WO2022250009A1

WO2022250009A1 - Method for assisting differentiation of lewy body disease and alzheimer's disease, biomarker, and reagent kit

Info

Publication number: WO2022250009A1
Application number: PCT/JP2022/021093
Authority: WO
Inventors: 隆宏西部; 直子今若
Original assignee: 富士フイルム株式会社
Priority date: 2021-05-26
Filing date: 2022-05-23
Publication date: 2022-12-01
Also published as: JPWO2022250009A1

Abstract

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

Description

Methods, Biomarkers, and Reagent Kits to Help Differentiate Lewy Body Disease from Alzheimer's Disease

The present invention relates to methods, biomarkers, and reagent kits to help distinguish between Lewy body disease and Alzheimer's disease.

Alzheimer's disease (AD) is one of the causative diseases of dementia with memory impairment and one or more cognitive dysfunctions (aphasia, lapse, agnosia, executive dysfunction). The brains of Alzheimer's disease patients are characterized by numerous senile plaques formed by accumulation of amyloid β (Aβ) protein and neurofibrillary tangles formed by accumulation of phosphorylated tau protein, mainly in the cerebral cortex and hippocampus. It is thought to occur due to neuronal cell death, synapse loss, and acetylcholine decrease.

Parkinson's disease (PD) is a progressive degenerative disorder of the extrapyramidal system that presents with four symptoms: resting tremor, muscle rigidity, akinesia, and postural reflex dysfunction. It is believed that nerve cells in the substantia nigra in the brain degenerate and the body becomes unable to move smoothly due to lack of dopamine in the striatum. Patients with Parkinson's disease are complicated with cognitive impairment at a high rate, and when cognitive impairment becomes severe and interferes with daily life, it is called Parkinson's disease dementia (PDD).

Lewy body disease (LBD) is a disease concept encompassing all pathologies characterized by the presence of Lewy bodies. Known Lewy body diseases include dementia with Lewy bodies (DLB), Parkinson's disease (PD), Parkinson's disease-type dementia (Parkinson's disease with dementia, PDD), and the like. Dementia with Lewy bodies (DLB) is characterized by progressive cognitive decline that interferes with daily activities, plus one or more symptoms of parkinsonism (resting tremor, muscle rigidity, immobility, impaired postural reflexes). ), cognitive fluctuations with marked changes in attention or clarity, recurrent and structured visual hallucinations, and the presence of two or more core features of REM sleep behavior disorder. there is In Parkinson's disease, Lewy bodies are found in neurons in the substantia nigra and locus coeruleus of the midbrain, which belongs to the brainstem. Lewy bodies tend to be seen in Lewy body dementia (LDB) and Parkinson's disease dementia (PDD) have similar symptoms. , Lewy body dementia is diagnosed if cognitive symptoms are present before the onset of parkinsonism or within 1 year after the onset of parkinsonism.

A test method for dementia includes the Mini-Mental State Examination (MMSE), and the subject is suspected to have dementia when the MMSE score is 23 or less out of 30 points. As a method for diagnosing Alzheimer's disease, anti-NCAM antibody and anti-L1CAM antibody are used to concentrate nerve-derived extracellular vesicles in the blood, and Aβ42, Tau, phosphorylated Tau, etc. contained in the above nerve-derived extracellular vesicles are detected. Methods for determining Alzheimer's disease have been reported as indicators (Patent Document 1, Patent Document 2, Non-Patent Document 1, Non-Patent Document 2). In addition, as a method for diagnosing Parkinson's disease, anti-NCAM antibody or anti-L1CAM antibody is used to concentrate neuron-derived extracellular vesicles in the blood, and α-synuclein, etc. contained in the extracellular vesicles is used as an index to detect Parkinson's disease. There have been reports of methods for determining the (Patent Document 1, Patent Document 2, Non-Patent Document 1, Non-Patent Document 2).

Japanese Patent Publication No. 2016-535283 Japanese Patent Publication No. 2017-525976

However, no biomarkers are known that can distinguish between Lewy body disease and Alzheimer's disease. In view of the above situation, an object of the present invention is to provide a method, a biomarker, and a reagent kit that assist in distinguishing between Lewy body disease and Alzheimer's disease.

The present inventors examined whether specific extracellular vesicles can serve as biomarkers for assisting in the differentiation between dementia with Lewy bodies and Alzheimer's disease. As a result, the present inventors found that, among extracellular vesicles, extracellular vesicles with CD9 or extracellular vesicles with phosphatidylserine and CD9 help distinguish between dementia with Lewy bodies and Alzheimer's disease. The inventors have newly found that it can be a biomarker for the purpose, and have completed the invention.

That is, the inventors have found that the above problems can be achieved by the following configuration.
[1] measuring the amount of extracellular vesicles with CD9 or the amount of extracellular vesicles with phosphatidylserine and CD9 in a biological sample derived from a subject suspected of having Lewy body disease or Alzheimer's disease; Lewy body disease and Lewy body disease, including determining Lewy body disease and Alzheimer's disease in a subject using the amount of extracellular vesicles having CD9 or the amount of extracellular vesicles having phosphatidylserine and CD9 as an index A method to aid in the differentiation of Alzheimer's disease.
[2] The method for assisting the differentiation between Lewy body disease and Alzheimer's disease according to [1], wherein the Lewy body disease is Parkinson's disease dementia, Lewy body dementia, or/and Parkinson's disease.
[3] The determination is to determine Parkinson's disease dementia or Lewy body dementia when the amount of extracellular vesicles is greater than the first reference value, or / and the extracellular small The method for assisting discrimination between Lewy body disease and Alzheimer's disease according to [2], wherein Parkinson's disease is determined when the amount of cysts is equal to or less than a second reference value.
[4] The Lewy body disease is Parkinson's disease dementia or Lewy body dementia, and the amount of extracellular vesicles is measured by measuring the amount of extracellular vesicles having CD9. Lewy body disease and Alzheimer's disease according to any one of claims [1] to [3], wherein the determination is performed using the amount of extracellular vesicles having CD9 as an index. A method to aid in the differentiation of diseases.
[5] The Lewy body disease is Parkinson's disease dementia or Lewy body dementia, and the measurement of the amount of extracellular vesicles measures the amount of extracellular vesicles having the phosphatidylserine and CD9. Lewy small according to any one selected from [1] to [3], wherein the determination is to determine the amount of extracellular vesicles having the phosphatidylserine and CD9 as an indicator A method to assist in distinguishing between physical illness and Alzheimer's disease.
[6] the Lewy body disease is Parkinson's disease, the measurement of the amount of the extracellular vesicles is to measure the amount of the extracellular vesicles having the CD9, and the determination is the presence of the CD9 The method for assisting in distinguishing between Lewy body disease and Alzheimer's disease according to any one of [1] to [3], wherein the amount of extracellular vesicles present is determined as an index.
[7] Levy according to [4], wherein the determination is to determine Parkinson's disease dementia or Lewy body dementia when the amount of extracellular vesicles is greater than the first reference value A method to assist in distinguishing body disease from Alzheimer's disease.
[8] The determination is to determine that the patient has Alzheimer's disease when the amount of extracellular vesicles is equal to or less than a first reference value and greater than a second reference value, [4] or A method for assisting in distinguishing between Lewy body disease and Alzheimer's disease according to [7].
[9] Levy according to [5], wherein the determination is to determine Parkinson's disease dementia or Lewy body dementia when the amount of extracellular vesicles is greater than the first reference value A method to assist in distinguishing body disease from Alzheimer's disease.
[10] The determination is to determine that the patient has Alzheimer's disease when the amount of extracellular vesicles is equal to or less than a first reference value and greater than a second reference value, [5] or A method for assisting in distinguishing between Lewy body disease and Alzheimer's disease according to [9].
[11] The discrimination between Lewy body disease and Alzheimer's disease according to [6], wherein the determination is to determine Parkinson's disease when the amount of extracellular vesicles is equal to or less than the second reference value. how to assist.
[12] The determination is to determine that the patient has Alzheimer's disease when the amount of extracellular vesicles is equal to or less than a first reference value and greater than a second reference value, [6] or A method for assisting in distinguishing between Lewy body disease and Alzheimer's disease according to [11].
[13] A reagent kit for assisting differentiation between Lewy body disease and Alzheimer's disease, containing a substance having affinity for CD9.
[14] The reagent kit for assisting differentiation between Lewy body disease and Alzheimer's disease according to [13], further comprising a substance having affinity for phosphatidylserine.
[15] The Lewy body disease is Parkinson's disease dementia, Lewy body dementia, or / and Parkinson's disease, [13] or [14] to help differentiate between Lewy body disease and Alzheimer's disease reagent kit for.
[16] A biomarker to help differentiate between Lewy body disease and Alzheimer's disease, comprising extracellular vesicles with CD9 or extracellular vesicles with phosphatidylserine and CD9.
[17] The biomarker for assisting the differentiation between Lewy body disease and Alzheimer's disease according to [16], wherein the Lewy body disease is Parkinson's disease dementia, Lewy body dementia, or/and Parkinson's disease.

Furthermore, the present inventors have found that extracellular vesicles with CD9, or extracellular vesicles with phosphatidylserine and CD9, to help distinguish Parkinson's disease dementia or Lewy body dementia from Parkinson's disease. We also found that it can be used as a biomarker, and completed the following invention.
[1A] The amount of extracellular vesicles with CD9 or extracellular vesicles with phosphatidylserine and CD9 in a biological sample from a subject with Parkinson's disease dementia or Lewy body dementia or suspected Parkinson's disease Dementia with Parkinson's disease or Lewy body dementia in a subject by measuring the amount of vesicles, the amount of extracellular vesicles having the CD9, or the amount of extracellular vesicles having the phosphatidylserine and CD9 as an index A method to help differentiate between Parkinson's disease dementia or Lewy body dementia and Parkinson's disease, comprising determining Parkinson's disease with Parkinson's disease.
[2A] In the determination, the amount of extracellular vesicles is the third reference value (the amount of the biomarker of the present invention for differentiating Parkinson's disease dementia or Lewy body dementia from Parkinson's disease, For example, the value set to the second reference value or more and the first reference value or less) is to be determined as Parkinson's disease dementia or Lewy body dementia, or / and the extracellular The method for assisting the differentiation between Parkinson's disease dementia or Lewy body dementia and Parkinson's disease according to [1A], wherein Parkinson's disease is determined when the amount of vesicles is equal to or less than a third reference value. .
[3A] A reagent kit for assisting the differentiation between Parkinson's disease dementia or Lewy body dementia and Parkinson's disease, containing a substance having affinity for CD9.
[4A] The reagent kit for assisting the differentiation between Parkinson's disease dementia or Lewy body dementia and Parkinson's disease according to [3A], further comprising a substance having an affinity for phosphatidylserine.
[5A] A biomarker to help differentiate between Parkinson's disease dementia or Lewy body dementia and Parkinson's disease, comprising extracellular vesicles with CD9 or extracellular vesicles with phosphatidylserine and CD9.

INDUSTRIAL APPLICABILITY According to the present invention, discrimination between Lewy body disease and Alzheimer's disease can be assisted. Specifically, it can help differentiate between Parkinson's disease dementia, Lewy body dementia, and/or Parkinson's disease and Alzheimer's disease.

FIG. 1 is a box and whisker graph evaluating plasma specimens of Alzheimer's disease patients and Parkinson's disease dementia patients or Lewy body dementia patients using the amount of exosomes having CD9 as an index. FIG. 2 is a box and whisker graph evaluating plasma samples from patients with Alzheimer's disease and Parkinson's disease or dementia with Lewy bodies using the amount of exosomes having CD9 and phosphatidylserine as indices.

In this specification, when the upper limit and lower limit of a range are indicated, A to B are from A to B, unless otherwise specified. In addition, the term "measurement" as used herein includes quantitative, semi-quantitative, and qualitative.

Extracellular vesicles in the present invention are cell-derived small membrane vesicles composed of a lipid bilayer membrane. In the present invention, the diameter of extracellular vesicles can be measured, for example, by a nanoparticle tracking analysis method (NTA method) using a nanoparticle analysis system (Nanosite). The diameter of extracellular vesicles refers to the number average particle size. Examples of the extracellular vesicles include those having a diameter of 20 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 50 nm to 500 nm, particularly preferably 50 nm to 200 nm. As described in, for example, Nature Reviews Immunology 9, 581-593 (August 2009), "Obesity Research" Vol.13 No.2 2007 Topics Naoto Aoki, etc., the extracellular vesicles have their origin and small membrane size. There are those classified variously according to the size of cells and the like. Specific examples include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, apoptotic bodies, adiposomes, etc. Exosomes and microvesicles are preferred, and exosomes are more preferred.

The exosomes are small membrane vesicles composed of a lipid bilayer membrane derived from cells, for example, those having a diameter of 50 nm to 200 nm, preferably 50 nm to 150 nm, 50 nm to 100 nm is more preferred. Exosomes are thought to be derived from late endosomes.

The above-mentioned microvesicles are small membrane vesicles derived from cells and composed of a lipid bilayer membrane. is more preferred. Microvesicles are believed to originate from cell membranes.

The extracellular vesicles may be contained in a biological sample derived from a subject, or may be isolated from a biological sample derived from a subject, isolated from a biological sample derived from a subject preferably.

Any biological sample derived from the subject may be used as long as it can contain extracellular vesicles. Examples include serum, plasma, whole blood, blood-derived samples such as buffy coat, cerebrospinal fluid, urine, saliva, and semen. , thoracic effusion, cerebrospinal fluid, tear fluid, mucus, lymph, ascites, pleural fluid, amniotic fluid, bladder lavage fluid, bronchoalveolar lavage fluid and the like, preferably blood-derived samples or cerebrospinal fluid. , serum, plasma, or cerebrospinal fluid is more preferred, serum or plasma is more preferred, and plasma is particularly preferred. In addition, blood-derived samples are more useful because the burden of sample collection on subjects is light. The above-mentioned biological samples derived from subjects, for example, even if they are directly collected from subjects, should not be subjected to pretreatment such as recovery, concentration, purification, isolation, dilution with buffers, etc., and filtration sterilization. may These pretreatments may be appropriately carried out according to conventional methods. Hereinafter, the biological sample derived from the subject may be abbreviated as "biological sample".

The method for isolating extracellular vesicles from the biological sample is not particularly limited and may be carried out according to a conventional method. Examples of methods for isolating extracellular vesicles from the biological sample include affinity methods (e.g., PS affinity method), differential centrifugation methods (e.g., pellet down method, sucrose cushion method, density gradient centrifugation method, etc.). ultracentrifugation), immunoprecipitation, chromatography (e.g., ion exchange chromatography, gel permeation chromatography), density gradient method (e.g., sucrose density gradient method), electrophoresis (e.g., organelle electrophoresis) method), magnetic separation method (e.g. magnetic activated cell sorting (MACS) method), ultrafiltration concentration method (e.g. nanomembrane ultrafiltration concentration method), percoll gradient isolation method, method using microfluidic device , PEG precipitation method, etc., and the affinity method because it can obtain extracellular vesicles with a high degree of purification, or the differential centrifugation method because it is possible to recover theoretically without bias, the affinity method or Ultracentrifugation is more preferred, and affinity methods are particularly preferred. Among affinity methods, the PS affinity method, which is affinity purification for phosphatidylserine, is preferred. The affinity method and the differential centrifugation method may be performed, for example, according to the method described in International Publication No. 2016/088689. These isolation methods may be used alone or in combination of two or more. Also, isolation by one isolation method may be repeated two or more times.

A subject in the present invention is a subject suspected of having Alzheimer's disease or Lewy body disease (e.g., Lewy body dementia, Parkinson's disease dementia, Parkinson's disease), for example, Alzheimer's disease or Lewy body disease. Humans who are suspected of being at risk of developing the disease.

<Biomarkers to help differentiate between Lewy body disease and Alzheimer's disease>
The biomarker for assisting the differentiation between Lewy body disease and Alzheimer's disease of the present invention (hereinafter sometimes abbreviated as the biomarker of the present invention) is an extracellular vesicle having CD9, or an extracellular vesicle having phosphatidylserine and CD9 Contains vesicles. Extracellular vesicles in the biomarkers of the present invention are the same as those described above, and preferred ones are also the same.

<Method to help distinguish between Lewy body disease and Alzheimer's disease>
The method of the present invention for assisting in the discrimination between Lewy body disease and Alzheimer's disease (hereinafter sometimes abbreviated as the discrimination assisting method of the present invention) comprises extracellular vesicles containing CD9 in a biological sample derived from a subject. or measuring the amount of extracellular vesicles having phosphatidylserine and CD9 (hereinafter sometimes abbreviated as a measuring step), the amount of extracellular vesicles having CD9, or the phosphatidylserine and CD9 Determining Lewy body disease and Alzheimer's disease in a subject using the amount of extracellular vesicles having

The biological sample, test subject, and extracellular vesicles in the method for assisting identification of the present invention are the same as those described above, and preferred ones are also the same.

The "amount" in the method for assisting identification of the present invention includes mass and concentration. In addition, the above "quantity" includes measured values that have a correlation with mass and concentration (e.g., absorbance, absorbance change amount, transmitted light, transmitted light change amount, fluorescence intensity, fluorescence intensity change amount, luminescence amount, luminescence amount change amount, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change amount, refractive index, refractive index change amount, etc.).

Measurement of the amount of extracellular vesicles having CD9, measurement of the amount of extracellular vesicles having phosphatidylserine and CD9 (measurement of the amount of the biomarker of the present invention) in the identification assistance method of the present invention is generally performed in this field There is no particular limitation as long as the method is performed. Measurement of the amount of the biomarker of the present invention includes, for example, a measurement method utilizing the affinity between a substance having affinity for the biomarker of the present invention and the biomarker of the present invention, a mass spectrometry method, and a method combining these methods. be done. Examples of substances having affinity for the biomarkers of the present invention include substances having affinity for CD9 and phosphatidylserine possessed by the biomarkers of the present invention, substances having affinity for phosphatidylserine, Substances that specifically bind to the biomarkers of the invention are preferred.

The substance having an affinity for CD9 may be a substance that binds to CD9, preferably a substance that specifically binds to CD9. Examples of substances having affinity for CD9 include antibodies that bind to CD9 (hereinafter sometimes abbreviated as anti-CD9 antibodies) and lectins that bind to sugar chains of CD9. Examples thereof include proteins that bind to CD9, nucleic acids (aptamers) that bind to CD9, and the like. Proteins that bind to CD9 are preferred, and anti-CD9 antibodies are more preferred.

The substance having an affinity for phosphatidylserine may be any substance that binds to phosphatidylserine, preferably a substance that specifically binds to phosphatidylserine. Examples of substances having an affinity for phosphatidylserine include proteins that bind to phosphatidylserine and nucleic acids (aptamers) that bind to phosphatidylserine. Proteins that bind to phosphatidylserine include preferable. Examples of proteins that bind to phosphatidylserine include antibodies that bind to phosphatidylserine (hereinafter sometimes abbreviated as anti-PS antibodies) and phosphatidylserine affinity proteins. preferable. Anti-PS antibodies include, for example, anti-phosphatidylserine antibody 1H6 (Merck & Co.). Phosphatidylserine affinity proteins include, for example, Tim1 (T-cell immunoglobulin-mucin domain-containing molecule 1, T-cellimmunoglobulin-mucin-domain1), Tim2 (T-cell immunoglobulin-mucin domain-containing molecule 2, T-cellimmunoglobulin-mucin -domain2), Tim3 (T-cell immunoglobulin-mucin domain-containing molecule 3, T-cellimmunoglobulin-mucin-domain3), Tim4 (T-cell immunoglobulin-mucin domain-containing molecule 4, T-cellimmunoglobulin-mucin-domain4) Protein; AnnexinV; Tim proteins are preferably Tim1 and Tim4, more preferably Tim4. Only one type of substance having an affinity for phosphatidylserine may be used, or two or more types may be used, and it is preferable to use only one type.

The substance having an affinity for CD9 and the substance having an affinity for phosphatidylserine may be commercially available products or those appropriately prepared by conventional methods. Moreover, the anti-CD9 antibody and the anti-PS antibody may be either polyclonal antibodies or monoclonal antibodies, and these may be used alone or in appropriate combination. In addition, the anti-CD9 antibody and the anti-PS antibody are not only immunoglobulin molecules themselves (intactimmunoglobulin), but also fragments thereof that are capable of binding to antigens Fab, F(ab')2, F( Ab') and other fragment antibodies, single chain antibodies (singlechainFv), synthetic antibodies such as diabodies, triabodies, tetrabodies, and the like may also be used. In addition, when these antibodies are prepared, they may be prepared, for example, according to the method described in "Immunoassay" (edited by Biochemical Assay Study Group, Kodansha, 2014).

The substance having affinity for CD9 and the substance having affinity for phosphatidylserine may be labeled with a labeling substance. Examples of the labeling substance include enzymes such as peroxidase, microperoxidase and alkaline phosphatase; radioactive isotopes such as ^99m Tc, ¹³¹ I, ¹²⁵ I, ¹⁴ C, ³ H, ³² P and ³⁵ S; Fluorescent substances such as thiocyanate (FITC), 4-methylumbelliferone, HiLyte, Alexa, CyDye or rhodamine, or derivatives thereof, luminescent substances such as luciferin, luminol, ruthenium complexes, phenol, naphthol, or anthracene, or These derivatives have properties as spin labeling agents represented by substances that absorb in the ultraviolet region and compounds with an oxyl group such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl. , colloidal gold, nanoparticles such as quantum dots, etc., and enzymes and fluorescent substances are preferred. Measurement of these labeling substances may be performed according to a known measurement method according to the labeling substance. In addition, a secondary affinity substance (for example, secondary antibodies) may also be used. The secondary affinity substance is preferably labeled with a labeling substance, more preferably a secondary antibody labeled with a labeling substance. The labeling substances are the same as those described above, and preferred ones are also the same. The method for labeling the primary affinity substance and the secondary affinity substance is not particularly limited. Indirect labeling via the ligand and receptor may also be used. As an indirect labeling method, for example, one of avidins and biotins is bound to a substance having affinity for CD9 or a substance having affinity for phosphatidylserine (primary affinity substance). and a labeling method that utilizes the affinity between avidins and biotins using a labeling substance bound to the other one of avidins and biotins. Biotins include biotin, iminobiotin, desthiobiotin, biocytin, biotin sulfoxide and the like, with biotin being preferred. Avidins include avidin, tamapidine, tamapidine 2, streptavidin and the like, with streptavidin being preferred. A method of binding either a ligand or a receptor (e.g., avidins and biotins) to the substance having affinity for CD9 or the substance having affinity for phosphatidylserine (primary affinity substance), labeling A method for binding the remaining one of the ligand and the receptor (eg, avidins and biotins) to the substance may be carried out according to a conventional method. The method of indirectly labeling the secondary affinity substance is the same as the method of indirectly labeling the primary affinity substance, and specific examples and preferable examples are also the same.

The substance having affinity for CD9 and the substance having affinity for phosphatidylserine may be immobilized on a solid phase, and the substance having affinity for phosphatidylserine may be immobilized on a solid phase. Those that are immobilized are preferred.

Examples of the solid phase include synthetic polymer compounds such as latex, polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber; Examples include porous glass, frosted glass, ceramics, alumina, silica gel, activated carbon, and inorganic substances such as metal oxides. Also, two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples thereof include microtiter plates (ELISA plates), beads, tubes (microtubes), particles, dedicated trays integrally formed with many tubes, disc-shaped pieces, test tubes, and the like. is mentioned. The method for immobilizing the substance having affinity for CD9 and the substance having affinity for phosphatidylserine on the solid phase is not particularly limited as long as it is a method commonly used in this field. can be done according to

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

Examples of the measurement method using the affinity with the biomarker of the present invention include sandwich methods (e.g., sandwich ELISA method, AlphaLISA ^TM method (exoscreen ^TM method), fluorescence resonance energy transfer (FRET) method, bioluminescence resonance Energy transfer (BRET method, etc.), competitive method, agglutination method (immuno-nephelometry, immuno-nephelometry), immunochromatography, capillary electrophoresis, Western blot method, surface plasmon resonance method (SPR method), etc. immunoassay methods using antibodies that bind to the biomarkers of , and sandwich methods (eg, sandwich ELISA method, AlphaLISA ^™ method (Exoscreen ^™ method)) are preferred. As the measurement principle, either the homogeneous method or the heterogeneous method may be used. Immunological assay methods classified by labels are not particularly limited, and examples include enzyme-linked immunosorbent assay (ELISA method), enzyme immunoassay method (EIA method), radioimmunoassay method (RIA method), fluorescent enzyme immunoassay method method (FEIA method), fluorescence immunoassay method (FIA method), chemiluminescence enzyme immunoassay method (CLEIA method), chemiluminescence immunoassay method (CLIA method), electrochemiluminescence immunoassay method (ECLIA method), etc. , enzyme-linked immunosorbent assay (ELISA), and chemiluminescence enzyme immunoassay (CLEIA) are preferred. In addition, when using a substance having affinity for the biomarker of the present invention other than the antibody that binds to the biomarker of the present invention, it may be performed according to the immunoassay method described above. and a method of performing the immunoassay using a substance other than the antibody that binds to the tetraspanin, and a substance other than the anti-PS antibody that has an affinity for the phosphatidylserine. Preferred methods include those similar to the immunoassay methods described above. As the method for measuring the biomarker of the present invention, the immunoassay method described above and a method according to the immunoassay method described above are preferable.

Specifically, the amount of the biomarker of the present invention may be measured according to the method described in WO 2016/088689, and all descriptions in the above publication are incorporated herein.

Specifically, for example, (1) the biomarker of the present invention in a biological sample and the first affinity substance of the biomarker of the present invention (hereinafter referred to as the first may be abbreviated as an affinity substance) and a second affinity substance of the biomarker of the present invention (hereinafter sometimes abbreviated as a second affinity substance), and the biomarker in the biological sample is a step of forming a complex comprising a biomarker of the invention, a first affinity substance, and a second affinity substance (hereinafter sometimes abbreviated as "complex formation step"); and (2) A preferred method includes a method (sandwich method) including a step of measuring the amount of the complex (hereinafter sometimes abbreviated as a "complex amount measurement step").

In the complex forming step, the biological sample and the first affinity substance are brought into contact to form a first complex composed of the biomarker of the present invention in the biological sample and the first affinity substance. 1 step, contacting the first complex and the second affinity substance to form a second complex composed of the first complex and the second affinity substance It preferably includes two steps.

When measuring extracellular vesicles having CD9 as a biomarker of the present invention, the first affinity substance and the second affinity substance are substances having affinity for CD9, and specific examples are Preferred examples are the same as those described above. Moreover, the combination of the first affinity substance and the second affinity substance is not particularly limited, and the combination of both the first affinity substance and the second affinity substance is preferably an anti-CD9 antibody. Moreover, even if the first affinity substance and the second affinity substance are the same, they are different (for example, the first affinity substance and the second affinity substance do not compete with each other). good too.

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

The order of contacting the first affinity substance, the second affinity substance, and the biological sample in the complex formation step is not particularly limited. Contact with an affinity substance is preferred. Specifically, when measuring extracellular vesicles having phosphatidylserine and CD9 as biomarkers of the present invention, the biological sample is contacted with a substance having an affinity for phosphatidylserine as the first affinity substance. After contacting, it is preferable to contact a substance having an affinity for CD9 as a second affinity substance. Further, when measuring extracellular vesicles having CD9 as a biomarker of the present invention, after contacting a biological sample with a substance having an affinity for CD9 as a first affinity substance, a second As an affinity substance for CD9, it is preferable to contact a substance having affinity for CD9.

More specifically, in the complex formation step, for example, when measuring extracellular vesicles having CD9 as a biomarker of the present invention, an anti-CD9 antibody immobilized on a solid phase is contacted with a biological sample. , forming a first complex between the anti-CD9 antibody and extracellular vesicles having phosphatidylserine and CD9 in the biological sample, contacting the first complex with the anti-CD9 antibody, and forming the first complex A second complex is formed between the body and the anti-CD9 antibody. Further, when measuring extracellular vesicles having phosphatidylserine and CD9 as biomarkers of the present invention, an anti-PS antibody or Tim protein (preferably Timl or Tim4, more preferably Tim4) immobilized on a solid phase and a living organism The sample is contacted to form a first complex between the anti-PS antibody or the Tim protein and extracellular vesicles having phosphatidylserine and CD9 in the biological sample, and the first complex and the anti-CD9 antibody to form a second complex between the first complex and the anti-CD9 antibody.

After forming the complex, it is preferable to perform a washing operation (B/F separation) at least before the step of measuring the amount of the complex. Specifically, for example, a washing operation may be performed after forming the first complex or/and after forming the second complex in the above method. It is preferable to perform a washing operation (B/F separation), and then perform a washing operation (B/F separation) after forming a second complex.

In the complex amount measurement step, the amount of the complex (second complex) containing the first affinity substance, the second affinity substance, and the biomarker of the present invention obtained in the complex formation step is measured. Any method may be used as long as it is a step of measuring the amount of the complex, for example, the first or the second affinity substance may be measured. In addition, specifically, the complex amount measurement step includes, for example, (1) a method using a second affinity substance that is directly or indirectly labeled with a labeling substance, or (2) further directly or indirectly with a labeling substance. A secondary affinity substance that binds to an indirectly labeled second affinity substance (primary affinity substance) (e.g., a second affinity substance that is directly or indirectly labeled with a labeling substance) It is preferable to measure the second complex obtained in the complex formation step by a method using a secondary antibody that binds to the substance. In addition, in the complex amount measurement step, it is preferable to perform a washing operation (B/F separation) before detecting the labeling substance.

More specifically, the complex amount measurement step includes, for example, (1) an anti-CD9 antibody directly or indirectly labeled with a labeling substance, or (2) further directly or indirectly labeled with a labeling substance, The second complex obtained in the complex formation step is preferably measured by a method using a secondary antibody that binds to the anti-CD9 antibody (primary affinity substance). In addition, in the complex amount measurement step, it is preferable to perform a washing operation (B/F separation) before detecting the labeling substance.

Measurement of the amount of the biomarker of the present invention is performed by, as one aspect of the sandwich method, a proximity-based measurement method (e.g., AlphaLISA ^TM Act), etc. As the proximity-based measurement method, specifically, for example, the first affinity substance and the second affinity substance are immobilized on different first beads and second beads (acceptors or donors). It is implemented using the One of the first bead and the second bead is a donor, and a photosensitizer contained in the donor releases singlet oxygen upon excitation. The other of the first bead and the second bead is the acceptor. Forming a complex of the first bead to which the first affinity substance is bound, the biomarker of the present invention, and the second bead to which the second affinity substance is bound, and applying excitation light to the complex Irradiation causes singlet oxygen to be released from the donor photosensitizer. Singlet oxygen, which reaches the acceptor only when the complex is formed and the first and second beads are in close proximity, causes the acceptor to initiate a chemiluminescent reaction and detect the resulting light. By doing so, the amount of the biomarkers of the invention can be measured. In addition, the amount of the biomarker of the present invention may be measured by the Exoscreen ^™ method (Yoshioka et al., NatCommun, 2014), which is an improved version of the AlphaLISA ^™ method, or the like.

It has an affinity for the amount of the biological sample, the amount (concentration) of the protein in the biological sample, the amount (concentration) and the number of particles of the biomarker of the present invention in the biological sample, and the biomarker of the present invention to be reacted with these. The amount (concentration) of the substance, the labeling substance, the labeling method, and the like may be appropriately set according to the type of biological sample, the required measurement sensitivity, the measurement method and measurement device to be used, and the like. In addition, the amount (concentration) and the like of the biomarker of the present invention in a biological sample may be calculated by creating a calibration curve using standard products. Examples of the standard product include extracellular vesicles containing phosphatidylserine and CD9, extracellular vesicles containing CD9, and the like.

The method of the present invention for assisting in the discrimination between Lewy body disease and Alzheimer's disease (distinction assisting method of the present invention) comprises determining Lewy body disease and Alzheimer's disease in a subject using the amount of the biomarker of the present invention as an index. including. That is, the method for assisting differentiation of the present invention includes providing the amount of the biomarker of the present invention as an index for determining Lewy body disease and Alzheimer's disease in a subject. The above determination includes, for example, determining whether a subject suspected of having Lewy body disease or Alzheimer's disease has a high possibility of having Lewy body disease or Alzheimer's disease (the subject has Lewy body disease determination of whether there is a high possibility that the subject is suffering from Alzheimer's disease), determination of the possibility of suffering from Lewy body disease or Alzheimer's disease Determination of the possibility of having the disease, such as determination (determination of the possibility of the subject suffering from Lewy body disease, determination of the possibility of the subject suffering from Alzheimer's disease). Further, as the above determination, for example, determination of whether or not a subject suspected to be at risk of developing Lewy body disease or Alzheimer's disease has a high risk of developing Lewy body disease or Alzheimer's disease (the subject has Lewy body disease determination of whether the risk of developing a disease is high, determination of whether the subject has a high risk of developing Alzheimer's disease), determination of the presence or absence of the risk of developing Lewy body disease or Alzheimer's disease (the subject has Lewy bodies Determination for evaluating onset risk, such as determination of presence/absence of risk of developing disease, determination of presence/absence of risk of subject developing Alzheimer's disease. The above determination includes determining whether or not there is a high possibility of suffering from Lewy body disease or Alzheimer's disease in a subject suspected of having Lewy body disease or Alzheimer's disease, It is preferable to determine whether or not there is a possibility that In addition, by determining Lewy body disease and Alzheimer's disease in a subject, the possibility of having the disease in the subject and changes in the risk of developing the disease may be monitored over time.

More specifically, the identification assisting method of the present invention uses the amount of the biomarker of the present invention as an index to determine Lewy body dementia or Parkinson's disease-type dementia, Alzheimer's disease, or Parkinson's disease in a subject. include. That is, the method for assisting differentiation of the present invention provides the amount of the biomarker of the present invention as an index for determining dementia with Lewy bodies or Parkinson's disease, Alzheimer's disease, or Parkinson's disease in a subject. include. For the determination, for example, dementia with Lewy bodies or dementia with Parkinson's disease, Alzheimer's disease, or Parkinson's disease in a subject suspected of dementia with Lewy bodies or Parkinson's disease, Alzheimer's disease, or Parkinson's disease Determination of whether the subject is likely to be affected (determining whether the subject is likely to be suffering from Lewy body dementia or Parkinson's disease dementia, whether the subject is suffering from Alzheimer's disease determination of whether the subject is likely to have Parkinson's disease), Lewy body dementia or Parkinson's disease dementia, Alzheimer's disease, or Parkinson's disease Determination of the possibility of suffering (determination of the possibility of the subject suffering from Lewy body dementia or Parkinson's disease dementia, determination of the possibility of the subject suffering from Alzheimer's disease Determination of the possibility that the subject has Parkinson's disease). In addition, as the above determination, dementia with Lewy bodies or dementia with Parkinson's disease, Alzheimer's disease, or in subjects suspected of developing Parkinson's disease, dementia with Lewy bodies or Parkinson's disease, Alzheimer's disease, Alternatively, determination of whether the risk of developing Parkinson's disease is high (determination of whether the subject has a high risk of developing dementia with Lewy bodies or Parkinson's disease, determination of whether the subject has a high risk of developing Alzheimer's disease determination of whether the subject has a high risk of developing Parkinson's disease), determination of the presence or absence of risk of developing Lewy body dementia or Parkinson's disease dementia, Alzheimer's disease, or Parkinson's disease ( Determination of whether the subject is at risk of developing dementia with Lewy bodies or Parkinson's disease, determination of whether the subject is at risk of developing Alzheimer's disease, determination of whether the subject is at risk of developing Parkinson's disease, etc. Determination for evaluating the risk of developing Lewy body dementia or Parkinson's disease-type dementia, Alzheimer's disease, or in subjects suspected of having Parkinson's disease, Lewy body dementia or Parkinson's disease-type dementia, Alzheimer's disease , or determination of whether there is a high possibility of suffering from Parkinson's disease, determination of the possibility of suffering from Lewy body dementia or Parkinson's disease dementia, Alzheimer's disease, or Parkinson's disease is preferable .

Determination of Lewy body disease and Alzheimer's disease in the method for assisting differentiation of the present invention is performed, for example, by measuring the amount of the biomarker of the present invention in a subject-derived biological sample obtained by measuring the amount of the biomarker of the present invention. , by comparing with a predetermined reference value (cutoff value). As the criteria, for example, a first reference value for determining Parkinson's disease dementia or Lewy body dementia and Alzheimer's disease, or / and a second reference value for determining Alzheimer's disease and Parkinson's disease You can set it. The first reference value is a larger value (the amount of the biomarker of the present invention) than the second reference value. By using the first reference value, if the amount of the biomarker of the present invention is greater than the first reference value, the subject has Parkinson's disease dementia or Lewy body dementia (or simply Lewy body disease ), or it can be determined that there is a high risk of developing or at risk of developing. By using the second reference value, the possibility that the subject suffers from Parkinson's disease (or simply Lewy body disease) when the amount of the biomarker of the present invention is equal to or less than the second reference value It can be determined that there is a high or possible risk of developing a disease, or that there is a high risk of developing it. Further, by using the first reference value and the second reference value, when the amount of the biomarker of the present invention is equal to or less than the first reference value and greater than the second reference value, the subject has Alzheimer's disease. It can be determined that there is a high possibility or possibility of suffering from the disease, or that the risk of developing the disease is high or that there is a risk of developing the disease.

Determination of Lewy body disease and Alzheimer's disease in the method for assisting differentiation of the present invention is performed using only the first reference value described above, in subjects suspected of having Parkinson's disease dementia or Lewy body dementia, or Alzheimer's disease. Dementia with disease type or dementia with Lewy bodies and Alzheimer's disease alone may be determined. Specifically, when the amount of the biomarker of the present invention is greater than the first reference value, it is highly likely or possible that the subject suffers from Parkinson's disease dementia or Lewy body dementia. Alternatively, it is determined that the risk of onset is high or there is a risk of onset, and the subject is likely to have Alzheimer's disease when the amount of the biomarker of the present invention is equal to or less than the first reference value It can be determined that there is, or that there is a high risk of onset or there is a risk of onset. In addition, when the amount of the biomarker of the present invention is equal to or less than the first reference value and greater than the second reference value, it is highly likely or possible that the subject suffers from Alzheimer's disease, or has developed Alzheimer's disease. It is preferable to determine that the risk is high or that there is a risk of onset. Determination of Lewy body disease and Alzheimer's disease in the method for assisting differentiation of the present invention is performed only by determining Alzheimer's disease and Parkinson's disease in a subject suspected of having Alzheimer's disease or Parkinson's disease using only the second reference value. good too. Specifically, when the amount of the biomarker of the present invention is equal to or less than the second reference value, there is a high possibility that the subject suffers from Parkinson's disease, or there is a high risk of developing Parkinson's disease. It is determined that there is a risk, and if the amount of the biomarker of the present invention is equal to or less than the first reference value, it is likely or possible that the subject suffers from Alzheimer's disease, or the risk of developing Alzheimer's disease is high, or It can be determined that there is an onset risk. In addition, when the amount of the biomarker of the present invention is equal to or less than the first reference value and is greater than the second reference value, it is highly likely or possible that the subject suffers from Alzheimer's disease, or has developed Alzheimer's disease. It is preferable to determine that the risk is high or that there is a risk of onset.

The first reference value is the amount of the biomarker of the present invention preset for determining Parkinson's disease dementia or Lewy body dementia and Alzheimer's disease. The method for determining the first reference value is not particularly limited. For example, it is obtained from patients with or at risk of developing Parkinson's disease dementia or dementia with Lewy bodies and patients with or at risk of developing Alzheimer's disease. The amount of the biomarker of the present invention contained in the obtained biological sample is measured, and using the obtained amount of the biomarker of the present invention, statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis) can be determined. Biological samples obtained from a patient or person at risk of developing Parkinson's disease or dementia with Lewy bodies and a patient or person at risk of developing Alzheimer's disease are preferably similar biological samples. The second reference value is preset for determining Alzheimer's disease and Parkinson's disease. It can be determined by the same method as the first reference value, except that biological samples obtained from patients with or at risk of developing Parkinson's disease and from patients with or at risk of developing Alzheimer's disease are used. In addition, it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, etc. in setting the reference value. The predetermined reference value can be determined, for example, so that the sensitivity is 60% or more, preferably 70% or more, more preferably 80% or more, and 90% or more. Particularly preferred, for example, the specificity can be determined to be 60% or higher, preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher.

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

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

The reagent kit for aid in differentiation of the present invention contains a secondary affinity substance (e.g., secondary antibody) that binds to the substance having affinity for CD9 (primary affinity substance), or/and the phosphatidylserine. It may further contain a secondary affinity substance (for example, a secondary antibody) that binds to the substance (primary affinity substance) that has an affinity for the antibody, and the secondary affinity substance is labeled with a labeling substance. It is preferable to have In addition, the labeling of the substances having affinity (the primary affinity substance and the secondary affinity substance) in the identification aid reagent kit of the present invention is performed by directly or indirectly labeling the substance having affinity with a labeling substance. May be labeled. The substances having affinity (the primary affinity substance and the secondary affinity substance), the labeling substance, and the labeling method in the identification aid reagent kit of the present invention are the same as those of the identification aid method of the present invention. Specific examples and preferable ones are also the same.

The concentration (amount) of the substance having affinity for CD9 and the substance having affinity for phosphatidylserine (primary affinity substance) in the identification aid reagent kit of the present invention depends on the measurement method, It may be appropriately set within the range normally used in this field. The concentration of the substance having affinity for CD9 and/or the substance having affinity for phosphatidylserine (primary affinity substance) when immobilized on a solid phase is, for example, 10 ~20,000 ng/mL, preferably 100 to 10,000 ng/mL. preferable. In addition, reagents commonly used in this field, for example, buffers, reaction accelerators, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc., are added to substances having affinity for CD9. Or/and it may be coexisted with a substance having an affinity for the phosphatidylserine. These concentrations and pH may also be appropriately selected from the range usually used in this field.

The identification aid reagent kit of the present invention may comprise reagents necessary for measuring the biomarker of the present invention, in addition to the substance having affinity for CD9. Such reagents include, for example, detergents, sample diluents, reagents for detecting labeling substances, reagents for directly or indirectly binding labeling substances to the aforementioned affinity substances, and reagents for binding avidins or biotins to the aforementioned affinity substance or labeling substance. The concentration, pH, etc. of these reagents may also be appropriately selected from the ranges commonly used in this field.

The reagent kit for aid in differentiation of the present invention is a standard used to create a standard curve for extracellular vesicles having CD9, or used to create a standard curve for extracellular vesicles having CD9 and phosphatidylserine. may include standard products that are Examples of the standard product include extracellular vesicles having CD9 and extracellular vesicles having phosphatidylserine. The standard may be in a solution state, a frozen state, a dried state, or a freeze-dried state. In addition, reagents commonly used in this field, such as buffers, reaction accelerators, sugars, proteins, salts, stabilizers such as surfactants, preservatives, etc., may coexist with the standard product. These concentrations and pH may also be appropriately selected from the range usually used in this field.

The identification aid reagent kit of the present invention may include an attached document and an instruction manual. As the package insert or instruction manual, for example, measuring the amount of the biomarker of the present invention in a biological sample derived from a subject or / and Alzheimer's disease and Parkinson's disease using the amount of the biomarker of the present invention as an index Package inserts, instruction manuals, etc. that describe assistance in identification (identification assistance method of the present invention) are included. These package inserts and instruction manuals may be described separately in a plurality of items, or may be collectively described in a single item.

Specific examples of the identification aid reagent kit of the present invention include the following.
<1> (A) a kit containing a substance having affinity for CD9;
<1-1> (A-1) a substance having an affinity for the CD9 immobilized on the solid phase, and (B-1) a substance having an affinity for the labeled CD9 kit;
<1-2> (A-1) a substance having an affinity for the CD9 immobilized on the solid phase, (B) a substance having an affinity for the CD9, and (C) a labeled A kit containing a substance having affinity for "(B) the substance having affinity for CD9" (for example, an antibody (secondary antibody) against the substance (B) having affinity for CD9) ;
<2> A kit containing (D) the substance having affinity for phosphatidylserine and (A) the substance having affinity for CD9;
<2-1> (D-1) containing a substance having affinity for the phosphatidylserine immobilized on the solid phase and (A-2) a substance having affinity for the labeled CD9 kit;
<2-2> (D-1) a substance having an affinity for the phosphatidylserine immobilized on the solid phase, (A) a substance having an affinity for the CD9, and (E) a labeled and a substance having affinity for "(A) the substance having affinity for CD9" (for example, an antibody (secondary antibody) against the substance (A) having affinity for CD9). kit;
<2-3> (D-2) Contains a substance having affinity for the above-mentioned labeled phosphatidylserine and (A-1) a substance having affinity for the above-mentioned CD9 immobilized on a solid phase kit;
<2-4> (D) a substance having an affinity for the phosphatidylserine, (F) having an affinity for the labeled "(D) a substance having an affinity for the phosphatidylserine" a substance (for example, (D) an antibody (secondary antibody) against a substance having affinity for phosphatidylserine) and (A-1) a substance having affinity for CD9 immobilized on a solid phase; Kit including. Among them, <1>, <1-1>, <1-2>, <2>, <2-1>, or <2-2> is preferable. In addition, the labeled substances with affinity are those in which one of ligands and receptors such as avidins and biotins are bound to unlabeled substances with affinity, and ligands such as avidins and biotins. A kit may be constructed in which the remaining one of the receptors is bound to a labeling substance.

More specifically, the following are more preferred examples of the identification aid reagent kit of the present invention.
[1] (A) kit containing anti-CD9 antibody;
[1-1] A kit containing (A-1) an anti-CD9 antibody immobilized on a solid phase and (B-1) a labeled anti-CD9 antibody;
[1-2] (A-1) anti-CD9 antibody immobilized on solid phase, (B) anti-CD9 antibody, and (C) antibody against labeled "(B) anti-CD9 antibody" (secondary antibody) a kit containing;
[2] (D) a kit comprising an anti-PS antibody or a phosphatidylserine affinity protein (preferably Tim protein, preferably Tim1, Tim4, more preferably Tim4) and (A) an anti-CD9 antibody;
[2-1] (D-1) an anti-PS antibody immobilized on a solid phase or a phosphatidylserine affinity protein (preferably Tim protein, preferably Tim1 or Tim4, more preferably Tim4) and (A-2) a kit containing a labeled anti-CD9 antibody;
[2-2] (D-1) an anti-PS antibody immobilized on a solid phase or a phosphatidylserine affinity protein (preferably Tim protein, preferably Tim1 or Tim4, more preferably Tim4), (A) anti-CD9 A kit containing the antibody, (E) a labeled anti-CD9 antibody is preferred. Labeled antibodies or phosphatidylserine affinity proteins are those in which one of ligands and receptors such as avidins and biotins are bound to each unlabeled antibody phosphatidylserine affinity protein, and avidins and biotins. A kit may be constructed in which the other one of the ligand and the receptor is bound to a labeling substance. Also, each antibody and phosphatidylserine affinity protein may be a solution (reagent) containing, for example, a concentration of 10 to 5000 ng/mL, preferably a solution (reagent) containing a concentration of 100 to 500 ng/mL.

According to the present invention, data (judgment results) for assisting discrimination between Lewy body disease and Alzheimer's disease in a subject can be obtained. When it is determined that the subject has a high possibility of suffering from Alzheimer's disease or may be suffering from Alzheimer's disease according to the present invention, or that the subject has a high risk of developing Alzheimer's disease or has a risk of developing Alzheimer's disease , For example, diagnostic methods using imaging devices such as interviews, amyloid PET diagnosis, MMSE tests, for example, decrease of Aβ42 in cerebrospinal fluid, increase of Tau or phosphorylated Tau, etc. Recommended in dementia disease treatment guidelines Tests related to Alzheimer's disease biomarkers and the like in which the subject is present, and tests using Alzheimer's disease biomarker candidate substances may be further performed, and the results of these tests and the like may be taken into account to diagnose Alzheimer's disease in the subject. In addition, a drug for delaying the progress of Alzheimer's disease, such as a cholinesterase inhibitor, or a therapeutic drug may be administered, or surgery may be performed. In addition, when it is determined that the subject is likely to have or may be suffering from Parkinson's disease according to the present invention, or that the subject has a high risk of developing Parkinson's disease or is at risk of developing Parkinson's disease For example, tests for Parkinson's disease recommended in the clinical practice guidelines for Parkinson's disease, such as interviews, MIBG myocardial scintigraphy, and dopamine transporter scintigraphy, and tests using candidate substances for Parkinson's disease biomarkers may be further performed. In consideration of these results, etc., a diagnosis of Parkinson's disease in the subject can be made. In addition, a drug such as levodopa (L-dopa) that delays the progression of Parkinson's disease or a therapeutic drug may be administered, or surgery may be performed. In addition, according to the present invention, the subject is likely to have or may be suffering from Parkinson's disease dementia or Lewy body dementia, or the subject has Parkinson's disease dementia or Lewy body dementia Antidementia drugs such as cholinesterase inhibitors and levodopa (L-dopa), etc. The dosage of the anti-Parkinson's disease therapeutic agent may be adjusted, for example, the dosage may be lower than that for a Parkinson's disease patient, or no dosage may be given.

Since the method, biomarker, and reagent kit for assisting in distinguishing between Lewy body disease and Alzheimer's disease of the present invention can determine Lewy body disease and Alzheimer's disease in a subject, Useful.

EXAMPLES The present invention will be specifically described below based on examples and comparative examples, but the present invention is not limited by these examples.

Example 1. Evaluation of Alzheimer's disease specimens and Parkinson's disease dementia or Lewy body dementia specimens using the amount of exosomes having CD9 as an index Measurement of exosomes by anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method AUC and p-value were calculated.
(1) Preparation of calibrator
Using MagCapture (registered trademark) Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "A kit"), exosomes are isolated from the COLO201 cell culture supernatant according to the instruction manual attached to the A kit. was isolated, and the exosomes were eluted using the eluent attached to the A kit. Using a protein assay BCA kit (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), the protein concentration in the obtained exosome solution was measured by the BCA method (bicinchoninic acid method). Then, the resulting exosome solution, PS Capture Exosome ELISA Kit, Streptavidin HRP (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "B kit") using the reaction buffer attached, proteins measured by the BCA method Based on the concentration, dilute to 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0, and 20.0ng/mL, respectively, and prepare an 8-point COLO201 cell culture supernatant-derived exosome dilution series (hereinafter referred to as "Calibrator") was obtained.
(2) Pretreatment of samples for measurement
EDTA plasma from 10 samples of Alzheimer's disease (AD) patients purchased from PrecisionMed, Parkinson's disease dementia or Lewy body dementia (PDD/DLB, MMSE ≤ 23) 5 samples was centrifuged at 10,000 g for 20 minutes each After separation, the supernatant was recovered. The obtained supernatants were each diluted 100-fold using the Reaction Buffer attached to the A kit to obtain a "specimen diluent for measurement".
(3) Measurement by ELISA Using the anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method, the anti-CD9 antibody is immobilized on a solid phase and the anti-CD9 antibody is used as a detection antibody. It was measured. Reagents attached to the B kit were used except for the detection antibody and the solid phase immobilization plate. (i) Preparation of biotin-labeled anti-CD9 mouse monoclonal antibody Anti-CD9 mouse monoclonal antibody (1K, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was reduced with 1.6 mM DTT, and then biotin-PEAC5-maleimide (Dojindo Laboratories) (manufactured) at 37°C for 1.5 hours to prepare a biotin-labeled anti-CD9 mouse monoclonal antibody.
(ii) Preparation of anti-CD9 antibody-immobilized plate
After diluting the WashingBuffer (10x) attached to the B kit 10 times with purified water (distilled water), add 1/100 volume to the diluted solution obtained ExosomeBindingEnhancer (100x) attached to the B kit did. The resulting solution is referred to as "washing solution (1x)". Anti-CD9 mouse monoclonal antibody (lK) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted with 50 mM MOPS (pH 7.5) to a concentration of 10 μg/mL, and 100 μL was added to wells of a 96-well microplate (Nunc). and incubated overnight in the refrigerator. After washing the wells three times with TBST (TrisBufferedSaline, pH7.4), 300μL of TBS (TrisBufferedSaline, pH7.4) containing 10mg/mL Block Ace was added and incubated overnight in a refrigerator to obtain an anti-CD9 antibody immobilized plate. rice field.
(iii) Measurement of CD9-bearing exosomes Each well of the anti-CD9 antibody-immobilized plate was washed three times with 300 to 350 μL of washing solution (1×). Next, the sample diluent for measurement of Alzheimer's disease patients prepared in (2) (10 samples n = 2, 20 wells), Parkinson's disease dementia or Lewy body dementia patients (5 samples n = 2, 10 wells) , 100 μL each of the calibrator prepared in (2) (n=2 for 8 points, 16 wells) and ReactionBuffer (n=2, 2 wells) attached to the B kit as a blank were dispensed into each well of the plate. Then, a plate seal was affixed to the plate, and the reaction was allowed to proceed at room temperature for 2 hours while stirring at about 500 rpm using a microplate shaker. After the reaction, the reaction solution was discarded, and each well was washed 3 times with 300 to 350 μL of washing solution (×1). The biotin-labeled anti-CD9 mouse monoclonal antibody was diluted to a final concentration of 250 ng/mL using the ReactionBuffer attached to the B kit to obtain a biotin-labeled antibody reaction solution. 100 μL of the resulting biotin-labeled antibody reaction solution was dispensed into each well, a plate seal was affixed, and reaction was allowed to proceed at room temperature for 1 hour while stirring at about 500 rpm using a microplate shaker. After the reaction, the reaction solution was discarded, and each well was washed 3 times with 300 to 350 μL of washing solution (×1). 1/100 amount of HRP-conjugatedStreptavidin (100×) was added to the ReactionBuffer attached to the B kit and mixed well to prepare an HRP-labeled streptavidin reaction solution (1×). 100 μL of the resulting HRP-labeled streptavidin reaction solution (1×) was dispensed into each well, a plate seal was attached, and the mixture was reacted at room temperature for 2 hours while stirring at about 500 rpm using a microplate shaker. . After completion of the reaction, the reaction solution was discarded, and each well was washed 5 times with 300 to 350 μL of washing solution (1×). 100 μL of TMB (3,3′,5,5′-tetramethylbenzidine) solution attached to the B kit which had been returned to room temperature was dispensed into each well and stirred for about 1 minute using a microplate shaker. After that, a plate seal was affixed, and reaction was allowed to stand at room temperature (20 to 25°C) for 30 minutes. After that, add 100 μL of the StopSolution attached to the B kit that has been returned to room temperature to each well, stir for about 5 seconds using a microplate shaker, and immediately use a 96-well microplate reader (Tecan, Safire2). Absorbance at 450 nm and sub-wavelength 620 nm were measured using The value obtained by subtracting the sub-wavelength 620 nm absorbance value from the 450 nm absorbance value is the “absorbance value”, and the value obtained by subtracting the blank absorbance value from the absorbance value of the diluted sample for measurement is calculated as the “corrected sample absorbance value”. . Then, a standard curve was created from the absorbance value obtained by subtracting the blank absorbance value from the absorbance value of the COLO201 cell culture supernatant-derived exosome dilution series (calibrator) and the protein concentration of the calibrator. Using the standard curve, the corrected sample absorbance value was converted to protein concentration, and the obtained converted value was multiplied by the sample dilution rate to obtain the "sample measured value" [ng/mL].
(4) Calculation of AUC
Based on the specimen measurement values obtained in (3), Alzheimer's disease was determined by Wilcoxon/Kruska1-Wallis test (rank sum) using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA) A significant difference test was performed between the patient and the patient with Parkinson's disease dementia or Lewy body dementia, and the p-value was calculated. In addition, based on the sample measurement values obtained in (3), logistic regression analysis was performed using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA), and the obtained receiver operating characteristic curve The area under the curve (AUC) was calculated from (ROC curve). The results obtained are shown in Table 1 below and FIG. In the figure, the vertical axis indicates the sample measurement value, the horizontal axis AD indicates the results of Alzheimer's disease patients, and PDD/DLB indicates the results of Parkinson's disease dementia or Lewy body dementia patients, respectively.

Example 2. Evaluation of Alzheimer's disease specimens and Parkinson's disease-type dementia or Lewy body dementia specimens using the amount of exosomes containing PS (phosphatidylserine) and CD9 as an index Instead of "anti-CD9 antibody immobilization plate" Sandwich ELISA of Tim4-anti-CD9 antibody was performed in the same manner as in Example 1, except that the Tim4-immobilized plate attached to "PSCaptureExosome ELISA Kit, StreptavidinHRP (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., B kit above)" was used. Measure exosomes by the method, perform a significant difference test between Alzheimer's disease (AD) sample values and Parkinson's disease dementia or Lewy body dementia (PDD/DLB), and calculate AUC and p value The results obtained are shown in Table 1 below and FIG.

Comparative Example 1. Evaluation of Alzheimer's disease specimens and Parkinson's disease dementia or Lewy body dementia specimens using the amount of exosomes having CD63 as an index Instead of anti-CD9 antibody as an antibody to be immobilized on an antibody-immobilized plate Anti-CD63 antibody (3-13) (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was used, and PS Capture Exosome ELISA Kit and Streptavidin HRP (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd., the above "B kit" were used as detection antibodies. ) Exosomes are measured by the anti-CD63 antibody-anti-CD63 antibody sandwich ELISA method in the same manner as in Example 1 except that the biotin-labeled anti-CD63 antibody attached to ) is used, and the measured value of the specimen of Alzheimer's disease A significant difference test was performed on the specimen measurement values of Parkinson's disease dementia or Lewy body dementia, and the P value was calculated. The results obtained are shown in Table 1 below.

Comparative Example 2. Evaluation of Alzheimer's disease specimens and Parkinson's disease dementia or Lewy body dementia specimens using the amount of exosomes having CD81 as an index Instead of anti-CD9 antibody as an antibody to be immobilized on an antibody-immobilized plate Anti-CD81 mouse monoclonal antibody (17B1) (manufactured by Tomiju Film Wako Pure Chemical Industries, Ltd.) was used, and the above anti-CD81 antibody was biotin-labeled in the same manner as in Example 1 as the detection antibody, except that By the same method as in Example 1, exosomes are measured by a sandwich ELISA method of anti-CD81 antibody-anti-CD81 antibody, and the significance of the specimen measurement values of Alzheimer's disease and Parkinson's disease dementia or Lewy body dementia Difference tests were performed and p-values were calculated. The results obtained are shown in Table 1 below.

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

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

From Table 1, plasma from Alzheimer's disease patients and Parkinson's disease dementia or Lewy body dementia patients are used as samples, and Alzheimer's disease and Parkinson's disease dementia or Lewy body dementia are measured using the amount of exosomes having CD9 as an index. The AUC was 0.949 when disease was evaluated, and 0.90 when the amount of exosomes with phosphatidylserine and CD9 was used as an index. On the other hand, when the amount of exosomes having CD63, the amount of exosomes having CD81, the amount of exosomes having phosphatidylserine and CD63, or the amount of exosomes having phosphatidylserine and CD81 were used as indicators, there was no statistically significant difference. rice field. From these results, by using the amount of exosomes having CD9 and the amount of exosomes having phosphatidylserine and CD9 as indicators, it was found that Alzheimer's disease and Parkinson's disease dementia or Lewy body dementia can be determined. In particular, it was found that the amount of exosomes having CD9 can be determined with high accuracy.

Example 3. Evaluation of Alzheimer's disease specimens and Parkinson's disease specimens using the amount of exosomes having CD9 as an index As Alzheimer's disease patient specimens, "EDTA plasma of 18 Alzheimer's disease patients purchased from PrecisionMed" was used, and " The same procedure as in Example 1 except that "EDTA plasma of 5 Parkinson's disease patients purchased from PrecisionMed" (MMSE is 27 or more) was used instead of "specimen of Parkinson's disease dementia or dementia with Lewy bodies". According to the method, exosomes were measured by sandwich ELISA method of anti-CD9 antibody-anti-CD9 antibody, significant difference test was performed between Alzheimer's disease specimen measurement value and Parkinson's disease specimen measurement value, and AUC and P value were calculated. The results obtained are shown in Table 2 below.

Comparative Example 5. Evaluation of Alzheimer's disease specimens and Parkinson's disease specimens using the amount of exosomes containing CD63 as an index As Alzheimer's disease patient specimens, "EDTA plasma of 18 Alzheimer's disease patients purchased from PrecisionMed" was used, and " The same procedure as in Comparative Example 1 except that "EDTA plasma from 5 Parkinson's disease patients purchased from PrecisionMed" (MMSE is 27 or more) was used instead of "Parkinson's disease dementia or Lewy body dementia specimen". According to the method, exosomes were measured by sandwich ELISA method of anti-CD63 antibody-anti-CD63 antibody, significant difference test was performed between Alzheimer's disease specimen measurement value and Parkinson's disease specimen measurement value, and p-value was calculated. The results obtained are shown in Table 2 below.

Comparative Example 6. Evaluation of Alzheimer's Disease Specimens and Parkinson's Disease Specimens Using the Amount of Exosomes Having CD81 as an Index By the same method as in Comparative Example 2, except that "EDTA plasma from 5 Parkinson's disease patients purchased from PrecisionMed" (MMSE is 27 or more) was used instead of "specimen of type dementia or dementia with Lewy bodies" , Exosomes were measured by sandwich ELISA method of anti-CD81 antibody-anti-CD81 antibody, significant difference test was performed between Alzheimer's disease specimen measurement value and Parkinson's disease specimen measurement value, and p-value was calculated. The results obtained are shown in Table 2 below.

From Table 2, AUC was 0.819 when Alzheimer's disease and Parkinson's disease were evaluated using plasma samples derived from Alzheimer's disease patients and Parkinson's disease patients, respectively, and using the amount of exosomes having CD9 as an index. On the other hand, when the amount of exosomes having CD63 or the amount of exosomes having CD81 was used as an indicator, there was no statistically significant difference. From these results, it was found that Alzheimer's disease and Parkinson's disease can be determined by using the amount of exosomes having CD9 as an index.

Example 4. Evaluation of specimens of Parkinson's disease dementia or Lewy body dementia (MMSE of 23 or less) and Parkinson's disease (MMSE of 27 or more) using the amount of exosomes having CD9 as an index "Parkinson's disease type Dementia or Lewy body dementia specimens"," Parkinson's disease dementia or Lewy body dementia patients (PDD / DLB, MMSE is 23 or less) 5 specimens of EDTA plasma", Alzheimer's disease patient specimens Sandwich ELISA method of anti-CD9 antibody-anti-CD9 antibody was performed in the same manner as in Example 1, except that instead, "EDTA plasma from 8 specimens of Parkinson's disease patients (MMSE ≥ 27) purchased from PrecisionMed" was used. By measuring the exosomes, the significant difference test of the specimen measured value of Parkinson's disease dementia or Lewy body dementia and the specimen measured value of Parkinson's disease was performed, AUC and p value were calculated. The results obtained are shown in Table 3 below.

Example 5. Evaluation of specimens of Parkinson's disease dementia or Lewy body dementia (MMSE of 23 or less) and Parkinson's disease (MMSE of 27 or more) using the amount of exosomes having PS and CD9 as an index " Using EDTA plasma from 5 specimens of Parkinson's disease dementia or Lewy body dementia (PDD/DLB, MMSE less than 23) as specimens of Parkinson's disease dementia or Lewy body dementia, Alzheimer's disease Sandwich ELISA of Tim4-anti-CD9 antibody in the same manner as in Example 2, except that "EDTA plasma from 8 specimens of Parkinson's disease patients (MMSE is 27 or more) purchased from PrecisionMed" was used instead of patient specimens. Exosomes were measured by the method, the significance test was performed between the measured values of Parkinson's disease dementia or Lewy body dementia and the measured values of Parkinson's disease samples, and AUC and p value were calculated. The results obtained are shown in Table 3 below.

From Table 3, plasma from patients with Parkinson's disease dementia or Lewy body dementia (MMSE is 23 or less) and Parkinson's disease patients (MMSE is 27 or more) are used as samples, exosomes having CD9 and PS and CD9 When evaluating Parkinson's disease dementia or Lewy body dementia (MMSE 23 or less) and Parkinson's disease (MMSE 27 or more) using the amount of exosomes as an index, the AUC was 1.000. From these results, by using the amount of exosomes with CD9 or the amount of exosomes with PS and CD9 as an index, Parkinson's disease dementia or Lewy body dementia (MMSE is 23 or less) and Parkinson's disease (MMSE) is 27 or more) can be determined.

Claims

measuring the amount of extracellular vesicles with CD9 or the amount of extracellular vesicles with phosphatidylserine and CD9 in a biological sample from a subject suspected of having Lewy body disease or Alzheimer's disease;
Determining Lewy body disease and Alzheimer's disease in a subject using the amount of extracellular vesicles having CD9 or the amount of extracellular vesicles having phosphatidylserine and CD9 as an index,
A method to help distinguish between Lewy body disease and Alzheimer's disease.
2. The method of claim 1, wherein the Lewy body disease is Parkinson's disease dementia, Lewy body dementia, or/and Parkinson's disease.
The determination is to determine Parkinson's disease dementia or Lewy body dementia when the amount of the extracellular vesicles is greater than the first reference value, or / and the amount of the extracellular vesicles 3. The method for assisting discrimination between Lewy body disease and Alzheimer's disease according to claim 2, wherein Parkinson's disease is determined when is equal to or less than a second reference value.
The Lewy body disease is Parkinson's disease dementia or Lewy body dementia, and the measurement of the amount of extracellular vesicles is to measure the amount of extracellular vesicles having the CD9,
2. The method for assisting in distinguishing between Lewy body disease and Alzheimer's disease according to claim 1, wherein the determination is based on the amount of extracellular vesicles containing CD9 as an indicator.
The Lewy body disease is Parkinson's disease dementia or Lewy body dementia,
measuring the amount of extracellular vesicles is measuring the amount of extracellular vesicles having the phosphatidylserine and CD9;
2. The method for assisting in distinguishing between Lewy body disease and Alzheimer's disease according to claim 1, wherein the determination is performed using the amount of extracellular vesicles containing phosphatidylserine and CD9 as an index.
wherein the Lewy body disease is Parkinson's disease;
measuring the amount of the extracellular vesicles is measuring the amount of the extracellular vesicles having the CD9,
The determination is to determine the amount of extracellular vesicles having the CD9 as an index,
The method of claim 1 for assisting in distinguishing between Lewy body disease and Alzheimer's disease.
A reagent kit for assisting differentiation between Lewy body disease and Alzheimer's disease, containing a substance having affinity for CD9.
8. The reagent kit for assisting the differentiation between Lewy body disease and Alzheimer's disease according to claim 7, wherein said Lewy body disease is Parkinson's disease dementia, Lewy body dementia, or/and Parkinson's disease.
A biomarker to help differentiate between Lewy body disease and Alzheimer's disease, comprising extracellular vesicles with CD9 or extracellular vesicles with phosphatidylserine and CD9.
10. The biomarker for assisting the differentiation between Lewy body disease and Alzheimer's disease according to claim 9, wherein the Lewy body disease is Parkinson's disease dementia, Lewy body dementia, or/and Parkinson's disease.