Mild cognitive impairment detection marker and use thereof

Abstract

The present disclosure provides a technique relating to mild cognitive impairment detection using a skin blotting sample. In this disclosure, the mild cognitive impairment detection uses a mild cognitive impairment detection marker containing at least one selected from the group consisting of Aβ - (39 + 37 + 36) / ALB, Aβ - (39 + 37) / ALB, and Aβ - (37 + 36) / ALB as a detection index.

Description

Markers for detecting mild cognitive impairment and their use

[0001] A technique for detecting mild cognitive impairment using skin blotting samples is disclosed.

[0002] Dementia is defined as "a syndrome that usually results from chronic or progressive brain disease and consists of impairments in numerous higher brain functions such as memory, thinking, orientation, comprehension, calculation, learning, language, and judgment" (Non-Patent Literature 1). The prevalence of dementia among elderly people aged 65 and over in Japan is estimated at 3.8-11.0%, while the number of people affected worldwide (2001) is estimated at 24.3 million. Furthermore, a significant increase of 4.6 million people per year is projected. When dementia develops, it is thought that the quality of life (QOL) of the patient themselves will decline and the burden of care on family members and others providing care will increase. In Japan, the "Basic Law on Dementia" was enacted in 2023, and dementia prevention has become a social imperative.

[0003] Mild Cognitive Impairment (MCI), according to the Ministry of Health, Labour and Welfare, is a condition in which forgetfulness is the primary symptom, but there is little impact on daily life, and it cannot be diagnosed as dementia. It is characterized by (1) memory impairment that cannot be explained solely by age or education level, (2) complaints of forgetfulness from the individual or their family, (3) overall cognitive function being within the normal range, (4) independence in daily living activities, and (5) not being dementia (Non-Patent Literature 2). A follow-up study by Manly et al. over 18-24 months showed that 22% of MCI cases progressed to dementia, 47% remained unchanged, and 31% improved (Non-Patent Literature 3). Furthermore, a systematic review by Bruscoli et al. reported an annual risk of progression from MCI to dementia of 10% (Non-Patent Literature 4). In other words, MCI is a reversible condition, and early intervention based on MCI screening is considered effective in preventing dementia.

[0004] The examination methods for MCI can be broadly divided into three categories. The first is an interview-style tool. For the purpose of screening MCI, which was difficult to diagnose with conventional tools, MoCA (The Montreal Cognitive Assessment) has been developed (Non-Patent Document 5). The identification sensitivity of MCI is 18% for MMSE (Mini-Mental State Examination), while MoCA has a very high accuracy of 90%. As a Japanese version, MoCA-J has been developed and its effectiveness in Japanese elderly people has been proven (Non-Patent Document 6). However, this tool is not suitable for population screening because it requires one-on-one interviews between medical staff and patients, and it is difficult to conduct the examination if there are visual or auditory impairments. The second is the imaging diagnosis method, and methods such as PET (Positron Emission Tomography) and MRI (Magnetic Resonance Image) are used in this method (Non-Patent Documents 7 and 8). However, these require large-scale facilities and specialized technicians. The third is the biochemical examination that analyzes biological samples such as cerebrospinal fluid and blood. However, the cerebrospinal fluid examination is highly invasive, and although the blood examination is less invasive, its accuracy is not sufficient (Non-Patent Documents 9 and 10). From the above, it can be said that the conventional examination methods for MCI are not suitable for population screening in terms of invasiveness, diagnostic accuracy, simplicity, cost, time, etc.

[0005] World Health Organization. International statistical classification of diseases and related health problems. 10th Revision. Geneva: World Health Organization. 1993. Ministry of Health, Labour and Welfare. Mild Cognitive Impairment. e-Health Net. 2023 / 11 / 6 [https: / / www.e-healthnet.mhlw.go.jp / information / dictionary / alcohol / ya-033.html]. Manly JJ, Tang MX, Schupf N, et al. Frequency and Course of Mild Cognitive Impairment in a Multiethnic Community. Ann Neurol. 2008; 63(4): 494-506. Bruscoli M, Lovestone S. Is MCI really just early dementia? A systematic review of conversion studies. Int Psychogeriatr. 2004; 16(2): 129-140. Nasreddine ZS, Phillips NA, Be’dirian V, et al. The Montreal Cognitive Assessment, MoCA: A brief screening tool for mild cognitive impairment. J Am Geriatr Soc. 2005; 53(4): 695-699. Fujiwara Y, Suzuki H, Yasunaga M, et al. Brief screening tool for mild cognitive impairment in older Japanese: Validation of the Japanese version of the Montreal Cognitive Assessment. Geriatr Gerontol Int. 2010; 10(3): 225-232.Hane FT, Robinson M, Lee BY, et al. Recent Progress in Alzheimer's Disease Research, Part 3: Diagnosis and Treatment. J Alzheimers Dis. 2017; 57(3): 645-665.Jack CR Jr, Wiste HJ, Vemuri P, et al. Brain beta-amyloid measures and magnetic resonance imaging atrophy both predict time-to-progression from mild cognitive impairment to Alzheimer's disease. Brain. 2010; 133(11): 3336-3348.Bjerke M, Engelborghs S. Cerebrospinal fluid biomarkers for early and differential Alzheimer's disease diagnosis. J Alzheimers Dis. 2018; 62(3): 1199-1209.Planche V, Bouteloup V, Pellegrin I, et al. Validity and performance of blood biomarkers for Alzheimer disease to predict dementia risk in a large clinic-based cohort. Neurology. 2023; 100(5): e473-e484.Minematsu T, Horii M, Oe M, et al. Skin blotting: A noninvasive technique for evaluating physiological skin status. Adv Skin Wound Care. 2014; 27: 272-9.Friedel M, Thompson IAP, Kasting G, et al.Opportunities and challenges in the diagnostic utility of dermal interstitial fluid. Nat Biomed Eng. 2023. Online ahead of print. [https: / / www.nature.com / articles / s41551-022-00998-9] .Chevallet M, Diemer H, Van Dorssealer A, et al. Toward a better analysis of secreted proteins: the example of the myeloid cells secretome. Proteomics. 2007; 7(11): 1757-1770.Nuan J, Small DH. Regulation of APP cleavage by alpha-, beta- and gamma-secretases. FEBS Lett. 2000; 483(1): 6-10.Shankar GM, Li S, Mehta TH, et al. Amyloid β-protein dimers isolated directly from Alzheimer brains impair syn-aptic plasticity and memory. Nat Med. 2008; 14: 837-842.Viola KL, Klein WL. Amyloid β oligomers in Alzheimer’s disease pathogenesis, treatment, and diagnosis. Acta Neuropathol. 2015; 129(2): 183-206.Adolfsson O, Pihlgren M, Toni N, et al. An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβbinding properties promotes neuroprotection and glial engulfment of Aβ. J Neurosci. 2012; 32(28): 9677-9689.Yang T, Li S, Xu H, et al. Large soluble oligomers of Amyloid β-protein from Alzheimer brain are far less neuroactive than the smaller oligomers to which they dissociate. J Neurosci. 2017; 37(1): 152-163.Fukukmoto H, Tokuda T, Kasai T, et al. High-molecular-weight β-amyloid oligomers are elevated in cerebrospinal fluid of Alzheimer patients. FASEB J. 2010; 24: 2716-2726.Zou L, Chan KH, Chu LW, et al. Plasma amyloid-β oligo-mers level is a biomarker for Alzheimer’s disease diagnosis. Biochem Biophys Res Commun. 2012; 423(4): 697-702.Hatashita S, Yamasaki H. Diagnosed mild cognitive impairment due to Alzheimer’s disease with PET biomarkers of beta amyloid and neuronal dysfunction. Plos One. 2013; 8(6): e66877.Babic’ LekoM, Borovecki F, Dejanovic’ N, et al. Predictive value of cerebrospinal fluid Visinin-like protein-1 levels for Alzheimer's disease early detection and differential diagnosis in patients with mild cognitive impairment. J Alzheimers Dis. 2016; 50(3): 765-778.

[0006] As mentioned above, since MCI is a reversible condition, it is believed that dementia can be effectively prevented if early intervention (appropriate treatment, etc.) is possible based on the results of MCI screening. To realize MCI screening, it is necessary to develop an MCI testing technology that can be performed non-invasively and easily. The object of this invention is to provide an MCI testing technology that can be performed non-invasively and easily.

[0007] In their investigation to solve the aforementioned problems, the present inventors focused on skin blotting. Skin blotting is a method of extracting biomolecules contained in the skin and interstitial fluid by applying a skin blotting membrane to the skin surface and fixing them to the membrane (Non-Patent Literature 11). Here, the interstitial fluid is derived from plasma and also contains biomolecules from the blood (Non-Patent Literature 12).

[0008] As a result of diligent research, the inventors have found that MCI testing can be easily performed by measuring the amount of specific amyloid oligomers and albumin in a sample collected by skin blotting (skin blotting sample), and using the ratio of these amounts calculated from that measurement as an indicator (marker).

[0009] Embodiments such as the following are provided: (I) Method for obtaining Aβ oligomers by skin blotting (I-1) A method for obtaining at least one selected from the group consisting of 39 kDa oligomers, 37 kDa oligomers, and 36 kDa oligomers of amyloid β by performing skin blotting on a subject.

[0010] (II) Mild Cognitive Impairment Detection Markers (MCI Detection Markers) (II-1) MCI detection markers comprising at least one selected from the group consisting of Aβ-(39+37+36) / ALB, Aβ-(39+37) / ALB, and Aβ-(37+36) / ALB; where, Aβ is amyloid β, ALB is albumin, Aβ-(39+37+36) / ALB is the ratio of the total amount of amyloid β 39 kDa oligomer (hereinafter, "Aβ-39"), 37 kDa oligomer (hereinafter, "Aβ-37"), and 36 kDa oligomer (hereinafter, "Aβ-36") to the amount of albumin, Aβ-(39+37) / ALB is the ratio of the total amount of Aβ-39 and Aβ-37 to the amount of albumin, and Aβ-(37+36) / ALB represents the ratio of the total amount of Aβ-37 and Aβ-36 to the amount of albumin, respectively. The meanings of these terms are the same in (III) to (V) below.

[0011] (III) Kit for detecting mild cognitive impairment (MCI detection kit) (III-1) A kit for detecting MCI, comprising a skin blotting membrane, a substance that binds to ALB, and a substance that binds to Aβ. (III-2) The MCI detection kit according to (III-1), wherein the substance that binds to ALB is an antibody against ALB or an antigen-binding fragment thereof, and the substance that binds to Aβ is an antibody against Aβ or an antigen-binding fragment thereof.

[0012] (IV) Method for using a mild cognitive impairment detection marker (MCI detection marker) (IV-1) A method for using an MCI detection marker to assist in the diagnosis of MCI in a subject, wherein the MCI detection marker is at least one selected from the group consisting of Aβ-(39+37+36) / ALB, Aβ-(39+37) / ALB, and Aβ-(37+36) / ALB obtained from a skin blotting sample of the subject.

[0013] (V) Method for obtaining indicators to assist in the diagnosis of mild cognitive impairment (method for obtaining indicators to assist in the diagnosis of MCI) (V-1) A method for obtaining indicators to assist in the diagnosis of MCI in a subject, comprising the steps of (1) and (2) below: (1) A step of measuring at least one selected from the group consisting of Aβ-39, Aβ-37, and Aβ-36, and ALB from a skin blotting sample of the subject; (2) A step of calculating at least one numerical value selected from the group consisting of Aβ-(39+37+36) / ALB, Aβ-(39+37) / ALB, and Aβ-(37+36) / ALB from the measured values ​​obtained in step (1). (V-2) The method described in (V-1) further comprises the following steps: (3) A step of comparing the numerical values ​​of the subjects obtained in step (2) with the cutoff value set by ROC analysis and grouping the subjects according to the following criteria: (a) If the numerical value of a subject is less than the cutoff value, the subject does not qualify as MCI or is highly unlikely to qualify (non-MCI group); (b) If the numerical value of a subject is equal to or greater than the cutoff value, the subject qualifies as MCI or is highly likely to qualify (MCI group). This method is also a method of obtaining and providing an index to assist in the diagnosis of MCI in subjects. Furthermore, this method is also a method of assisting in the diagnosis of MCI in subjects. Furthermore, this method is also a method of screening (selecting) subjects into the non-MCI group and the MCI group. This method is performed without the judgment or intervention of a physician and therefore constitutes a non-medical act. Subjects classified as the "MCI group" by this method can obtain a definitive diagnosis by further examination by a physician.

[0014] This disclosure provides a non-invasive and easy-to-implement MCI testing technology. As one embodiment of the MCI testing technology, a marker (MCI detection marker) used to detect MCI using a skin blotting sample is provided. As another embodiment of the MCI testing technology, a kit (MCI detection kit) suitably used to detect MCI using a skin blotting sample is provided. Furthermore, as yet another embodiment of the MCI testing technology, a method for detecting MCI using a subject's skin blotting sample is provided. And yet another embodiment of the MCI testing technology, a method for assisting MCI determination using a subject's skin blotting sample is provided.

[0015] In the example, (a) the measurement site (skin patch application site) where skin blotting was performed is shown. (b) The structure of the skin patch used for skin blotting is shown. In the example, (a) the staining image of Aβ (Amyloid β) oligomers and (b) the staining image of ALB (Albumin), which is the internal standard, are shown, measured by Western blotting on samples taken from the MCI group by skin blotting. The following three indicators show the intergroup comparison between the MCI group and the normal group: a) Aβ-(39+37+36) / ALB: ratio of the total amount of Aβ oligomers of 39, 37, and 36 kDa to the amount of ALB, b) Aβ-(39+37) / ALB: ratio of the total amount of Aβ oligomers of 39 and 37 kDa to the amount of ALB, c) Aβ-(37+36) / ALB: ratio of the total amount of Aβ oligomers of 37 and 36 kDa to the amount of ALB. The accuracy of MCI identification based on the three indicators above is shown.

[0016] Explanation of Terms: The following definitions of terms used in this specification are provided below. Unless otherwise specified, the following definitions of terms apply throughout this specification.

[0017] "MCI" is an abbreviation for mild cognitive impairment. As mentioned above, MCI is a condition in which forgetfulness is the main symptom, but it has little impact on daily life and cannot be diagnosed as dementia. Specifically, it is a condition in which (1) there is memory impairment that cannot be explained solely by age or education level, (2) the person or their family complains of forgetfulness, (3) overall cognitive function is within the normal range, (4) daily living activities are performed independently, and (5) it is not dementia. Three methods are known for testing MCI (interview method [MoCA (including MoCA-J)], imaging diagnostics, and biochemical tests using cerebrospinal fluid or blood), and this disclosure includes MCI determined by any of these methods.

[0018] A "subject" is a person who is to be tested. This person can be of any age or gender. Subjects include individuals with MCI (MCI patients), individuals suspected of having MCI based on their symptoms (MCI suspected individuals), and individuals who are neither MCI patients nor MCI suspected individuals (healthy individuals).

[0019] Skin blotting is a technique that non-invasively removes and fixes components from skin tissue by applying a membrane to the skin surface. The membrane used for this purpose is called a "blotting membrane," and the sample collected by skin blotting is called a "skin blotting sample." For example, by applying a blotting membrane moistened with an aqueous medium to the skin surface for about 10 minutes, components of the interstitial fluid (proteins, etc.) can be non-invasively extracted and fixed onto the blotting membrane.

[0020] A "skin blotting membrane" (hereinafter also simply referred to as "blotting membrane") is a membrane used for skin blotting. The membrane is composed of a water-insoluble substrate and can adsorb target proteins and standard proteins when moistened with an aqueous medium. Although not limited, examples of fiber materials include cellulose esters such as nitrocellulose, nylon, polyvinylidene fluoride (PVDF), and cellulose acetate, polyesters such as polyethylene terephthalate, and polyolefin resins such as polyethylene, polypropylene, and polystyrene. Blotting membranes composed of these materials can be used with or without charge, but it is preferable that they have polar functional groups on their surface so that they can generate an attractive force based on electrostatic interaction with the polarity of the target protein and standard protein. If necessary, cationic functional groups (e.g., primary amino groups, secondary amino groups, tertiary amino groups, quaternary ammonium groups, quaternary phosphonium groups, etc.) and / or anionic functional groups (e.g., hydroxyl groups, carboxyl groups, sulfo groups, sulfate groups, phosphate groups, silanol groups, etc.) may be introduced into the substrate. Examples of blotting membranes having polar functional groups on their surface include nitrocellulose membranes and nylon membranes. Commercially available nitrocellulose membranes and nylon membranes used for blotting proteins, nucleic acids, etc., can be used.

[0021] The pore size of the blotting membrane is not limited, but examples include 0.1 μm to 0.5 μm, preferably 0.2 μm to 0.45 μm. The blotting membrane is not limited, but for ease of use, it may be pre-cut into the shape to be used. Examples of cut shapes include rectangles and circles. In the case of a rectangle, the size can be appropriately selected from a range of 5 mm to 20 mm in both length and width.

[0022] Examples of the "aqueous medium" used to moisten the blotting membrane include water, physiological saline, and phosphate-buffered saline. Preferably, it is physiological saline or phosphate-buffered saline. Its pH is preferably adjusted to a range of pH 4.5 to 8.0, and more preferably to a range of pH 6.5 to 7.5. The blotting membrane may be stored in an airtight bag or container after being pre-soaked in such an "aqueous medium." It may also be sterilized.

[0023] "Amyloid-beta" or "Aβ" is human-derived amyloid-beta. Aβ is a protein of approximately 37 to 43 amino acids that was identified as a major component of cerebrovascular amyloid angiopathy and senile plaques that accumulate in the brains of Alzheimer's disease patients through biochemical analysis. Aβ is cleaved from the precursor protein, Amyloid-beta precursor protein (APP), by a two-step cleavage by β-secretase and γ-secretase and secreted outside the cell. It then undergoes degradation through various pathways outside the cell. In other words, Aβ is a degradation fragment of APP, and multiple molecular species exist depending on the number of amino acids. For example, the main molecular species of Aβ known to be Aβ40, which ends in the 40th amino acid (Val), and Aβ42, which ends in the 42nd amino acid (Ala), are known. "Aβ" is a general term for the degradation fragments of APP, consisting of various molecular species.

[0024] The "target protein" is an oligomer of Aβ. This Aβ oligomer includes oligomers with a molecular weight of approximately 36 kDa (36 kDa oligomer; also referred to herein as "Aβ-36"), oligomers with a molecular weight of approximately 37 kDa (37 kDa oligomer; also referred to herein as "Aβ-37"), and oligomers with a molecular weight of approximately 39 kDa (39 kDa oligomer; also referred to herein as "Aβ-39"). Since the molecular weight of Aβ is approximately 4 kDa, these Aβ oligomers can be octamers to decamers of the aforementioned Aβ. The molecular weight of these Aβ oligomers can be determined by separation by electrophoresis using a gradient gel (polyacrylamide gel) with a concentration gradient in the range of 4 to 20%, and by immunostaining, as shown in the examples described later.

[0025] These Aβ oligomers are proteins collected from the subject's skin by skin blotting, as shown in the examples described later, and can be analyzed and identified using fractionation methods that utilize differences in molecular weight and immunostaining methods that use anti-Aβ antibodies. Any fractionation method that can separate Aβ oligomers based on differences in molecular weight of at least 36 kDa, 37 kDa, and 39 kDa is acceptable, and examples include polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography.

[0026] Any antibody capable of recognizing Aβ (including each molecular species) that constitutes the Aβ oligomer can be used as an "anti-Aβ antibody," and commercially available anti-Aβ antibodies can be used for convenience. Examples of commercially available anti-Aβ antibodies are not limited, but include, for example, β-Amyloid(D54D2)XP Rabbit mAb (manufactured by Cell Signaling Technology, Inc.) ("XP" is a registered trademark of CST). This anti-Aβ antibody is a rabbit monoclonal antibody (IgG) that has been confirmed to specifically recognize and bind to the human Aβ molecular species Aβ-37, Aβ-38, Aβ-39, Aβ-40, and Aβ-42. Another example of a commercially available anti-Aβ antibody is anti-amyloid β antibodies (BAN50, BNT77, BA27, BC05) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). These anti-Aβ antibodies are mouse monoclonal antibodies that have been confirmed to specifically recognize and bind to human Aβ molecular isoforms Aβ-40, β-42, and / or β-43. Of these, BAN50 is a human Aβ N-terminal specific antibody, and BNT77 is an Aβ central portion (11-28a.a.) antibody; both are reactive with Aβ-40, β-42, and β-43. BA27 is an Aβ40 C-terminal specific antibody and is reactive with Aβ-40, and BC05 is an Aβ42 / 43 C-terminal specific antibody and is reactive with Aβ-42 and Aβ-43. Note that these commercially available anti-Aβ antibodies are just examples and are not limited to these.

[0027] The anti-Aβ antibody may be an unlabeled antibody, or it may be a labeled antibody labeled with any labeling agent (e.g., fluorescent dyes such as fluorescein isothiocyanate (FITC), Alexa, Fluor® dye, Cys dye; or labeling enzymes such as alkaline phosphatase (AP, ALP) or peroxidase (HRP, POD)). If the anti-Aβ antibody is an unlabeled antibody, immunohistochemistry can be performed using a secondary antibody labeled with a fluorescent substance or enzyme along with the anti-Aβ antibody.

[0028] The "standard protein" is human albumin (ALB). As shown in the examples described later, albumin can be collected from the subject's skin by skin blotting and specifically analyzed using an anti-ALB antibody. The anti-ALB antibody can be any antibody that can specifically recognize and bind to human albumin, and commercially available anti-ALB antibodies can be used for convenience. For example, the anti-ALB antibody used in the examples (Anti-Albumin Antibody: Bethyl Laboratories, Inc.) is a goat-derived polyclonal antibody that has been confirmed to specifically recognize and bind to human albumin. However, the commercially available anti-ALB antibody is just one example and is not limited to this. The anti-ALB antibody may be an unlabeled antibody, but for the sake of simplicity in immunohistochemistry, it is preferable to use a labeled antibody labeled with any of the above-mentioned labeling agents.

[0029] An "MCI detection marker" or "marker" is used to detect whether a subject belongs to the MCI group or the non-MCI group, using a skin blotting sample from the subject as the test sample. "MCI detection" means detecting whether a subject belongs to the MCI group or the non-MCI group. Examples of MCI detection markers include "Aβ-(39+37+36) / ALB", "Aβ-(39+37) / ALB", and "Aβ-(37+36) / ALB".

[0030] In the formula "Aβ-(39+37+36) / ALB" (hereinafter also referred to as "marker 1"), "Aβ-(39+37+36)" represents the total amount of Aβ-39, Aβ-37, and Aβ-36 in the test sample. Furthermore, "ALB" represents the amount of albumin in the test sample. In other words, marker 1 represents the ratio (quantity ratio) of the total amount of Aβ-39, Aβ-37, and Aβ-36 to the total amount of albumin contained in the test sample.

[0031] In "Aβ-(39+37) / ALB" (hereinafter also referred to as "marker 2"), "Aβ-(39+37)" represents the total amount of Aβ-39 and Aβ-37 in the test sample. "ALB" represents the amount of albumin in the test sample. In other words, marker 2 represents the ratio (amount ratio) of the total amount of Aβ-39 and Aβ-37 to the amount of albumin contained in the test sample.

[0032] In "Aβ-(37+36) / ALB" (hereinafter also referred to as "marker 3"), "Aβ-(37+36)" represents the total amount of Aβ-37 and Aβ-36 in the test sample. "ALB" represents the amount of albumin in the test sample. In other words, marker 3 represents the ratio (amount ratio) of the total amount of Aβ-37 and Aβ-36 to the amount of albumin contained in the test sample.

[0033] "Aβ-(39+37+36)", "Aβ-(39+37)", "Aβ-(37+36)", and "ALB" each represent the amount of protein contained in the test sample, and the "amount ratio" refers to the ratio of each protein amount. However, since the amount ratio is a relative ratio, it can also be calculated using another value that reflects the amount of protein. Specifically, the intensity of the bands detected by Western blotting can be read using an image analysis tool such as ImageJ, converted to protein amounts, and then the amount ratio can be determined, or the amount ratio can be determined based on the intensity of the bands without converting to protein amounts.

[0034] The "MCI group" includes subjects who have MCI (MCI patients) and subjects who may have MCI. The definitive determination of whether or not a subject is an MCI patient (non-MCI patient) is made by a physician. Subjects who may have MCI include subjects who are suspected of having MCI but are not definitively diagnosed as MCI patients (MCI suspected positives). In other words, the "MCI group" includes both MCI patients and MCI suspected positives.

[0035] On the other hand, the "non-MCI group" includes subjects who do not have MCI (non-MCI patients) and subjects who may not have MCI. Subjects who may not have MCI include subjects who are not suspected of having MCI but are confirmed to have MCI (MCI false negatives). In other words, the "non-MCI group" includes both non-MCI patients and MCI false negatives.

[0036] "Diagnosis of MCI" includes a definitive determination made by a physician based on the subject's condition and information, using methods such as interviews, imaging diagnostics, and biochemical tests. An MCI patient is a patient who has been determined to have MCI by a physician. On the other hand, a non-MCI patient includes not only healthy individuals who do not have symptoms of MCI, but also individuals who have symptoms of MCI but have been determined by a physician not to be MCI patients. "Assistance in the diagnosis of MCI" includes actions that assist in the aforementioned definitive determination, and this includes actions that acquire information that may be useful in diagnosing MCI, and actions that provide such information to physicians, etc. Such actions are considered non-medical acts.

[0037] (I) Method for obtaining Aβ oligomers by skin blotting By performing skin blotting, at least one oligomer selected from the group consisting of 39 kDa oligomers, 37 kDa oligomers, and 36 kDa oligomers of Aβ, as well as albumin (ALB), can be obtained from the skin of a subject. The skin blotting, subject, Aβ, 39 kDa oligomer, 37 kDa oligomer, and 36 kDa oligomer are as described above and in the examples. These Aβ oligomers are useful as "target proteins" used for MCI detection, as described later. Specifically, as will be described later, the relative ratio of the amount of Aβ oligomers in a sample collected by skin blotting to the amount of albumin (ALB) (standard protein) in the same sample ("Aβ-(39+37+36) / ALB", "Aβ-(39+37) / ALB", and "Aβ-(37+36) / ALB") can be used as a marker to detect whether a subject belongs to the MCI group or the non-MCI group.

[0038] (II) MCI detection markers and methods of use thereof As "MCI detection markers," "Aβ-(39+37+36) / ALB," "Aβ-(39+37) / ALB," and "Aβ-(37+36) / ALB" are provided. "Aβ-(39+37+36) / ALB" (marker 1) can be determined by measuring Aβ-39, Aβ-37, Aβ-36, and ALB contained in a subject's skin blotting sample, and dividing the total amount of Aβ-39, Aβ-37, and Aβ-36 by the amount of ALB. "Aβ-(39+37) / ALB" (marker 2) can be determined by measuring Aβ-39, Aβ-37, and ALB contained in a subject's skin blotting sample, and dividing the total amount of Aβ-39 and Aβ-37 by the amount of ALB. "Aβ-(37+36) / ALB" (marker 3) can be determined by measuring Aβ-37, Aβ-36, and ALB contained in the subject's skin blotting sample, and dividing the total amount of Aβ-37 and Aβ-36 by the amount of ALB.

[0039] The methods for Aβ-39, Aβ-37, Aβ-36, and ALB can be any method capable of measuring the amount of these proteins, for example, immunological assays using antibodies that recognize and bind to these proteins. Examples of conventionally known immunological assays include immunoassays (RIA: Radio Immunoassay, EIA: Enzyme Immunoassay, FIA: Fluoro Immunoassay, CLIA: Chemiluminescent Immunoassay, CLEIA: Chemiluminescent Enzyme Immunoassay), dot blotting (including Western blotting), latix agglutination (including ELISA), and immunochromatography. These immunological assays can be appropriately selected and used depending on the specificity of the antibody used.

[0040] For example, using specific antibodies that specifically recognize and bind to Aβ-39, Aβ-37, Aβ-36, and ALB, respectively, allows for the simultaneous and convenient measurement of the amounts of Aβ-39, Aβ-37, Aβ-36, and ALB in a skin blotting sample using immunochromatography.

[0041] Furthermore, when antibodies against Aβ (monomer) (anti-Aβ antibodies) are used instead of specific antibodies against Aβ-39, Aβ-37, and Aβ-36, Western blotting is a suitable example. In this case, the skin blotting sample is subjected to electrophoresis using a polyacrylamide gel to separate each protein based on the difference in molecular weight. After this separation, the sample is transferred to a hydrophobic membrane, and each protein is measured using anti-Aβ antibody and anti-ALB antibody as primary antibodies. Specifically, the primary antibodies (anti-Aβ antibody, anti-ALB antibody) are reacted with the membrane, and after washing, a secondary antibody labeled with an arbitrary labeling agent is reacted with the membrane. By utilizing the activity corresponding to the labeling agent, each protein can be detected and quantified by chemiluminescence or colorimetric method. By using an antibody labeled with an arbitrary labeling agent as the primary antibody, the use of a secondary antibody can be omitted.

[0042] Furthermore, when using anti-Aβ antibodies as antibodies against Aβ-39, Aβ-37, and Aβ-36, the skin blotting sample can be subjected to a fractionation method such as gel filtration chromatography to separate Aβ-39, Aβ-37, and Aβ-36 based on their molecular weight, and then subjected to the aforementioned immunological assay method (excluding Western blotting) to obtain the respective protein amounts.

[0043] The antibodies used in immunological assays (anti-Aβ antibodies, anti-ALB antibodies) can be any antibodies capable of detecting Aβ-39, Aβ-37, Aβ-36, and ALB, and to that extent, the type of antibody (monoclonal or polyclonal) is not considered. Antigen-binding fragments of monoclonal antibodies such as Fab, F(ab'), F(ab')2, and Fv can also be used.

[0044] These MCI detection markers (Marker 1, Marker 2, and Marker 3) are all used to detect whether a subject belongs to the MCI group or the non-MCI group (MCI detection) using the subject's skin blotting sample as the test sample. Therefore, by using the MCI detection markers, it becomes possible to non-invasively and simply detect whether a subject belongs to the MCI group or the non-MCI group by the skin blotting method. For this purpose, the MCI detection markers (Marker 1, Marker 2, and Marker 3) may be used individually or in combination of two or more.

[0045] The MCI detection marker is used as a marker to detect whether a subject belongs to the MCI group or the non-MCI group (MCI detection) using the subject's skin blotting sample as the test sample. The result of this MCI detection can be used to assist in the diagnosis of MCI, which is carried out as necessary.

[0046] (III) Kit for MCI Detection The kit for MCI detection is a kit suitably used to detect MCI using a subject's skin blotting sample. As the kit for MCI detection, there is provided a kit containing at least a membrane for skin blotting, a substance that binds to ALB, and a substance that binds to Aβ as a set.

[0047] The substance that binds to ALB (ALB-binding substance) may be any substance that can specifically recognize and bind to human-derived ALB. Preferably, an antibody against ALB (anti-ALB antibody) can be exemplified. In this regard, it may be a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment. The anti-ALB antibody can be simply a commercially available one. The antibody may be an unlabeled antibody or a labeled antibody labeled with any labeling agent.

[0048] As the substance that binds to Aβ (Aβ-binding substance), any substance that can recognize and bind to human-derived Aβ may be used. Preferably, an antibody against Aβ (anti-Aβ antibody) can be exemplified. In this regard, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment may be used. As shown in the examples described later, an anti-Aβ antibody (a monoclonal antibody in the examples) can recognize and bind to various Aβ oligomers (Aβ-39, Aβ-37, Aβ-36). The antibody may be an unlabeled antibody or a labeled antibody labeled with an arbitrary labeling agent. The kit for detecting MCI may contain the ALB-binding substance and the Aβ-binding substance in a state held on a substrate.

[0049] In addition to the membrane for skin blotting, the substance that binds to ALB, and the substance that binds to Aβ, the kit for detecting MCI can contain an arbitrary substance. Examples of such substances include, but are not limited to, substances used to treat the membrane for skin blotting and recover proteins (e.g., stripping buffer solution, protein precipitant, etc.), substances used in electrophoresis (acrylamide electrophoresis gel (4-20%), protein denaturant (sodium dodecyl sulfate, etc.), gel buffer, electrophoresis buffer, membrane for transferring electrophoresis results, etc.), and materials used in immunological measurements (e.g., secondary antibody (which may be labeled), enzyme, chromogenic agent, detection reagent, buffer, washing solution, etc.). Furthermore, it may include a written description such as an instruction manual, a method for evaluating detection results, or a method for subsequent processing (operation).

[0050] The kit for detecting MCI can be preferably used to detect whether a subject belongs to the MCI group or the non-MCI group using the subject's skin blotting sample. By using the kit for detecting MCI, it is possible to easily obtain an index for assisting in the diagnosis of MCI, which will be described later.

[0051] (IV) Method for obtaining indicators to aid in MCI diagnosis The method for obtaining indicators to aid in MCI diagnosis comprises the following steps (1) and (2). Furthermore, it may include step (3). (1) A step of measuring at least one selected from the group consisting of Aβ-39, Aβ-37, and Aβ-36, as well as ALB, from a skin blotting sample of a subject (measurement step). (2) A step of calculating at least one selected from the group consisting of Aβ-(39+37+36) / ALB (marker 1), Aβ-(39+37) / ALB (marker 2), and Aβ-(37+36) / ALB (marker 3) from the measured values ​​obtained in step (1) (marker value calculation step). (3) A step in which the subjects are grouped according to the following criteria by comparing the subject values ​​obtained in step (2) with the cutoff values ​​set by ROC analysis: (a) If the subject's value is less than the cutoff value, the subject does not qualify as MCI or is highly unlikely to qualify (non-MCI group); (b) If the subject's value is equal to or greater than the cutoff value, the subject qualifies as MCI or is highly likely to qualify (MCI group).

[0052] The following describes each step. (1) Measurement step In this measurement step, a skin blotting sample collected from a subject by skin blotting is used as the test sample, and at least one selected from the group consisting of Aβ-39, Aβ-37, and Aβ-36, as well as ALB, are measured. The measurement method can be the immunological measurement method described above, and if necessary, this can be combined with a fractionation method based on molecular weight.

[0053] (2) Step for calculating marker values ​​In this step, at least one marker value (test value) is calculated from the measured values ​​obtained in step (1) above, selected from the group consisting of marker 1, marker 2, and marker 3, according to the measured values. Marker 1 can be calculated by dividing the sum of the measured values ​​of Aβ-39, Aβ-37, and Aβ-36 by the measured value of ALB. Marker 2 can be calculated by dividing the sum of the measured values ​​of Aβ-39 and Aβ-37 by the measured value of ALB. Marker 3 can be calculated by dividing the sum of the measured values ​​of Aβ-37 and Aβ-36 by the measured value of ALB.

[0054] (3) Grouping Process In this process, the subject's numerical value (test value) for each MCI detection marker (markers 1, 2, and 3) calculated from the skin blotting sample is compared with a pre-set cutoff value corresponding to each MCI detection marker, and the subject is grouped according to the following criteria: (a) If the test value is less than the cutoff value, the subject does not have MCI or is highly unlikely to have MCI (non-MCI group). (b) If the test value is equal to or greater than the cutoff value, the subject has MCI or is highly likely to have MCI (MCI group).

[0055] The cutoff value can be set by analysis using the ROC (Receiver Operating Characteristic) curve (ROC analysis). The ROC curve is created by measuring Aβ-39, Aβ-37, Aβ-36, and ALB in skin blotting samples from MCI patients and skin blotting samples from non-MCI patients, calculating the sensitivity and specificity of each MCI detection marker (markers 1, 2, and 3) from the measured values, and plotting these values ​​on a coordinate system with specificity (1 - specificity) on the horizontal axis and sensitivity on the vertical axis.

[0056] When setting a cutoff value using an ROC curve, it can be determined by balancing sensitivity and specificity. For example, the cutoff value could be the value of the point that is the shortest distance from the upper left corner of the ROC curve, or it could be the value of the point furthest from the shaded line (a straight line drawn from the lower left corner to the upper right corner of the figure; for example, the dotted line in each figure of Figure 5) where the Area Under the Curve (AUC) is 0.5000 (Youden's index).

[0057] While not limited, the cutoff value for marker 1 can be set in the range of 0.35 to 0.53. Preferably, it can be set in the range of 0.45 to 0.51, and more preferably in the range of 0.475 to 0.485. The cutoff value for marker 2 can be set in the range of 0.26 to 0.39. Preferably, it can be set in the range of 0.27 to 0.33, and more preferably in the range of 0.295 to 0.310. Furthermore, the cutoff value for marker 3 can be set in the range of 0.34 to 0.38. Preferably, it can be set in the range of 0.325 to 0.37, and more preferably in the range of 0.350 to 0.360.

[0058] The cutoff value is a numerical value set based on the ROC curve created according to the measurement method adopted. Therefore, when selecting the cutoff value, it is desirable to create the ROC curve in advance according to the measurement method adopted.

[0059] Depending on the type of MCI detection marker, if the test value is less than the aforementioned cutoff value, the subject can be determined to belong to the non-MCI group. On the other hand, if the test value is equal to or greater than the cutoff value, the subject can be determined to belong to the MCI group.

[0060] For example, in the measurement method used in the embodiment described later, setting the cutoff value of marker 1 to 0.47991 yields a sensitivity of 88.9%, specificity of 81.8%, and AUC of 80.8%; setting the cutoff value of marker 2 to 0.30282 yields a sensitivity of 88.9%, specificity of 72.7%, and AUC of 78.8%; and setting the cutoff value of marker 3 to 0.3555 yields a sensitivity of 88.9%, specificity of 81.8%, and AUC of 84.8%. In all cases, the attributes of the subject's MCI can be determined with high accuracy.

[0061] The cutoff values ​​for each marker are not limited to those listed above, and can be appropriately selected and set from the created ROC curve according to the desired accuracy. However, setting the cutoff value too high tends to increase the probability that MCI patients are included in the non-MCI group (increasing false negatives), and conversely, setting the cutoff value too low tends to increase the probability that non-MCI patients are included in the MCI group (increasing false positives).

[0062] The results obtained in the classification process can be used as an indicator to assist in the diagnosis of MCI in subjects. In other words, it becomes possible to diagnose MCI in subjects using these results as an auxiliary indicator (reference information). Furthermore, this selection process makes it possible to screen (select) subjects into the non-MCI group and the MCI group. With this method, subjects classified as the "MCI group" (and also subjects classified as the "non-MCI group") can obtain a definitive diagnosis by undergoing a medical examination.

[0063] In this specification, the terms “contains” and “includes” include the meanings of “consisting of” and “substantially consisting of.”

[0064] The present invention will be described below using experimental examples to aid in understanding its structure and effects. However, the present invention is not limited in any way by these experimental examples. The following experiments were conducted at room temperature (25 ± 5°C) and atmospheric pressure unless otherwise specified. Unless otherwise specified, "%" below means "mass percent" and "parts" means "parts by mass".

[0065] The following experiment was conducted with the approval of the Ishikawa Prefectural College of Nursing Ethics Committee (2023-147). (A) Experimental Method 1. Participants This experiment included all participants in the follow-up survey of the eSports project. The eSports project was conducted from July to September 2023, based on a comprehensive cooperation agreement between Ishikawa Prefectural College of Nursing and Kahoku City, and was primarily led by the Department of Community Nursing. This project aimed to verify the dementia and frailty prevention effects of eSports and targeted elderly people aged 65 and over who were able to attend the research facility. However, those with acute illnesses, malignant tumors, infectious diseases, heart problems, body temperature of 38°C or higher, anemia, and skin sensory impairment or skin abnormalities were excluded. Participants experienced eSports four times during an intervention period of approximately one month after the baseline survey, and then participated in the follow-up survey. This project is a before-and-after comparative study in which cognitive function and electroencephalograms are measured in the baseline survey and follow-up survey.

[0066] 2. As basic attributes of the survey items, age and sex were collected through interviews. MoCA-J was measured to determine MCI, and amyloid β (Aβ) was measured as a candidate MCI marker and albumin (ALB) as an internal standard by skin blotting. Aβ is the most commonly used MCI marker in blood tests (Non-Patent Literature 10). It is known that Aβ is degraded from amyloid precursor protein by β- and γ-secretase to become Aβ oligomers, which cause neurotoxicity (Non-Patent Literature 12).

[0067] 3. Assessment of Mild Cognitive Impairment In the MoCA-J, a score of 25 or less is classified as MCI (Non-Patent Literature 6). In this experiment, one researcher met with each participant individually in a private room, asked questions verbally while showing them a questionnaire, and the participant filled out the questionnaire with their answers.

[0068] 4. Skin Blotting Skin blotting was performed using a modified protocol from a previously reported study (Non-Patent Literature 11). The measurement site was the medial side of the forearm (midpoint between the medial epicondyle and the styloid process) (see Figure 1a). If the measurement site was moist due to sweating, it was gently wiped with a dry paper towel before skin blotting. For skin blotting, a skin patch was used, consisting of two 10 mm square nitrocellulose membranes (Bio-Rad) made by layering 8 mm square filter paper, fixed with medical tape (Nitoms), and with cellophane tape (35 x 20 mm) applied to the filter paper side as a topcoat (see Figure 1b). One drop of saline solution was added to each nitrocellulose membrane to soak the membrane and filter paper, and after removing excess saline solution, the patch was applied to the measurement site on the skin with the nitrocellulose membrane side in contact and fixed for 10 minutes. The skin patches were then peeled off the skin and collected, and stored at 4°C until protein could be recovered.

[0069] 5. Protein Recovery and Analysis Two sets of nitrocellulose membranes were removed from the skin patches and each was immersed in 1 mL of exfoliation buffer solution [150 mM NaCl, 0.3% Triton X-100 in 10 mM phosphate buffer] and vigorously stirred for 10 seconds. The nitrocellulose membranes were then removed and the protein was recovered from the remaining solution by trichloroacetic acid precipitation (Non-Patent Literature 13). The pellet (precipitated recovered protein) was dried by evaporating the acetone and dissolved in 15 μL of sample buffer [Laemmli sample buffer (Bio-Rad) with phosphatase inhibitor cocktail (Nacalai Tesque) and 2-Mercaptoethanol (Bio-Rad) added, final SDS concentration 2%] to be used as the test sample.

[0070] Protein analysis was performed by Western blotting. Specifically, the test samples were subjected to 4-20% acrylamide gel electrophoresis (200V, 30 minutes) to separate the proteins, which were then transferred to a polyvinylidene difluoride membrane (Bio-Rad) by semi-dry blotting. Protein Ladder One Plus, Triple-color for SDS-PAGE (Nacalai Tesque) was used as the molecular weight marker. Subsequently, the target protein (Aβ) and internal standard protein (ALB) were measured by immunohistochemical double staining.

[0071] For the detection of the target protein, an anti-β-Amyloid antibody (β-Amyloid (D54D2) XP Rabbit mAb, Cell Signaling Technology [CST], 1:4000 dilution) was used as the primary antibody. In addition, a peroxidase-labeled anti-rabbit IgG antibody (Peroxidase AffiniPure F(ab')2 fragment Donkey anti-rabbit IgG (H+L) (min X Bov, Ck, Gt, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot), Jackson Immuno Research, 1:1000 dilution) and a peroxidase chemiluminescent substrate (Merck) were used as secondary antibodies, and imaging was performed using a chemiluminescence imaging system (liponics).

[0072] Next, the gel was quenched by immersion in a 15% hydrogen peroxide solution for 30 minutes, and then detected and recorded using alkaline phosphatase-labeled anti-albumin antibody (Goat anti-Human Albumin Antibody IgG Fraction, Bethyl Laboratories, 1:500 dilution) and alkaline phosphatase luminescent substrate (SurModics).

[0073] All reactions and washes in the immunohistochemical staining were performed using a vacuum-assisted immunohistochemical staining apparatus (SNAP id 2, Merck).

[0074] The stained images of the target protein and internal standard protein bands, separated and detected by electrophoresis, were converted to Grace x Kale images using image analysis software (Image J, National Institute of Health), and the detection intensity of each band was measured. The molecular weight of each band was analyzed using CS analyzer 4 (ATTO).

[0075] 6. Statistical Analysis SPSS Statistics (29.0.1.0) was used for statistical analysis. Participants were classified into the MCI group (those with a MoCA-J score of 25 or less) and the normal group (those with a score of 26 or more). Descriptive statistics were performed on the attributes of the participants and their MoCA-J scores for each group. For intergroup comparisons, normality was examined using the chi-square test for categorical variables and the Shapiro-Wilk test for continuous variables, after which independent t-tests or Mann-Whitney U tests were used, with a significance level of 0.05.

[0076] Multiple indices were established based on the detection intensity of each band obtained by Western blotting (see Table 2). The correlation with the MoCA-J score was analyzed using Spearman's rank correlation coefficient, and for indices with a significance probability of less than 0.1, the association with MCI was analyzed using binary logistic regression analysis with forced input of age and sex. After comparing the groups of indices that showed a significant association (p<0.05) using the Mann-Whitney U test, receiver operating characteristic (ROC) analysis was performed, and the identification accuracy was indicated using the area under the curve (AUC). Furthermore, the value that maximized the sum of sensitivity and specificity was set as the cutoff value.

[0077] (B) Experimental Results 1. Overview of Participants and MCI In the eSports project, 20 people participated in the follow-up survey. In this experiment, all 20 people were included in the analysis. Of these, 9 were in the MCI group and the rest were in the normal group. Table 1 shows an overview of the participants in both groups.

[0078]

[0079] As shown in Table 1, there were no significant differences in age or sex between the MCI group and the normal group. On the other hand, there was a significant difference in the median (interquartile range) of the MoCA-J score between the two groups (p<0.001).

[0080] 2. MCI Markers Figure 2 shows the staining images of Aβ (Figure 2(a)) and the staining image of ALB (Figure 2(b)), an internal standard, measured by Western blotting on samples collected by skin blotting from the MCI group and the normal group (10 randomly selected individuals out of a total of 20). ALB was detected around 38 kDa in all samples. Three distinct bands were detected for Aβ, and their molecular weights were estimated to be 39 kDa, 37 kDa, and 36 kDa, respectively (Figure 2(a)).

[0081] Aβ is a 37-42 residue peptide obtained by cleaving amyloid-beta precursor protein with β- and γ-secretase (Non-Patent Literature 14). Aβ forms various soluble oligomers and has been reported to be involved in the development of Alzheimer's disease (Non-Patent Literature 15 and 16). Adolfsson et al. created a neutralizing antibody for Aβ and verified its binding ability by Western blotting, showing that it can recognize monomers from approximately 4 kDa to high molecular weight Aβ oligomers up to approximately 70 kDa (Non-Patent Literature 17). In the stained Western blotting image shown in this paper, three bands can be observed in the 30-40 kDa range, similar to the present experiment. From this, it is considered that the Aβ molecular species shown by the three bands obtained from the skin blotting sample in the above experiment are 8-10 mers of Aβ.

[0082] High molecular weight Aβ oligomers are generally considered to have lower neurotoxicity than low molecular weight Aβ oligomers, but Yang et al. have reported that factors that destabilize high molecular weight Aβ oligomers are exacerbating factors of Alzheimer's disease (Non-Patent Literature 18). Furthermore, it has been shown that the concentration of high molecular weight Aβ oligomers in the cerebrospinal fluid and plasma of Alzheimer's disease patients is high (Non-Patent Literature 19, 20).

[0083] Based on the above, it is considered that the Aβ oligomer detected from the skin blotting samples in this experiment is related to MCI and therefore can serve as an indicator for MCI identification.

[0084] Therefore, we analyzed the correlation between the MoCA-J score and the indices shown in Table 2, using the detection intensity of the three Aβ bands and the ALB band (reflecting the respective protein concentrations).

[0085]

[0086] As a result, the significance probabilities for Aβ-37 / Aβ-(39+37+36), Aβ-(39+37+36) / ALB, Aβ(39+37) / ALB, and Aβ-(37+36) / ALB were less than 0.1. When these were analyzed using binomial logistic regression analysis adjusted for age and sex, significant associations were found between the MoCA-J score and the three indicators Aβ-(39+37+36) / ALB, Aβ-(39+37) / ALB, and Aβ-(37+36) / ALB, as shown in Table 3.

[0087]

[0088] Figure 3 shows the results of comparing these three indicators between the MCI group and the normal group. As shown in Figure 3, significant differences were observed in all of them (Aβ-(39+37+36) / ALB: p=0.020, Aβ(39+37) / ALB: p=0.031, Aβ-(37+36) / ALB: p=0.007).

[0089] Figure 4 shows the results of ROC analysis to determine the MCI identification accuracy for each indicator. The AUC, cutoff value, and the sensitivity and specificity at those values ​​are shown in Table 4, and all of them demonstrated high accuracy.

[0090]

[0091] Fujiwara et al. reported that the accuracy of MoCA-J for MCI detection was AUC 0.95, sensitivity 0.93, and specificity 0.89 (Non-Patent Literature 6). Hatashita et al. reported that the accuracy of PET scans observing Aβ aggregation and neurodegeneration for identifying MCI was sensitivity 96.6% and specificity 42.1% (Non-Patent Literature 21). According to a report by Babic' Leko et al., the accuracy of cerebrospinal fluid (CSF) examination for identifying MCI was sensitivity 0.77 and specificity 1.0 (Non-Patent Literature 22), and Planche et al. reported that the accuracy of blood tests for identification was AUC 0.74 (Non-Patent Literature 10).

[0092] While the accuracy of Aβ testing using skin blotting samples for identifying MCI is not as high as that of MoCA-J, it is not inferior to PET scans, cerebrospinal fluid tests, and blood tests. Furthermore, because it is a simple and non-invasive test, it is a suitable method for screening.

[0093] The results suggest that MCI can be easily screened by measuring 39kDa isoforms of Aβ as target proteins, and / or 39kDa isoforms, as well as ALB as a standard protein, using skin blotting samples from subjects, and evaluating the three indicators (markers) mentioned above.

[0094] Using skin blotting samples for MCI detection and screening, it is possible to detect MCI non-invasively and easily, and it is expected that early intervention will enable the prevention of dementia.

Claims

1. A method for obtaining at least one oligomer selected from the group consisting of 39 kDa oligomers, 37 kDa oligomers, and 36 kDa oligomers of amyloid-beta by performing skin blotting on a subject.

2. A mild cognitive impairment detection marker comprising at least one selected from the group consisting of Aβ-(39+37+36) / ALB, Aβ-(39+37) / ALB, and Aβ-(37+36) / ALB; where Aβ is amyloid-beta, ALB is albumin, Aβ-(39+37+36) / ALB is the ratio of the total amount of 39kDa oligomers, 37kDa oligomers, and 36kDa oligomers of amyloid-beta to the amount of albumin, Aβ-(39+37) / ALB is the ratio of the total amount of 39kDa oligomers, 37kDa oligomers, and 36kDa oligomers of amyloid-beta to the amount of albumin, and Aβ-(37+36) / ALB represents the ratio of the total amount of amyloid-beta 37kDa oligomers and 36kDa oligomers to the amount of albumin, respectively.

3. A mild cognitive impairment detection kit containing a skin blotting membrane, an albumin-binding substance, and an amyloid-beta-binding substance.

4. The mild cognitive impairment detection kit according to claim 3, wherein the substance that binds to albumin is an antibody against albumin or an antigen-binding fragment thereof, and the substance that binds to amyloid-beta is an antibody against amyloid-beta or an antigen-binding fragment thereof.

5. A method for using a mild cognitive impairment detection marker to assist in the diagnosis of mild cognitive impairment in a subject, wherein the mild cognitive impairment detection marker is at least one selected from the group consisting of Aβ-(39+37+36) / ALB, Aβ-(39+37) / ALB, and Aβ-(37+36) / ALB obtained from a skin blotting sample of the subject: where, Aβ is amyloid-beta, ALB is albumin, Aβ-(39+37+36) / ALB is the ratio of the total amount of 39kDa oligomers, 37kDa oligomers, and 36kDa oligomers of amyloid-beta to the amount of albumin, and Aβ-(37+36) / ALB represents the ratio of the total amount of amyloid-beta 37kDa oligomers and 36kDa oligomers to the amount of albumin, respectively.

6. A method for obtaining an index to assist in the diagnosis of mild cognitive impairment in a subject, comprising the following steps (1) and (2): (1) A step of measuring at least one selected from the group consisting of Aβ-39, Aβ-37, and Aβ-36, as well as ALB, from a skin blotting sample of the subject; (2) A step of calculating at least one selected from the group consisting of Aβ-(39+37+36) / ALB, Aβ-(39+37) / ALB, and Aβ-(37+36) / ALB from the measured values ​​obtained in step (1); where Aβ is amyloid-beta, ALB is albumin, Aβ-39, Aβ-37, and Aβ-36 are the 39kDa oligomer, 37kDa oligomer, and 36kDa oligomer of amyloid-beta, respectively. Aβ-(39+37+36) / ALB represents the ratio of the total amount of amyloid-beta 39kDa oligomers, 37kDa oligomers, and 36kDa oligomers to the amount of albumin, Aβ-(39+37) / ALB represents the ratio of the total amount of amyloid-beta 39kDa oligomers and 37kDa oligomers to the amount of albumin, and Aβ-(37+36) / ALB represents the ratio of the total amount of amyloid-beta 37kDa oligomers and 36kDa oligomers to the amount of albumin.

7. The method according to claim 6, further comprising the following steps: (3) a step of comparing the numerical values ​​of the subjects obtained in step (2) with the cutoff values ​​set by ROC analysis and grouping the subjects according to the following criteria: (a) if the numerical value of a subject is less than the cutoff value, the subject does not qualify as having mild cognitive impairment or is highly unlikely to qualify; (b) if the numerical value of a subject is equal to or greater than the cutoff value, the subject qualifies as having mild cognitive impairment or is highly likely to qualify.

Images