellular vesicle having phosphatidylserine and tetraspanin and the extracellular vesicle having tetraspanin in the biological sample, to mass or concentration, etc.

[0259] In addition, the measured value, the mass or concentration, etc. converted by the operation section, the ratio of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount of the extracellular vesicle having the tetraspanin can be stored in the memory or a storage device provided in a hard disk or the like.

[0260] The determination section in the PD diagnosis assistance device (II) is a section that determines Parkinson's disease using the ratio of the amount of the extracellular vesicle having phosphatidylserine and tetraspanin to the amount of the extracellular vesicle having tetraspanin calculated by the operation section as an index.

[0261] In addition, the determination section in the PD diagnosis assistance device (II) can be a section that discriminates whether the Parkinson's disease of the test subject has a dementia symptom or not.

[0262] The determination of Parkinson's disease in the PD diagnosis assistance device (II) or the predetermined reference value used in the determination, the determination method of the predetermined reference value, and the like are the same as those in the PD diagnosis assistance method (II), and the preferable contents or specific examples are also the same.

[0263] The predetermined reference value in the PD diagnosis assistance device (II) can be stored in the PD diagnosis assistance device (II) in advance or can be input from an input section of the PD diagnosis assistance device (II) at the time of determination.

[0264] The PD diagnosis assistance device (II) can further include an output section. In the output section, processing of displaying the determination result on a display device such as a display or outputting the determination result to a printing device such as a printer is performed.

[0265] According to the present application, data (determination result) for assisting a doctor in diagnosing Parkinson's disease of a test subject can be obtained.

[0266] When the subject according to the present application is determined to have Parkinson's disease, the physician can perform further examinations recommended in the guidelines for the diagnosis and treatment of Parkinson's disease, such as examinations related to biomarkers of Parkinson's disease, examinations using candidate substances for biomarkers of Parkinson's disease, taking into account the results of these examinations, and the like, and can perform a diagnosis related to Parkinson's disease of the subject.

[0267] Also, when the subject according to the present application is determined to have Parkinson's disease, the subject can be administered a drug for Parkinson's disease (a drug for delaying the progression of Parkinson's disease or a therapeutic drug) such as L-dopa or surgery.

[0268] When the subject is determined to have Parkinson's disease with symptoms of dementia, for example, a drug that can adversely affect the symptoms of dementia, such as an anticholinergic drug, is preferably not administered, and for example, a drug for dementia or mild cognitive impairment (a drug for delaying the progression of dementia or mild cognitive impairment or a therapeutic drug) such as a cholinesterase inhibitor or surgery is preferably administered.

[0269] When the subject is determined to have Parkinson's disease without symptoms of dementia, for example, a drug that can adversely affect the symptoms of dementia, such as an anticholinergic drug, can be administered.

[0270] Therefore, determining (distinguishing) whether the subject has Parkinson's disease with symptoms of dementia or Parkinson's disease without symptoms of dementia is effective for the determination of the treatment policy or diagnosis by the physician.

[0271] Industrial applicability

[0272] The method for assisting in the diagnosis of Parkinson's disease, the biomarker, the kit, and the device of the present application can assist in the diagnosis of Parkinson's disease, and are therefore effective in the field of clinical examinations. According to the present application, the diagnosis of Parkinson's disease can be assisted with high accuracy.

[0273] Examples

[0274] Hereinafter, the present application will be specifically described according to examples and comparative examples, but the present application is not limited by any of these examples. In the examples and comparative examples, "Parkinson's disease patients (MMSE greater than 27)" are subjects to be examined who are Parkinson's disease patients without symptoms of dementia, and "Parkinson's disease patients (MMSE of 27 or less)" are subjects to be examined who are Parkinson's disease patients with symptoms of dementia.

[0275] Example 1. Evaluation of Parkinson's disease subjects (MMSE greater than 27) with the amount of exosomes having PS and CD9 as an index

[0276] The AUC and p-value were calculated by sandwich ELISA of Tim protein-anti-CD9 antibody for the exosome measurement.

[0277] (1) Preparation of calibrators

[0278] Using MagCapture (registered trademark) Exosome Isolation Kit PS (manufactured by FUJIFILM Wako Pure Chemical Corporation, hereinafter referred to as "A kit"), exosomes were isolated from COLO201 cell culture supernatant according to the attached instruction manual of the kit, and the exosomes were dissolved using the dissolution solution attached to the kit. The protein concentration in the obtained exosome solution was measured by BCA method (bicinchoninic acid method) using a protein content measurement BCA kit (manufactured by FUJIFILM Wako Pure Chemical Corporation).

[0279] Next, based on the protein concentration measured by BCA method, the obtained exosome solution was diluted so as to become 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0, 20.0 ng / mL using Reaction Buffer attached to PS Capture Exosome ELISA Kit, Streptavidin HRP (PS Capture Exosome ELISA Kit, Streptavidin HRP) (manufactured by FUJIFILM Wako Pure Chemical Corporation, hereinafter referred to as "B kit"), and a dilution series of exosomes derived from COLO201 cell culture supernatant consisting of 8 concentrations (hereinafter, referred to as "calibrators") was obtained.

[0280] (2) Pretreatment of measurement subjects

[0281] After centrifuging 8 Parkinson's disease patient (MMSE greater than 27) subjects and 37 healthy person subjects purchased from PrecisionMed, Inc. at 10,000g for 20 minutes, the supernatant was recovered. The obtained supernatant was diluted 100 times using the Reaction Buffer attached to the A kit, and was set as "measurement subject diluent".

[0282] (3) Measurement based on ELISA method

[0283] A sandwich ELISA method of Tim4 protein-anti-CD9 antibody was performed by immobilizing the Tim4 protein to a solid phase and setting the anti-CD9 antibody as a detection antibody. Specifically, after reducing the anti-CD9 mouse monoclonal antibody (1K, manufactured by FUJIFILM Wako Pure Chemical Corporation) with 1.6 mM DTT, the biotin-labeled anti-CD9 mouse monoclonal antibody was prepared by reacting it with Biotin-PEAC5-maleimide (manufactured by DOJINDO LABORATORIES) at 37°C for 1.5 hours. In addition to the detection antibody, the reagents attached to the B kit were used.

[0284] First, the Washing Buffer (10x) attached to the B kit was diluted 10 times with purified water (distilled water), and 1 / 100 of the Exosome Binding Enhancer (100x) attached to the B kit was added to the obtained diluent. The obtained solution was set as "washing solution (1x)". Next, each well of the "Tim4 protein immobilized 96-well plate" attached to the B kit was washed 3 times with 300-350 μL of the washing solution (1x) each.

[0285] Next, the diluent of the Parkinson's disease patient (MMSE greater than 27) prepared in (2) (8 subjects n = 2, 16 wells), the diluent of the healthy subject prepared in (2) (37 subjects n = 2, 74 wells), the calibrators prepared in (2) (8 amounts n = 2, 16 wells), and the Reaction Buffer attached to the B kit as a blank medium (n = 2, 2 wells) were each dispensed in 100 μL into each well of the plate. Next, the plate was pasted with a sealing strip, and while stirring at about 500 rpm using a microplate shaker, it was reacted at room temperature for 2 hours. After the reaction, the reaction solution was discarded, and each well was washed 3 times with 300-350 μL of the washing solution (1x). Next, the biotin-labeled anti-CD9 mouse monoclonal antibody was diluted to a final concentration of 250 ng / mL using the Reaction Buffer attached to the B kit, and a biotin-labeled antibody reaction solution was obtained. The obtained biotin-labeled antibody reaction solution was each dispensed in 100 μL into each well, pasted with a sealing strip, and while stirring at about 500 rpm using a microplate shaker, it was reacted at room temperature for 1 hour. After the reaction, the reaction solution was discarded, and each well was washed 3 times with 300-350 μL of the washing solution (1x).

[0286] HRP-conjugated Streptavidin (100x) was added to the Reaction Buffer attached to the B kit in an amount of 1 / 100, and mixed well to prepare HRP-labeled streptavidin reaction solution (1x). The obtained HRP-labeled streptavidin reaction solution (1x) was each dispensed in 100 μL to each well, the sealing bar was attached, and the reaction was performed for 2 hours at room temperature while stirring at about 500 rpm using a microplate shaker. After the reaction was completed, the reaction solution was discarded, and each well was washed 5 times with washing solution (1x) of 300 to 350 μL.

[0287] Next, TMB (3,3',5,5'-tetramethylbenzidine) Solution attached to the B kit, which was returned to room temperature, was each dispensed in 100 μL to each well, and after stirring for about 1 minute using a microplate shaker, the sealing bar was attached, and a standing reaction was performed for 30 minutes at room temperature (20 to 25°C). Then, Stop Solution attached to the B kit, which was returned to room temperature, was each added in 100 μL to each well, and after stirring for about 5 seconds using a microplate shaker, the absorbance at 450 nm and the absorbance at a secondary wavelength of 620 nm were immediately measured using a 96-well plate reader (Tecan, Safire2), respectively. The value obtained by subtracting the absorbance at the secondary wavelength of 620 nm from the absorbance at 450 nm was set as the "absorbance value", and the value obtained by subtracting the absorbance of the blank medium from the absorbance of the sample to be measured was set as the "corrected sample absorbance value", and each was calculated. Then, a standard curve was prepared from the value obtained by subtracting the absorbance of the blank medium from the absorbance value of the dilution series (calibrator) of the exosome derived from the culture supernatant of COLO201 cells and the protein concentration of the calibrator. Using the standard curve, the corrected sample absorbance value was converted to the protein concentration, and the value obtained by multiplying the conversion value by the dilution rate of the sample to be measured was set as the "sample measurement value" [ng / mL].

[0288] (4) Calculation of AUC

[0289] Based on the sample measurement value obtained in (3), a significant difference test between the Parkinson's disease patients and the healthy persons was performed using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA) according to the Wilcoxon / Kruskal-Wallis test (rank sum), and the p value was calculated. Also, based on the sample measurement value obtained in (3), a logistic regression analysis was performed using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA), and based on the obtained receiver operating characteristic curve (ROC curve), the area under the curve value (AUC) was calculated.

[0290] The obtained results are shown in Table 1 below. Also, a box plot made on the basis of the subject measurement values is shown in Figure 1 The vertical axis in the figure indicates the subject measurement values, and the horizontal axis Control indicates the results of healthy persons, and PD indicates the results of Parkinson's disease patients (MMSE greater than 27).

[0291] Example 1. Evaluation of Parkinson's disease subjects (MMSE greater than 27) using the amount of exosomes having CD9 as an index

[0292] Instead of the Tim4 protein, an anti-CD9 antibody was immobilized to the solid phase, and otherwise, by the same method as in Example 1, and by an anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method, exosomes were measured, a significant difference test between Parkinson's disease patients (MMSE greater than 27) and healthy persons was performed, and AUC and p value were calculated.

[0293] A plate in which an anti-CD9 antibody was immobilized to the solid phase was prepared by the following method. Anti-CD9 mouse monoclonal antibody (1K) (manufactured by FUJIFILM Wako Pure Chemical Corporation) was diluted with 50 mM MOPS (pH 7.5) to a concentration of 10 μg / mL, 100 μL was added to the wells of a 96-well microplate (Nunc Corporation), and incubated overnight under refrigeration. After washing the wells 3 times with TBST (Tris Buffered Saline, pH 7.4), 300 μL of TBS (Tris Buffered Saline, pH 7.4) containing 10 mg / mL Block Ace was added, and the material incubated overnight under refrigeration was used as an antibody-immobilized plate.

[0294] The anti-CD9 antibody used as a detection antibody was an antibody biotin-labeled by the same method as in Example 1.

[0295] The obtained results are shown in Table 1 below. Also, a box plot made on the basis of the subject measurement values is shown in Figure 1 The vertical axis in the figure indicates the subject measurement values, and the horizontal axis Control indicates the results of healthy persons, and PD indicates the results of Parkinson's disease patients (MMSE greater than 27).

[0296] Example 2. Evaluation of Parkinson's disease subjects (MMSE greater than 27) using the amount of exosomes having PS and CD63 as an index

[0297] As the detection antibody, an anti-CD63 antibody was used instead of an anti-CD9 antibody, and otherwise, exosomes were measured by the same method as in Example 1 and by a sandwich ELISA method of Tim protein-anti-CD63 antibody, and a significant difference test between Parkinson's disease patients (MMSE greater than 27) and healthy persons was performed, and AUC and p value were calculated.

[0298] As the detection antibody, an anti-CD63 antibody was used instead of an anti-CD9 antibody, and otherwise, exosomes were measured by the same method as in Example 1 and by a sandwich ELISA method of Tim protein-anti-CD63 antibody, and a significant difference test between Parkinson's disease patients (MMSE greater than 27) and healthy persons was performed, and AUC and p value were calculated.

[0299] The results obtained are shown in Table 1 below.

[0300] Comparative Example 2. Evaluation of Parkinson's disease test subjects using CD63 as an index

[0301] As the detection antibody, an anti-CD63 antibody was used instead of an anti-CD9 antibody, and otherwise, exosomes were measured by the same method as in Example 1 and by a sandwich ELISA method of Tim protein-anti-CD63 antibody, and a significant difference test between Parkinson's disease patients (MMSE greater than 27) and healthy persons was performed, and AUC and p value were calculated.

[0302] As the detection antibody, an anti-CD63 antibody was used instead of an anti-CD9 antibody, and otherwise, exosomes were measured by the same method as in Example 1 and by a sandwich ELISA method of Tim protein-anti-CD63 antibody, and a significant difference test between Parkinson's disease patients (MMSE greater than 27) and healthy persons was performed, and AUC and p value were calculated.

[0303] The results obtained are shown in Table 1 below.

[0304] Example 3. Evaluation of Parkinson's disease dementia test subjects using the amount of exosomes having PS and CD81 as an index

[0305] As the detection antibody, an anti-CD81 mouse monoclonal antibody (17B1) (manufactured by FUJIFILM Wako Pure Chemical Corporation) was used instead of the anti-CD9 antibody, and other than this, exosomes were measured by the same method as in Example 1 and by a sandwich ELISA method of Tim protein-anti-CD81 antibody, and a significant difference test between Parkinson's disease patients (MMSE greater than 27) and healthy persons was performed, and AUC and p value were calculated.

[0306] The anti-CD81 antibody as the detection antibody was an antibody biotin-labeled by the same method as in Example 1.

[0307] The obtained results are shown in Table 1 below.

[0308] Comparative Example 3. Evaluation of Parkinson's disease subjects (MMSE greater than 27) using the amount of exosomes having CD81 as an index

[0309] Instead of the anti-CD9 antibody, an anti-CD81 mouse monoclonal antibody (17B1) (manufactured by FUJIFILM Wako Pure Chemical Corporation) was immobilized to a solid phase, and used as the detection antibody, and other than this, exosomes were measured by the same method as in Example 1 and by a sandwich ELISA method of anti-CD81 antibody-anti-CD81 antibody, and a significant difference test between Parkinson's disease patients (MMSE greater than 27) and healthy persons was performed, and AUC and p value were calculated.

[0310] A plate in which an anti-CD81 antibody was immobilized to a solid phase was prepared by the same method as in Comparative Example 1.

[0311] The anti-CD81 antibody as the detection antibody was an antibody biotin-labeled by the same method as in Example 1.

[0312] The obtained results are shown in Table 1 below.

[0313] Example 4. Evaluation of Parkinson's disease subjects (MMSE greater than 27) using the ratio of the amount of exosomes having PS and CD9 to the amount of exosomes having CD9 as an index

[0314] (A) / (B) (hereinafter, referred to as "corrected subject measurement value") was obtained by dividing the "subject measurement value" (value (A)) obtained in Example 1 (sandwich ELISA method of Tim protein-anti-CD9 antibody) by the "subject measurement value" (value (B)) obtained in Comparative Example 1 (sandwich ELISA method of anti-CD9 antibody-anti-CD9 antibody).

[0315] Based on the obtained corrected subject measurement values, a test for significant difference between the Parkinson's disease patients (MMSE greater than 27) and the healthy persons was performed by the same method as in Example 1(4), and AUC and p value were calculated.

[0316] The obtained results are shown in Table 1 below. And, a box plot made based on the corrected subject measurement values is shown in Figure 2 Fig. 1. In the figure, the vertical axis represents the corrected subject measurement values ((A) / (B)), and the horizontal axis Control represents the results of the healthy persons, and PD represents the results of the Parkinson's disease patients (MMSE greater than 27).

[0317] Example 5. Evaluation of Parkinson's disease subjects (MMSE greater than 27) with the ratio of the amount of exosomes having PS and CD63 to the amount of exosomes having CD63 as an index

[0318] (A) / (B) (corrected subject measurement value) was obtained by dividing the "subject measurement value" (value (A)) obtained in Example 2 (Tim protein-anti-CD63 antibody sandwich ELISA method) by the "subject measurement value" (value (B)) obtained in Comparative Example 2 (anti-CD63 antibody-anti-CD63 antibody sandwich ELISA method).

[0319] Based on the obtained corrected subject measurement values, a test for significant difference between the Parkinson's disease patients (MMSE greater than 27) and the healthy persons was performed by the same method as in Example 1(4), and AUC and p value were calculated.

[0320] The obtained results are shown in Table 1 below.

[0321] [Table 1]

[0322]

[0323] According to Table 1, when the plasma derived from the Parkinson's disease patients (MMSE greater than 27) having no symptoms of dementia and the healthy persons were respectively set as samples, and the Parkinson's disease was evaluated with the amount of exosomes having four times of transmembrane proteins of CD9, CD63, or CD81 as an index, AUC was 0.855, 0.848, 0.642, respectively.

[0324] On the other hand, when plasma derived from Parkinson's disease patients having no symptoms of dementia (MMSE is more than 27) and healthy persons were set as samples, and the amount of exosomes having phosphatidylserine to which tetraspanins of CD9, CD63 or CD81 and Tim protein bind was evaluated as an index for Parkinson's disease, AUC was 0.929, 0.899, 0.916, respectively, and it was found that Parkinson's disease (particularly, Parkinson's disease having no symptoms of dementia) can be detected with high accuracy.

[0325] Further, when the corrected subject measurement value ((A) / (B)) obtained by dividing the subject measurement value (value (A)) of exosomes having phosphatidylserine and CD63 by the subject measurement value (value (B)) of exosomes having CD63 was used as an index for Parkinson's disease, AUC was 0.892, and it was found that Parkinson's disease (particularly, Parkinson's disease having no symptoms of dementia) can be detected with high accuracy. Further, when the corrected subject measurement value ((A) / (B)) obtained by dividing the subject measurement value (value (A)) of exosomes having phosphatidylserine and CD9 by the subject measurement value (value (B)) of exosomes having CD9 was used as an index for Parkinson's disease, AUC was 0.973, and it was found that Parkinson's disease (particularly, Parkinson's disease having no symptoms of dementia) can be detected with extremely high accuracy.

[0326] Example 6. Evaluation of Parkinson's disease subjects (MMSE is 27 or less) using the ratio of the amount of exosomes having PS and CD9 to the amount of exosomes having CD9 as an index

[0327] "Subject measurement value" (value (A)) was obtained by using "EDTA plasma of 8 Parkinson's disease subjects (MMSE is 27 or less) and 30 healthy subjects purchased from Precision Med Co., Ltd.", and otherwise, by the same method as in Example 1, and by sandwich ELISA method of Tim protein-anti-CD9 antibody for exosome measurement.

[0328] Further, "subject measurement value" (value (B)) was obtained by using "EDTA plasma of 8 Parkinson's disease subjects (MMSE is 27 or less) and 30 healthy subjects purchased from Precision Med Co., Ltd.", and otherwise, by the same method as in Comparative Example 1, and by sandwich ELISA method of anti-CD9 antibody-anti-CD9 antibody for exosome measurement.

[0329] The (A) / (B) (hereinafter, referred to as "corrected subject measurement value") was obtained by dividing the "subject measurement value" (value (A)) by the "subject measurement value" (value (B)). Based on the obtained corrected subject measurement value, the difference significance test between the Parkinson's disease patients (MMSE of 27 or less) and the healthy persons was performed by the same method as that of Example 1 (4), and the AUC and the p value were calculated.

[0330] The obtained results are shown in Table 2 below. Also, the box plot made based on the corrected subject measurement value is shown in Figure 3 In the figure, the vertical axis indicates the subject measurement value, and the horizontal axis indicates the results of the healthy persons (Control), the results of the Parkinson's disease patients (MMSE of 27 or less) (PD (MMSE of 27 or less)), and the results of the Parkinson's disease patients (MMSE of more than 27) (PD (MMSE of more than 27)).

[0331] [Table 2]

[0332]

[0333] Example 7. Evaluation of Parkinson's disease subjects (MMSE of more than 27) using the amount of exosomes having PS and CD9 as an index

[0334] Using "EDTA blood plasma of 8 Parkinson's disease subjects (MMSE of more than 27) and 30 healthy subjects purchased from PrecisionMed Co.," other than this, the exosome measurement was performed by the same method as that of Example 1 and by the sandwich ELISA method of Tim protein-anti-CD9 antibody, the difference significance test between the Parkinson's disease subjects (MMSE of more than 27) and the healthy persons was performed, and the AUC and the p value were calculated.

[0335] The obtained results are shown in Table 3 below.

[0336] Comparative Example 4. Evaluation of Parkinson's disease subjects (MMSE of more than 27) using the amount of exosomes having CD9 as an index

[0337] Using "EDTA blood plasma of 8 Parkinson's disease subjects (MMSE of more than 27) and 30 healthy subjects purchased from PrecisionMed Co.," other than this, the exosome measurement was performed by the same method as that of Comparative Example 1 and by the sandwich ELISA method of anti-CD9 antibody-anti-CD9 antibody, the difference significance test between the Parkinson's disease subjects (MMSE of more than 27) and the healthy persons was performed, and the AUC and the p value were calculated.

[0338] The obtained results are shown in Table 3 below.

[0339] Example 8. Evaluation of Parkinson's disease subjects (MMSE of 27 or greater) using the ratio of the amount of exosomes having PS and CD9 to the amount of exosomes having CD9 as an index

[0340] By dividing the "subject measurement value" (value (A)) obtained in Example 7 (Tim protein-anti-CD9 antibody sandwich ELISA method) by the "subject measurement value" (value (B)) obtained in Comparative Example 4 (anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method), (A) / (B) (hereinafter, referred to as "corrected subject measurement value") was obtained.

[0341] According to the obtained corrected subject measurement value, by the same method as in Example 1 (4), a significant difference test between Parkinson's disease patients (MMSE of 27 or greater) and healthy persons was performed, and AUC and p value were calculated.

[0342] The obtained results are shown in Table 3 below. Also, a box plot made based on the corrected subject measurement value is shown in Figure 3 .

[0343] [Table 3]

[0344]

[0345] Example 9. Evaluation of Parkinson's disease subjects (MMSE of 27 or less) and Parkinson's disease subjects (MMSE of 27 or greater) using the ratio of the amount of exosomes having PS and CD9 to the amount of exosomes having CD9 as an index

[0346] Using "8 Parkinson's disease patient (MMSE of 27 or less) subjects purchased from PrecisionMed, Inc., and 8 Parkinson's disease patient (MMSE of 27 or greater) subjects purchased from PrecisionMed, Inc.", in addition to the same method as in Example 1, and by the Tim protein-anti-CD9 antibody sandwich ELISA method, the "subject measurement value" (value (A)) was obtained.

[0347] Also, using "8 Parkinson's disease patient (MMSE of 27 or less) subjects purchased from PrecisionMed, Inc., and 8 Parkinson's disease patient (MMSE of 27 or greater) subjects purchased from PrecisionMed, Inc.", in addition to the same method as in Comparative Example 1, and by the anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method, the "subject measurement value" (value (B)) was obtained.

[0348] The (A) / (B) (hereinafter, referred to as "corrected subject measurement value") was obtained by dividing the "subject measurement value" (value (A)) by the "subject measurement value" (value (B)). Based on the obtained corrected subject measurement value, the significant difference test between the Parkinson's disease patient (MMSE of 27 or less) and the Parkinson's disease subject (MMSE of more than 27) was performed by the same method as in Example 1(4), and the AUC and p value were calculated.

[0349] The obtained results are shown in Table 4 below. And, the box plot made based on the subject measurement value is shown in Figure 3 .

[0350] [Table 4]

[0351]

[0352] Example 10. Evaluation of Parkinson's disease subjects using the amount of exosome having PS and CD9 as an index

[0353] "EDTA blood plasma of 16 Parkinson's disease patients (8 subjects of MMSE of 27 or less and 8 subjects of MMSE of more than 27) and 30 healthy subjects purchased from Precision Med Co., Ltd." was used, and, other than that, the exosome measurement was performed by the same method as in Example 1 and by the sandwich ELISA method of Tim protein-anti-CD9 antibody, the significant difference test between the Parkinson's disease patient and the healthy person was performed, and the AUC and p value were calculated.

[0354] The obtained results are shown in Table 5 below. And, the box plot made based on the subject measurement value is shown in Figure 4 . In the figure, the vertical axis represents the subject measurement value, and the horizontal axis Control represents the results of the healthy person, and PD represents the results of the Parkinson's disease patient.

[0355] Comparative Example 7. Evaluation of Parkinson's disease subjects using the amount of exosome having CD9 as an index

[0356] "EDTA blood plasma of 16 Parkinson's disease patients (8 subjects of MMSE of 27 or less and 8 subjects of MMSE of more than 27) and 30 healthy subjects purchased from Precision Med Co., Ltd." was used, and, other than that, the exosome measurement was performed by the same method as in Comparative Example 1 and by the sandwich ELISA method of anti-CD9 antibody-anti-CD9 antibody, the significant difference test between the Parkinson's disease patient and the healthy person was performed, and the AUC and p value were calculated.

[0357] The obtained results are shown in Table 5 below.

[0358] Example 11. Evaluation of Parkinson's disease samples using the ratio of the amount of exosomes having PS and CD9 to the amount of exosomes having CD9 as an index

[0359] By dividing the "sample measurement value" (value (A)) obtained in Example 10 (sandwich ELISA method of Tim protein-anti-CD9 antibody) by the "sample measurement value" (value (B)) obtained in Comparative Example 7 (sandwich ELISA method of anti-CD9 antibody-anti-CD9 antibody), (A) / (B) (hereinafter, referred to as "corrected sample measurement value") was obtained.

[0360] Based on the obtained corrected sample measurement value, the difference significance test between Parkinson's disease patients and healthy persons was performed by the same method as in Example 1 (4), and AUC and p value were calculated.

[0361] The obtained results are shown in Table 5 below. Also, a box plot made based on the sample measurement value is shown in Figure 5 In the figure, the vertical axis indicates the corrected sample measurement value, and the horizontal axis indicates the results of healthy persons as Control and the results of Parkinson's disease patients as PD.

[0362] [Table 5]

[0363]

[0364] According to Table 2, when the plasma derived from Parkinson's disease patients having dementia symptoms (MMSE is 27 or less) and healthy persons were respectively set as samples and the corrected sample measurement value ((A) / (B)) obtained by dividing the sample measurement value of exosomes having phosphatidylserine and CD9 (value (A)) by the sample measurement value of exosomes having CD9 (value (B)) was evaluated, AUC was 0.804, and it was found that Parkinson's disease (in particular, Parkinson's disease having dementia symptoms) could be detected with high accuracy.

[0365] According to Table 3, when the plasma derived from Parkinson's disease patients having no dementia symptoms (MMSE is more than 27) and healthy persons were respectively set as samples and the corrected sample measurement value ((A) / (B)) obtained by dividing the sample measurement value of exosomes having phosphatidylserine and CD9 (value (A)) by the sample measurement value of exosomes having CD9 (value (B)) was evaluated, AUC was 0.950, and it was found that Parkinson's disease (in particular, Parkinson's disease having no dementia symptoms) could be detected with extremely high accuracy.

[0366] According to Table 4, when the plasma of the Parkinson's disease patients with dementia symptoms (MMSE of 27 or less) and the Parkinson's disease patients without dementia symptoms (MMSE of more than 27) were respectively set as samples and the corrected subject measurement value ((A) / (B)) obtained by dividing the subject measurement value of the exosome having phosphatidylserine and CD9 (value (A)) by the subject measurement value of the exosome having CD9 (value (B)) was evaluated, the AUC was 0.844, and it was found that the Parkinson's disease patients with dementia symptoms (MMSE of 27 or less) and the Parkinson's disease patients without dementia symptoms (MMSE of more than 27) could be distinguished with high accuracy. Furthermore, the correlation coefficient of the corrected subject measurement value ((A) / (B)) and MMSE was 0.7833, and a high correlation was observed.

[0367] According to Table 5, when the plasma derived from the Parkinson's disease patients (including the Parkinson's disease patients with dementia symptoms and the Parkinson's disease patients without dementia symptoms) and the healthy persons were respectively set as samples, and the amount of the exosome having CD9 was used as an index to evaluate the Parkinson's disease, the AUC was 0.764, and when the corrected subject measurement value ((A) / (B)) obtained by dividing the subject measurement value of the exosome having phosphatidylserine and CD9 (value (A)) by the subject measurement value of the exosome having CD9 (value (B)) was evaluated, the AUC was 0.877, and it was found that the Parkinson's disease could be detected with high accuracy.

Claims

1. Use of a combination of a substance having affinity for tetraspanins and a substance having affinity for phosphatidylserine in the manufacture of a diagnostic aid kit for Parkinson's disease, the kit being used to determine the amount of extracellular vesicles having tetraspanins and phosphatidylserine, wherein, The tetraspanin is selected from the group consisting of CD9, CD63, and CD81.

2. Use according to claim 1, wherein, The substance having affinity for phosphatidylserine is a T cell immunoglobulin • mucin-containing protein.

3. Use according to claim 1, wherein, The biological sample that is the measurement target of the kit is a blood sample or cerebrospinal fluid.

4. The use according to claim 1, wherein, The tetraspanin is CD9 or CD63.

5. The use according to any one of claims 1 to 4, wherein, The kit further comprises an extracellular vesicle having phosphatidylserine and a tetraspanin, and / or an extracellular vesicle having a tetraspanin as a standard.

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