Method for evaluating amyloid β accumulation level in brain

Abstract

This method for evaluating the amyloid β accumulation level in a brain comprises: a step for binding an exosome marker-specific binding substance (120) that specifically binds to an exosome marker (113) present on the surface of an exosome (110) and is labeled by a first nucleic acid fragment (121) to an exosome (110) obtained from a blood sample; a step for binding an amyloid β-specific binding substance (160) that specifically binds to amyloid β and is labeled by a second nucleic acid fragment (161) to the exosome (110) obtained from the blood sample; a step for using the first nucleic acid fragment (121) to detect the amount of the exosome (110) obtained from the blood sample; a step for using the second nucleic acid fragment (161) to detect the amount of the exosome (110) having amyloid β bound thereto; and a step for calculating the ratio of the amount of the exosome (110) having amyloid β bound thereto to the amount of the exosome (110) obtained from the blood sample.

Description

Methods for assessing the level of amyloid-β accumulation in the brain

[0001] The present invention relates to a method for assessing the level of amyloid-β accumulation in the brain. This application claims priority to Japanese Patent Application No. 2024-88258, filed May 30, 2024, the contents of which are incorporated herein by reference.

[0002] Alzheimer's disease is the most common type of dementia, accounting for 50 to 75% of all dementia cases, and treatments are currently being developed.

[0003] Alzheimer's disease is thought to be caused by neurodegeneration, where amyloid beta forms polymers outside of brain neurons, accumulates in the brain, and deposits in the brain, forming senile plaques.Next, a protein called tau becomes hyperphosphorylated within brain neurons, forming filaments that are deposited in the brain, forming neurofibrils, which then cause neurological disorders such as neuronal death and synaptic dysfunction.

[0004] For the treatment or prevention of Alzheimer's disease, it is effective to treat or prevent it when amyloid beta accumulates in the brain, before the tau-dependent neurological damage occurs in the brain. To achieve this, a method capable of measuring amyloid beta in the brain is required. However, the cerebrospinal fluid test currently used to measure amyloid beta is highly invasive, and PET imaging and methods for detecting amyloid beta in the blood by mass spectrometry require special equipment.

[0005] Exosomes are a type of extracellular vesicle, membrane vesicles with a diameter of approximately 40 to 150 nm secreted by cells. In recent years, it has been suggested that exosomes may play a role in transmitting information to distant cells and tissues. Exosomes contain various proteins, lipids, and nucleic acids, and are thought to induce functional and physiological changes when transported to other cells. Non-Patent Document 1 reports that amyloid beta binds to ganglioside GM1 present on the surface of exosomes in neurons, and that amyloid beta is transported by exosomes, accumulating in the brain and forming plaques. Non-Patent Document 2 also reports that amyloid beta was detected in exosomes isolated from the plasma of Alzheimer's patients.

[0006] On the other hand, quantitative detection of target molecules in biological samples is being carried out for early detection of diseases and prediction of the effectiveness of medication. Quantitative determination of nucleic acids such as DNA and RNA is carried out by real-time PCR and the like.

[0007] In recent years, there has been an increasing need for more accurate detection of target molecules for purposes such as earlier disease detection. As a method for detecting target molecules with high accuracy, for example, Non-Patent Document 3 describes a technology in which an enzyme reaction is carried out in a large number of microcompartments and a fluorescent signal is detected. This method is called digital measurement.

[0008] In digital measurement, a sample solution is divided into an extremely large number of minute solutions. The signal from each minute solution is then binarized, and the presence or absence of target molecules is determined, and the number of target molecules is measured. Digital measurement can significantly improve detection sensitivity and quantitativeness compared to conventional real-time PCR methods, etc.

[0009] In digital PCR, a type of digital measurement, a mixture of PCR reaction reagents and nucleic acids is diluted so that each microdroplet in a microcompartment contains zero to one template nucleic acid. In digital PCR, the volume of each microdroplet is preferably small in order to increase the sensitivity of nucleic acid amplification and to simultaneously amplify nucleic acids for multiple microdroplets. For example, Non-Patent Document 3 discloses a method using micro-sized droplets formed so that each well has a volume of several nanoliters.

[0010] Another digital measurement technique is digital Invasive Cleavage Assay (ICA). Patent Document 1 discloses a technique in which a DNA sample is amplified by a PCR reaction, the denatured PCR product is introduced into a device having microwells, and the DNA is detected by the Invader (registered trademark) method without amplifying the DNA during detection.

[0011] International Publication No. WO 2015 / 115635 International Publication No. WO 2023 / 080102

[0012] Lawrence Rajendran et al., Alzheimer's disease β-amyloid peptides are released in association with exosomes. Proc. Natl. Acad. Sci. USA 2006 Jul 25;103(30):11172-11177.Massimo S. Fiandaca et ai., Identification of preclinical Alzheimer's disease by a profile of pathogenic proteins in neurally derived blood exosomes: A case-control study. Alzheimer's & Dementia 11(2015)600-607Olmedillas-Lopez S., et al., Current and Emerging Applications of Droplet Digital PCR in Oncology. Mol Diagn Ther. 2017 Oct;21(5):493-510

[0013] Non-Patent Document 1 detects amyloid beta bound to exosomes released from cultured neurons, but this detection method involves isolating exosomes released from cultured neurons, immunostaining the exosome-bound amyloid beta, and detecting it using an electron microscope, and is not a method for rapidly detecting amyloid beta bound to exosomes derived from brain neurons in blood. Non-Patent Document 2 also detects amyloid beta in exosomes isolated from plasma, which is not a rapid detection method.

[0014] In Patent Document 2, the level of amyloid beta accumulation in the brain is evaluated by detecting the amount of amyloid beta bound to exosomes in the blood.

[0015] The present inventors have concluded that, because the amount of exosomes transported into the blood through the blood-brain barrier varies from person to person, simply detecting the amount of amyloid beta bound to exosomes in the blood is insufficient to accurately assess the level of amyloid beta accumulation in the brain; instead, measuring the ratio of exosomes bound to amyloid beta to total exosomes would enable a more accurate assessment of the level of amyloid beta accumulation in the brain, regardless of the amount of exosomes transported from the blood-brain barrier to the blood.

[0016] In contrast, Patent Document 2 does not measure the total amount of exosomes in the blood, which leads to errors in the measurement results of amyloid beta bound to exosomes. Furthermore, it is not known to measure the proportion of exosome particles bound to amyloid beta after measuring the total amount of exosomes.

[0017] The present invention has been made in view of the above circumstances, and aims to provide a technique that can accurately evaluate the level of amyloid β accumulation in the brain.

[0018] The present invention includes the following aspects: [1] A method for assessing the level of amyloid-β accumulation in the brain, comprising the steps of: binding exosomes obtained from a blood sample to an exosome marker-specific binding substance that specifically binds to an exosome marker present on the surface of exosomes and that is labeled with a first nucleic acid fragment; binding exosomes obtained from the blood sample to an amyloid-β-specific binding substance that specifically binds to amyloid-β and that is labeled with a second nucleic acid fragment; detecting the amount of exosomes obtained from the blood sample using the first nucleic acid fragment; detecting the amount of exosomes bound with amyloid-β using the second nucleic acid fragment; and calculating the ratio of the amount of exosomes bound with amyloid-β to the amount of exosomes obtained from the blood sample. [2] A method for evaluating the level of amyloid beta accumulation in the brain, comprising the steps of: binding exosomes derived from brain neurons obtained from a blood sample to an exosome marker-specific binding substance that specifically binds to an exosome marker present on the surface of exosomes and is labeled with a first nucleic acid fragment; binding exosomes derived from brain neurons obtained from the blood sample to an amyloid beta-specific binding substance that specifically binds to amyloid beta and is labeled with a second nucleic acid fragment; detecting the amount of exosomes derived from brain neurons using the first nucleic acid fragment; detecting the amount of exosomes derived from brain neurons and bound to amyloid beta using the second nucleic acid fragment; and calculating the ratio of the amount of exosomes derived from brain neurons and bound to amyloid beta to the amount of exosomes derived from brain neurons. [3] The method for evaluating the level of amyloid beta accumulation in the brain according to [2], further comprising the step of obtaining the exosomes derived from brain neurons from a blood sample prior to the step of binding the exosome marker-specific binding substance to the exosomes derived from brain neurons and the step of binding an amyloid beta-specific binding substance to the exosomes derived from brain neurons. [4] The method for evaluating the level of amyloid beta accumulation in the brain according to [3], wherein the step of obtaining the exosomes derived from brain neurons is performed using a substance that specifically binds to ganglioside GM1, a marker for the exosomes derived from brain neurons.[5] The method for evaluating the level of amyloid-β accumulation in the brain according to [4], wherein the substance is cholera toxin subunit B (CTB). [6] The method for evaluating the level of amyloid-β accumulation in the brain according to any one of [1] to [5], wherein the exosome marker is any one of CD9, CD63, CD8, and phosphatidylserine. [7] The method for evaluating the level of amyloid-β accumulation in the brain according to any one of [1] to [6], wherein the amyloid-β is amyloid-β monomer, amyloid-β protofibril, or N3pG Aβ. [8] The method for evaluating the level of amyloid-β accumulation in the brain according to any one of [1] to [7], wherein the amyloid-β-specific binding substance is an antibody or antibody fragment that specifically binds to amyloid-β. [9] The method for evaluating the level of amyloid-β accumulation in the brain according to any one of [1] to [8], wherein the amyloid-β-specific binding substance is a therapeutic drug for Alzheimer's disease that specifically binds to amyloid-β.

[10] The method for evaluating the level of amyloid beta accumulation in the brain according to any one of [1] to [9], wherein the step of detecting the amount of exosomes derived from brain neurons and the step of detecting the amount of exosomes derived from brain neurons to which amyloid beta is bound are performed by invasive cleavage assay.

[11] The method for evaluating the level of amyloid beta accumulation in the brain according to any one of [1] to

[10] , wherein the exosome marker-specific binding substance is an anti-CD9 antibody of clone number 479608.

[12] The method for evaluating the level of amyloid beta accumulation in the brain according to

[10] , wherein the exosomes are captured with magnetic beads, and then the exosome marker-specific binding substance and the amyloid beta-specific binding substance are bound to the exosomes, and the subsequent invasive cleavage assay is performed in a well with the magnetic beads removed from the exosomes.

[0019] According to the present invention, a technique can be provided that can accurately evaluate the level of amyloid β accumulation in the brain.

[0020] FIG. 1 is a schematic diagram illustrating a method for evaluating the accumulation level of amyloid beta in the brain according to a preferred embodiment of the present invention. FIG. 2 is a schematic table showing the relationship between the amount of exosomes transferred into the blood through the brain barrier and the progression of dementia. FIG. 3 is a schematic table showing the relationship between the ratio of the amount of exosomes bound to amyloid beta to the amount of exosomes transferred into the blood through the brain barrier and the progression of dementia. FIG. 4 is a schematic cross-sectional view showing an example of a fluidic device. FIG. 5 is a schematic cross-sectional view showing an example of a fluidic device. FIG. 6 is a schematic cross-sectional view showing an example of a fluidic device. FIG. 7 is a schematic cross-sectional view showing an example of a fluidic device. FIG. 8 is a schematic cross-sectional view showing an example of a fluidic device. FIG. 9 is a schematic diagram illustrating an example of an Invasive Cleavage Assay (ICA) method. FIG. 10 is a schematic diagram showing the flow of detecting exosomes when magnetic beads are contained in wells and the flow of detecting exosomes when magnetic beads are not contained in wells. FIG. 11 is a schematic explanatory diagram showing a brain neuron-derived exosome marker, amyloid beta, and the pattern of presence or absence of fluorescence due to the presence of the exosome marker. 16A and 16B are images of Western blot analysis results showing the presence or absence of CD9 in exosomes contained in the culture supernatant obtained by culturing N2a cells transfected with the APP gene and N2a cells not transfected with the APP gene for 24 hours.

[0044] FIG. 16B is an image of Western blot analysis results showing the presence or absence of amyloid beta in exosomes contained in the culture supernatant obtained by culturing N2a cells transfected with the APP gene and N2a cells not transfected with the APP gene for 24 hours.

[0045] FIG. 16C is a fluorescent image of exosomes derived from brain neurons.

[0046] FIG. 16D is a graph showing the relationship between the ratio of the fluorescence count shown in FIGS. 15 and 16A to the number of magnetic beads and the amount of exosomes in the reaction mixture supplied to the device.

[0047] FIG. 16E is a graph showing the results of detection of the total amount of exosomes when an anti-human CD9 antibody of clone number 77B was used.

[0048] FIG. 16F is a graph showing the results of detection of the total amount of exosomes when an anti-human CD9 antibody of clone number 1K was used. 1 is a graph showing the results of detecting the total amount of exosomes when anti-human CD63 antibody of clone number 3-13 was used.1 is a graph showing the detection results of the total exosome amount when CTB is used. FIG. 1 is a schematic diagram showing the mixing of exosomes obtained from the culture supernatant of N2a cells into which the APP gene has been introduced with N2a cells into which the APP gene has not been introduced. FIG. 2 is a graph showing the ratio of the green fluorescence count, which indicates the total exosome amount, and the red fluorescence count, which indicates the amount of exosomes bound to amyloid β, relative to the number of magnetic beads. FIG. 3 is an image showing the fluorescence count, which indicates the amount of Aβ protofibrils at each plasma concentration. FIG. 4 is an image showing the fluorescence count, which indicates the amount of ganglioside GM1 at each plasma concentration. FIG. 5 is an image showing the fluorescence count, which indicates the amount of CD9 at each plasma concentration.

[0021] Hereinafter, embodiments of the present invention will be described in detail, with reference to the drawings where necessary. In the drawings, identical or corresponding parts are designated by identical or corresponding reference numerals, and redundant explanations will be omitted. The dimensional ratios in the drawings may be exaggerated for the purpose of explanation, and do not necessarily correspond to the actual dimensional ratios.

[0022] A preferred embodiment of the present invention relates to a method for assessing the level of amyloid beta accumulation in the brain, comprising the steps of: (1) obtaining exosomes derived from brain neurons from a blood sample; (2) binding the exosomes obtained from the blood sample to an exosome marker-specific binding substance that specifically binds to an exosome marker present on the surface of the exosomes and is labeled with a first nucleic acid fragment; (3) binding the exosomes obtained from the blood sample to an amyloid beta-specific binding substance that specifically binds to amyloid beta and is labeled with a second nucleic acid fragment; (4) detecting the amount of exosomes derived from brain neurons using the first nucleic acid fragment; (5) detecting the amount of exosomes derived from brain neurons and bound to amyloid beta using the second nucleic acid fragment; and (6) calculating the ratio of the amount of amyloid beta-bound exosomes derived from brain neurons to the amount of exosomes derived from brain neurons. Examples of blood samples include whole blood, serum, plasma, or dilutions thereof. The assessment method according to this embodiment is described below.

[0023] FIG. 1 is a schematic diagram illustrating a method for assessing the level of amyloid β accumulation in the brain according to a preferred embodiment of the present invention.

[0024] As shown in FIG. 1 , in the evaluation method of this embodiment, in step (1), brain neuron-derived exosomes are captured and obtained using a solid phase 150 to which a substance (a brain neuron-derived exosome marker-specific binding substance) 151 that specifically binds to a marker indicating that the exosome is derived from a brain neuron is immobilized. Note that step (1) above is not limited to a capture method using the solid phase 150. Furthermore, in step (2), an exosome marker-specific binding substance 120 that specifically binds to an exosome marker 113 present on the surface of the exosome 110 and is labeled with a first nucleic acid fragment 121 is bound to the exosome 110 obtained from the blood sample. Furthermore, (3) an amyloid β-specific binding substance 160 that specifically binds to amyloid β111 and is labeled with a second nucleic acid fragment 161 is bound to the exosome obtained from the blood sample.

[0025] In this specification, amyloid β is not limited to amyloid β monomers but may also be amyloid β oligomers, and is a concept that includes amyloid β protofibrils, amyloid β fibrils, amyloid β plaques, and amyloid β in which one or more amino acid residues constituting amyloid β have been chemically modified. Modified amyloid β in which an amino acid residue has been chemically modified includes, but is not limited to, amyloid β in which the glutamic acid residue, the third amino acid residue from the N-terminus, has been pyroglutamylated, known as N3pG Aβ.

[0026] The blood sample subjected to steps (1) to (3) is the same among the above steps (1) to (3). In other words, steps (1) to (3) are performed on exosomes obtained from the same blood sample. The order of steps (1) to (3) is not particularly limited, and steps can be performed in any order. Steps (1) to (3) may also be performed simultaneously. In this embodiment, the explanation will proceed on the assumption that steps (2) and (3) are performed after step (1). Note that when steps (2) and (3) are performed prior to step (1), in steps (2) and (3), the exosome marker-specific binding substance 120 may bind to exosomes not derived from brain neurons contained in the blood sample, in addition to exosomes derived from brain neurons.

[0027] After steps (1) to (3) above, (4) the amount of brain neuron-derived exosomes in the blood sample is detected using the first nucleic acid fragment 121. Furthermore, after steps (1) to (3) above, (5) the amount of brain neuron-derived exosomes bound with amyloid beta in the blood sample is detected using the second nucleic acid fragment 161. The order of steps (4) and (5) is not particularly limited, and either step (4) or (5) may be performed first.

[0028] After steps (4) and (5), the ratio of the amount of exosomes detected in step (5) to the amount of exosomes detected in step (4) (hereinafter also referred to as the "first amyloid-β binding ratio") is calculated. The first amyloid-β binding ratio thus calculated can be used as an indicator of the level of amyloid-β accumulation in the brain. The first amyloid-β binding ratio includes percentages such as 30% as well as ratios such as 2:1.

[0029] The first amyloid-β binding fraction may be used, for example, to determine that a person has a high possibility of dementia when the first amyloid-β binding fraction calculated for an actual blood sample is equal to or greater than a predetermined threshold value. The same applies to the second amyloid-β binding fraction, which will be described in detail later.

[0030] FIG. 2 is a schematic table showing the relationship between the amount of exosomes 110 transported into the blood through the brain barrier and the progression of dementia. As shown in FIG. 2, the amount of exosomes 110 transported into the blood through the brain barrier varies from person to person. Even if the proportion of exosomes 110 bound to amyloid β111 is small, if the amount of exosomes 110 transported into the blood is large, the detected amount of exosomes 110 bound to amyloid β111 will be large. Furthermore, even if the proportion of exosomes 110 bound to amyloid β111 is large, if the amount of exosomes 110 transported into the blood is small, the detected amount of exosomes 110 bound to amyloid β111 ("complex amount" in the drawing) will be small.

[0031] Therefore, if the amount of exosomes 110 bound to amyloid β111 is simply detected, it is impossible to distinguish between the upper right column and the lower left column in the table shown in Figure 2 (see the two-way arrow in Figure 2), which creates the problem of not being able to accurately evaluate the accumulation level of amyloid β111 in the brain.

[0032] FIG. 3 is a schematic table showing the relationship between the ratio of the amount of exosomes 110 bound to amyloid β111 to the amount of exosomes 110 transported into the blood through the brain barrier and the progression of dementia.

[0033] As a result of examining the above-mentioned problems, the present inventors have discovered that by calculating the ratio of the amount of exosomes 110 bound to amyloid β111 in a blood sample to the total amount of exosomes in the blood sample, it is possible to distinguish the accumulation level of amyloid β111 in the brain between the upper right column and the lower left column (see the two-way arrows) in the tables shown in Figures 2 and 3, and thus it is possible to accurately evaluate the accumulation level of amyloid β111 in the brain (and therefore the progression of dementia).

[0034] In this embodiment, by performing the above step (1) of obtaining exosomes derived from brain neurons from a blood sample, the total amount of exosomes derived from brain neurons in the blood sample is detected in place of the total amount of exosomes in the blood sample in the above step (6), and the ratio of the amount of exosomes derived from brain neurons to amyloid beta in the blood sample relative to the total amount of exosomes is calculated; however, it is not necessarily necessary to perform the above step (1).

[0035] When step (1) above is performed, the amount of the denominator when calculating the above ratio can be reduced, and the proportion of exosomes bound to amyloid β in the blood sample can be increased, making it possible to more clearly distinguish between the cases shown in the upper right column and the lower left column in the tables shown in Figures 2 and 3. Alternatively, a sample of exosomes derived from brain neurons may be prepared without performing step (1) above, and then subjected to steps (2) to (6) above.

[0036] When the amount of total exosomes (not limited to those derived from brain neurons) in a blood sample is detected without performing the above step (1), the ratio of the amount of exosomes (not limited to those derived from brain neurons) bound to amyloid beta in the blood sample to the amount of total exosomes (hereinafter also referred to as the "second amyloid beta binding ratio") may be detected. Similar to the first amyloid beta binding ratio, the second amyloid beta binding ratio can also be used as an indicator of the level of amyloid beta accumulation in the brain.

[0037] When calculating the second amyloid-β binding ratio, the exosomes to which the exosome marker-specific binding substance and the amyloid-β-specific binding substance are bound in steps (2) and (3) above include not only exosomes derived from brain neurons but also other exosomes. In this case, it is thought that the amyloid-β-specific binding substance will not bind to exosomes not derived from brain neurons. Furthermore, when calculating the second amyloid-β binding ratio, the solid phase 150 to which the brain neuron-derived exosome marker-specific binding substance 151 is immobilized is not necessary.

[0038] Exosomes are membrane vesicles with diameters of approximately 40 to 150 nm secreted by most cells. They are found in living body fluids, including saliva, blood, urine, amniotic fluid, and malignant ascites, and are also secreted by cultured cells. In recent years, exosomes have been suggested to potentially play a role in transmitting information between distant cells and tissues. Exosomes contain various proteins, lipids, and nucleic acids, and are thought to induce functional and physiological changes when delivered to other cells. Specific examples include mediating adaptive immune responses against infectious pathogens and tumors, tissue repair, neurotransmission, and transporting pathogenic proteins. Amyloid beta, a potential causative factor in Alzheimer's disease, is transported by binding to exosomes derived from brain neurons and accumulates in the brains of Alzheimer's disease patients, forming plaques. Therefore, by using a substance that specifically binds to amyloid beta, it is possible to detect the amount of amyloid beta-bound exosomes derived from brain neurons.

[0039] The exosome marker 113 is, for example, a protein, a molecule believed to be contained in most exosomes. The type of exosome marker 113 is not particularly limited, but suitable examples include tetraspanins such as CD9, CD63, and CD81, as well as the lipid molecule phosphatidylserine.

[0040] Although the type of brain neuron-derived exosome marker 112 is not particularly limited, ganglioside GM1 is particularly suitable for use. In this embodiment, the description will proceed on the assumption that the brain neuron-derived exosome marker 112 is ganglioside GM1.

[0041] Examples of substances that specifically bind to ganglioside GM1 (brain neuron-derived exosome marker-specific binding substances 151) include cholera toxin subunit B (hereinafter also referred to as CTB), antibodies, antibody fragments, aptamers, etc. The brain neuron-derived exosome marker-specific binding substances 151 may be one type or two or more types. In this embodiment, the explanation will be given on the assumption that CTB is used as the substance 151 that specifically binds to ganglioside GM1, which is the brain neuron-derived exosome marker 112.

[0042] Examples of the solid phase 150 onto which the brain neuron-derived exosome marker-specific binding substance 151 is immobilized include particles, membranes, and substrates. Examples of particles include, but are not limited to, polymer particles, magnetic particles, and glass particles. The particles are preferably surface-treated to avoid nonspecific adsorption. Furthermore, particles having functional groups such as carboxyl groups on their surfaces are preferred for immobilizing the brain neuron-derived exosome marker-specific binding substance 151. More specifically, a product such as "Magnosphere LC300" manufactured by JSR Corporation can be used. In this embodiment, magnetic beads, a type of particle, are used as the solid phase 150.

[0043] Methods for immobilizing brain neuron-derived exosome marker-specific binding substances 151 on particle surfaces are not particularly limited, and include physical adsorption methods, chemical bonding methods, methods utilizing avidin-biotin binding, and methods utilizing binding between protein G or protein A and an antibody. Physical adsorption methods include methods in which brain neuron-derived exosome marker-specific binding substances 151 are immobilized on particle surfaces through hydrophobic interaction or electrostatic interaction. Chemical bonding methods include methods using a crosslinking agent. For example, when the particle surface has hydroxyl groups, the carboxyl groups of the brain neuron-derived exosome marker-specific binding substances 151 can be reacted with a crosslinking agent to form active esters, and then the hydroxyl groups can be reacted with the ester groups to immobilize the brain neuron-derived exosome marker-specific binding substances 151 on the particle surfaces. Furthermore, in order to avoid inhibiting the ability of brain neuron-derived exosome marker-specific binding substance 151 to recognize brain neuron-derived exosomes, it is preferable to provide a spacer between brain neuron-derived exosome marker-specific binding substance 151 and the particle surface.

[0044] A detection method using digital measurement can be suitably used to detect the amount of exosomes using the first nucleic acid fragment 121 and the second nucleic acid fragment. When steps (4) and (5) above are performed by digital measurement, it is preferable to use a fluidic device having a well array in which multiple wells are arranged.

[0045] (Fluidic Device) Figure 4 is a schematic cross-sectional view showing an example of a fluidic device. As shown in Figure 4, the fluidic device 200 includes a substrate 210 and a lid member 220 arranged opposite the substrate 210. The lid member 220 has a convex portion 221, and the tip of the convex portion 221 contacts the substrate 210. In the fluidic device 200, the well array 240 is integrally molded with the substrate 210 on one side of the substrate 210 and faces the lid member 220. The well array 240 has a plurality of wells 241. The lid member 220 may be welded or adhered to the substrate 210.

[0046] The wells 241 are open on the surface of the substrate 210. The shape, dimensions, and arrangement of the wells 241 are not particularly limited, but it is preferable that one brain neuron-derived exosome is introduced into one well 241. The wells 241 are preferably microwells with small volumes. For example, the volume of one well 241 may be approximately 10 fL to 100 pL. In the fluidic device 200, a plurality of wells 241 of the same shape and size constitute a well array 240. The term "same shape and size" means that the wells have the same shape and capacity to the extent required for digital measurement, and variations within the range of manufacturing errors are acceptable.

[0047] The diameter of well 241 may be, for example, about 1 to 10 μm, and the depth of well 241 may be, for example, about 1 to 10 μm. The arrangement of wells 241 is not particularly limited, and they may be arranged in a triangular lattice pattern, a square lattice pattern, or randomly arranged.

[0048] In the fluidic device 200, the presence of the convex portion 221 forms a space between the well array 240 and the cover member 220. This space constitutes a flow channel 230. The flow channel 230 functions as a path for transporting a liquid dispersed with exosomes derived from brain neurons, an exosome marker-specific binding substance labeled with a first nucleic acid fragment, an amyloid-β-specific binding substance labeled with a second nucleic acid fragment, and the like, as well as a sealing liquid (described below). The shape, structure, capacity, etc. of the flow channel 230 are not particularly limited, but the height of the flow channel 230 (the distance between the surface of the substrate 210 and the surface of the cover member 220 facing the substrate 210) may be, for example, 500 μm or less, for example, 300 μm or less, for example, 200 μm or less, or for example, 100 μm or less.

[0049] The convex portion 221 may be molded integrally with the lid member 220. The lid member 220 can be formed into a plate shape having the convex portion 221, for example, by molding a thermoplastic resin fluid using a molding die. Furthermore, the lid member 220 may be formed with an inlet port 222 and an outlet port 223 for a reagent.

[0050] When the lid member 220 has a protrusion 221, the lid member 220 and the substrate 210 are overlapped so that the protrusion 221 contacts the surface of the substrate 210 where the well 241 opens. As a result, the space between the lid member 220 and the substrate 210 becomes the flow path 230. The lid member 220 and the substrate 210 may be welded by laser welding or the like.

[0051] (Variation 1 of Fluidic Device) The fluidic device used in the detection method of this embodiment is not limited to the above-described fluidic device 200. Fig. 7 is a schematic cross-sectional view showing an example of a fluidic device. As shown in Fig. 7, fluidic device 500 includes substrate 210 and wall member 510. In fluidic device 500, well array 240 is molded integrally with substrate 210 on one side of substrate 210. Well array 240 has a plurality of wells 241.

[0052] The fluidic device 500 differs from the above-described fluidic device 200 mainly in that it does not have a cover member 220. Therefore, the fluidic device 500 does not have a flow path.

[0053] (Variation 2 of Fluidic Device) In the above-described fluidic device 200, the cover member 220 and the protrusion 221 are integrally molded. However, the cover member 220 and the protrusion 221 may be molded as separate bodies.

[0054] Furthermore, in the above-described fluidic device 200 and fluidic device 500, the well array 240 is integrally molded with the substrate 210 on one side of the substrate 210. However, the well array does not have to be integrally molded with the substrate 210. For example, the well array 240 molded separately from the fluidic device may be disposed on the substrate 210 of the fluidic device. Alternatively, a resin layer may be laminated on the surface of the substrate 210, and the well array may be formed in the resin layer by etching or the like.

[0055] (Fluidic Device Material) The substrate 210 is formed using, for example, a resin. The type of resin is not particularly limited, but a resin that is resistant to reagents and sealing liquid is preferable. Furthermore, if the signal to be detected is fluorescence, a resin with low autofluorescence is preferable. Examples of the resin include, but are not limited to, cycloolefin polymer, cycloolefin copolymer, silicone, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesin, amorphous fluororesin, etc.

[0056] A plurality of wells 241 may be formed on one surface in the thickness direction of the substrate 210. Methods for forming wells using resin include injection molding, thermal imprinting, and optical imprinting.

[0057] Alternatively, for example, a fluororesin may be laminated on the substrate 210 and processed by etching or the like to form a well array. As the fluororesin, for example, CYTOP (registered trademark) (Asahi Glass) or the like can be used.

[0058] Furthermore, when the fluidic device has a lid member 220, the material of the lid member 220 is preferably a resin with low autofluorescence, and may be, for example, a thermoplastic resin such as a cycloolefin polymer or a cycloolefin copolymer.

[0059] Furthermore, the cover member 220 may be made of a material that does not transmit light of wavelengths close to the wavelength detected during fluorescence observation of the signal, or may be made of a material that is completely opaque to light. For example, the cover member 220 may be made of a thermoplastic resin to which carbon, metal particles, or the like are added.

[0060] (Detection of Exosomes) Next, the method for detecting exosomes according to this embodiment will be described using the fluidic device 200 as an example, with reference to FIGS. 4 to 6 as the case may be.

[0061] <<Introduction Step>> First, as shown in FIG. 4 , a reagent solution L210 is introduced through the introduction port 222 of the fluidic device 200 and sent to the flow path 230. The reagent solution L210 of this embodiment is a liquid in which brain neuron-derived exosomes 110 obtained using the solid phase 150, an exosome marker-specific binding substance 120 labeled with a first nucleic acid fragment 121, and an amyloid β-specific binding substance 160 labeled with a second nucleic acid fragment 161 are dispersed, and also contains reagents for detecting the first nucleic acid fragment 121 and the second nucleic acid fragment 161. When amyloid β111 is bound to the brain neuron-derived exosomes 110, the amyloid β-specific binding substance 160 labeled with the second nucleic acid fragment 161 binds to the amyloid β111 bound to the brain neuron-derived exosomes 110. The above-mentioned components contained in the reagent solution L210 are mixed in advance in a sample tube or the like before being introduced into the fluidic device 200. It is not necessarily necessary to capture the brain neuron-derived exosomes 110 using the solid phase 150 prior to introducing the reagent solution L210 into the fluidic device 200. The solid phase 150 may be fixed in the fluidic device 200 (e.g., in the well 241), and the brain neuron-derived exosomes 110 contained in a blood-derived sample may be captured within the fluidic device 200. That is, the above step (1) may be performed within the fluidic device for detection or may be performed outside the fluidic device. Furthermore, prior to adding the brain neuron-derived exosomes 110 captured by the solid phase 150 to the reagent solution L210, the brain neuron-derived exosomes 110 may be removed from the solid phase 150, and then a complex containing the brain neuron-derived exosomes 110, the exosome marker-specific binding substance 120, and the amyloid β-specific binding substance 160 may be added to the reagent solution L210.

[0062] The reagent solution L210 sent to the flow channel 230 comes into contact with the well array 240. The reagent solution L210 is then contained inside the wells 241. As a result, the brain nerve cell-derived exosomes 110, the exosome marker-specific binding substance 120 labeled with the first nucleic acid fragment 121, the amyloid β-specific binding substance labeled with the second nucleic acid fragment 161, and reagents for detecting the first nucleic acid fragment 121 and the second nucleic acid fragment 161 are introduced into the wells 241.

[0063] The number of brain neuron-derived exosomes 110 introduced into one well 241 is not particularly limited, but preferably one or less, i.e., zero or one brain neuron-derived exosome 110, is introduced into one well 241. This allows amyloid β111 bound to the brain neuron-derived exosomes 110 to be detected on a single unit basis, enabling digital measurement. Furthermore, it is not necessary to introduce brain neuron-derived exosomes 110 into all wells of the well array.

[0064] The means for introducing the brain neuron-derived exosomes 110 into the wells 241 is not particularly limited, and an appropriate means can be selected depending on the selected brain neuron-derived exosomes 110. For example, a method can be used in which the brain neuron-derived exosomes 110 are allowed to settle within the fluidic device (within the flow channel) due to their own weight and then distributed to the wells 241. In this embodiment, the brain neuron-derived exosomes 110 are captured using the solid phase 150, and the brain neuron-derived exosomes 110 can be efficiently introduced into each well 241 due to the own weight of the solid phase 150 and the brain neuron-derived exosomes 110. Furthermore, as described above, the efficiency of introducing the brain neuron-derived exosomes 110 into the wells 241 can be improved by immobilizing the solid phase 150 (or a substance specifically binding to a brain neuron-derived exosome marker) to the wells 241 in advance and capturing the delivered brain neuron-derived exosomes 110.

[0065] As described above, when brain neuron-derived exosomes 110 are introduced into wells 241 using brain neuron-derived exosome marker-specific binding substances 151, it is preferable to form a conjugate between the brain neuron-derived exosome marker-specific binding substance 151 and brain neuron-derived exosomes 110 under conditions where zero or one brain neuron-derived exosome 110 is detected per brain neuron-derived exosome marker-specific binding substance (151). Furthermore, it is preferable that one well 241 is configured so that zero or one exosome marker-specific binding substance 120 and one amyloid β-specific binding substance 160 are introduced. This enables digital measurement.

[0066] Examples of the exosome marker-specific binding substance 120 and the amyloid β-specific binding substance 160 include antibodies, antibody fragments, and aptamers. Examples of antibody fragments include Fab and F(ab') ２ , Fab', single-chain antibody (scFv), disulfide-stabilized antibody (dsFv), dimeric V region fragment (diabody), CDR-containing peptide, etc. The antibody may be a monoclonal antibody or a polyclonal antibody. Alternatively, a commercially available antibody may be used.

[0067] As described above, amyloid β is not limited to amyloid β monomers, but may also be amyloid β protofibrils or N3pG Aβ. Therefore, amyloid β-specific binding substance 160 may be, for example, an antibody, antibody fragment, or aptamer that specifically binds to amyloid β protofibrils or N3pG Aβ.

[0068] Furthermore, the amyloid β-specific binding substance 160 may be a therapeutic drug for Alzheimer's disease that specifically binds to amyloid β. Examples of therapeutic drugs for Alzheimer's disease include, but are not limited to, lecanemab, which is an antibody that specifically binds to amyloid β protofibril (anti-amyloid β protofibril antibody), and donanemab, which is an antibody that specifically binds to N3pG Aβ (anti-N3pG Aβ antibody).

[0069] By using an Alzheimer's disease treatment drug as the amyloid beta-specific binding substance 160, it is possible to determine the proportion of amyloid beta or its polymers, which are the target of the treatment drug, contained in exosomes in a blood sample.

[0070] Furthermore, when amyloid β-specific binding substance 160 is an antibody that binds to amyloid β protein, the epitope of amyloid β-specific binding substance 160 is preferably a region consisting of the amino acid sequence of the 1st to 16th amino acid residues counting from the N-terminus of human amyloid β protein (see SEQ ID NO: 1). Furthermore, when exosome marker 113 is CD9 and exosome marker-specific binding substance 120 is an antibody that binds to CD9, the clone number of the antibody of exosome marker-specific binding substance 120 is preferably 479608.

[0071] Examples of methods for labeling the exosome marker-specific binding substance 120 and the amyloid β-specific binding substance 160 with nucleic acid fragments include a method using a crosslinker. The nucleic acid fragments may be labeled to the exosome marker-specific binding substance 120 and the amyloid β-specific binding substance 160 via a linker molecule. The linker is not particularly limited, and examples include polyethylene chains, hydrocarbon chains, peptides, and the like. The nucleic acid fragments may be DNA or RNA. They may also include artificial nucleic acids such as BNA and LNA.

[0072] Furthermore, for example, when antibody molecules are used as the exosome marker-specific binding substance 120 and the amyloid β-specific binding substance 160 and the antibody molecules are labeled with nucleic acid fragments, since nucleic acid fragments are relatively small molecules, for example, several to several tens of nucleic acid fragments can be labeled per antibody molecule. By increasing the number of nucleic acid fragments per antibody molecule, the time required to detect the exosome marker-specific binding substance 120 and the amyloid β-specific binding substance 160 can be further shortened. Furthermore, when antibody molecules are labeled with nucleic acid fragments, several to several hundred nucleic acid fragments may be labeled per antibody molecule. According to this embodiment, for example, the time required for the nucleic acid detection reaction (e.g., ICA reaction) is approximately 1 to 30 minutes, enabling rapid nucleic acid detection.

[0073] When brain neuron-derived exosomes 110 are contacted with exosome marker-specific binding substance 120 labeled with first nucleic acid fragment 121 and amyloid β-specific binding substance 160 labeled with second nucleic acid fragment 161, if amyloid β111 is bound to brain neuron-derived exosomes 110, the amyloid β111 bound to brain neuron-derived exosomes 110 binds to amyloid β-specific binding substance 160 labeled with second nucleic acid fragment 161, forming complex 100 shown in Fig. 1. Formation of complex 100 may be performed in well 241, or, for example, may be performed in a sample tube, and then reagent solution L210 containing complex 100 may be supplied to fluidic device 200.

[0074] <<Encapsulation step>> After introducing brain neuron-derived exosomes 110, an exosome marker-specific binding substance 120 labeled with a first nucleic acid fragment 121, an amyloid β-specific binding substance 160 labeled with a second nucleic acid fragment 161, and the like into well 241, a step of sealing the opening of well 241 may be carried out.

[0075] The method for sealing the opening of the well 241 is not particularly limited as long as it can prevent the liquid contained in one well 241 from mixing with the liquid contained in another well 241. For example, the opening of the well 241 may be sealed by covering it with a sealing liquid. Alternatively, the opening of the well 241 may be sealed by stacking a plate-like member such as a glass plate on it.

[0076] For example, as shown in FIG. 5 , a sealing liquid L220 is delivered from an inlet port 222 of the cover member 220 to a flow path 230 between the substrate 210 and the cover member 220. The sealing liquid L220 delivered to the flow path 230 comes into contact with the well array 240. The sealing liquid L220 then flushes out and replaces the reagent liquid L210 delivered to the flow path 230 that is not contained in the wells 241. As a result, the sealing liquid L220 individually seals each of the multiple wells 241 containing the reagent liquid L210 containing the exosomes 110 derived from brain neurons, and the wells 241 become independent reaction spaces (microcompartments 242). When the flow path 230 is filled with the sealing liquid L220, excess sealing liquid L220 is discharged from the discharge port 223. FIG. 6 shows a state in which all of the wells 241 of the well array 240 have been sealed with sealing liquid L220, forming sealed wells (microcompartments) 242.

[0077] Alternatively, a lipid may be dissolved in the reagent solution L210, and after feeding the sealing solution L220 into the channel 230, a liquid containing the lipid may be fed again to form a lipid bilayer membrane at the opening of the well 241, thereby sealing each of the multiple wells 241 individually with the lipid bilayer membrane to form sealed wells 242. Examples of lipids that form lipid bilayer membranes include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), and mixtures thereof.

[0078] Well 242 sealed by any of the sealing methods described above may contain an exosome marker-specific binding substance 120 labeled with a first nucleic acid fragment 121 and an amyloid beta-specific binding substance 160 labeled with a second nucleic acid fragment 161.

[0079] The sealing liquid is a liquid that can individually seal the liquids introduced into the multiple wells 241 so as not to mix with each other, thereby forming droplets (microdroplets), and is preferably an oily solution, more preferably an oil. Examples of oils that can be used include fluorine-based oils, silicone-based oils, hydrocarbon-based oils, and mixtures thereof. More specifically, products such as "FC-40" manufactured by Sigma can be used. FC-40 (CAS number: 86508-42-1) is a fluorinated aliphatic compound with a specific gravity of 1.85 g / mL at 25°C.

[0080] <Detection Step> Next, the first nucleic acid fragment 121 and the second nucleic acid fragment 161 are detected. Detection of the first nucleic acid fragment 121 refers to detection of exosomes. In the evaluation method of this embodiment for calculating the first amyloid-β binding ratio, exosomes derived from brain neurons are captured in advance using a brain neuron-derived exosome marker-specific binding substance 151. Therefore, detection of the first nucleic acid fragment 121 specifically refers to detection of exosomes derived from brain neurons. Detection of the second nucleic acid fragment 161 refers to detection of exosomes bound with amyloid-β. Detection of the first nucleic acid fragment 121 and the second nucleic acid fragment 161 is preferably performed using a signal amplification reaction. Examples of signal amplification reactions include invasive cleavage assays (ICA).

[0081] The ICA reaction is based on the principle that signal amplification proceeds through a cycle of two reactions: (I) complementary binding between nucleic acids, and (II) recognition and cleavage of the triple-stranded structure by an enzyme.

[0082] The ICA reaction is less susceptible to reaction cycle inhibition by impurities. Therefore, by using the ICA reaction, the amount of exosomes 110 derived from brain neurons and the amount of exosomes bound to amyloid beta can be accurately detected. When the ICA reaction is used for the signal amplification reaction, the reagent solution L210 contains the reaction reagents necessary for the ICA reaction. The detection of the first nucleic acid fragment 121 using the ICA reaction will be described in detail below, but the detection of the second nucleic acid fragment 161 is also performed in a similar manner.

[0083] Examples of reaction reagents required for the ICA reaction to detect the first nucleic acid fragment 121 include ICA reaction reagents such as a flap probe, an invasion probe, a flap endonuclease (FEN), a fluorescent substrate, etc. The flap probe and the invasion probe are nucleic acid fragments designed to hybridize to the first nucleic acid fragment 121 and form a flap structure with the first nucleic acid fragment 121.

[0084] Figure 10 is a schematic diagram illustrating an example of the ICA method. First, a flap probe 810 and an invasive probe 130 are hybridized to a first nucleic acid fragment 121. In the example of Figure 10, the flap probe 810 and the invasive probe 130 hybridize to the first nucleic acid fragment 121. As a result, a first flap site 811 is formed.

[0085] Next, when FEN is reacted with the first flap site 811, the first flap site 811 is cleaved to generate a nucleic acid fragment 811. Next, the nucleic acid fragment 811 hybridizes to a fluorescent substrate (nucleic acid fragment 820) to form a second flap site 821.

[0086] In the example of Figure 10, a fluorescent substance F is bound to the 5' end of nucleic acid fragment 820, and a quencher Q is bound to a few bases 3' from the 5' end of nucleic acid fragment 820. Subsequently, when FEN is reacted with second flap site 821, second flap site 821 is cleaved and nucleic acid fragment 821 is generated. As a result, fluorescent substance F is separated from quencher Q and a fluorescent signal is generated. By detecting this fluorescent signal, first nucleic acid fragment 121 can be detected.

[0087] The reagent solution L210 may be a liquid commonly used in biochemical analyses using fluidic devices, preferably an aqueous solution. The reagent solution L210 may also contain a surfactant or the like to facilitate sealing the liquid within the wells.

[0088] When an ICA reaction is used to detect the first nucleic acid fragment 121, if the first nucleic acid fragment 121 is present, an isothermal enzymatic reaction causes the fluorescent substance F to be released from the quenching substance Q, and a predetermined fluorescent signal is emitted in response to the excitation light.

[0089] For detecting the first nucleic acid fragment 121, a known appropriate method can be selected depending on the type of signal to be detected. For example, when observing a fluorescent signal, excitation light corresponding to the fluorescent substance is irradiated onto the well 242, and the fluorescence emitted by the fluorescent substance is observed. For example, as shown in FIG. 6 , a predetermined reaction is carried out in the sealed well 242, and the generated signal is observed. Well 242R is a well in which a signal was detected, and well 242 is a well in which a signal was not detected.

[0090] Next, the method of this embodiment will be described with reference to FIGS. 7 to 9, taking the case where the fluidic device 500 is used as an example.

[0091] First, as shown in Fig. 7, a reagent solution L210 is introduced into the fluidic device 500. The reagent solution L210 is the same as that described above and also contains reagents for detecting the first nucleic acid fragment 121 and the second nucleic acid fragment 161. In the reagent solution L210, the concentration of the brain nerve cell-derived exosomes 110 is preferably adjusted to a concentration such that one or less molecule of brain nerve cell-derived exosomes 110 is contained per well 241.

[0092] Next, as shown in Figure 8, a sealing liquid L220 is introduced into the fluidic device 500. The specific gravity of the sealing liquid L220 is greater than that of the reagent liquid L210. Therefore, the sealing liquid L220 sinks below the reagent liquid L210 that is not contained in the wells 241, and comes into contact with the well array 240. The sealing liquid L220 then individually seals each of the multiple wells 241 that contain the reagent liquid L210 containing exosomes derived from brain neurons, forming independent reaction spaces (microcompartments 242).

[0093] 9, a predetermined reaction is carried out in well 242, and the generated signal is observed. Well 242R is a well in which a signal is detected, and well 242 is a well in which a signal is not detected.

[0094] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention.

[0095] For example, it is also possible to detect the amount of exosomes derived from brain neurons without performing the step (1) above, and to calculate the first amyloid binding ratio by using a substance that is labeled with a nucleic acid fragment and that specifically binds to a brain neuron-derived exosome marker in step (2) above, instead of the exosome marker-specific binding substance that specifically binds to an exosome marker present on the surface of exosomes and that is labeled with a first nucleic acid fragment.

[0096] Furthermore, it is not necessarily necessary to accommodate exosomes 110 captured using a solid phase 150 such as magnetic beads in well 241 together with the solid phase 150; exosomes 110 labeled with the first nucleic acid fragment 121 and / or the second nucleic acid fragment may be removed from the solid phase 150 and placed in well 241 without the solid phase 150. Below, a detailed description is given of a method for removing exosomes 110 from magnetic beads, which are an example of solid phase 150, and placing them in well 241.

[0097] 11 is a schematic diagram showing the flow of detecting exosomes 110 when magnetic beads 150 are contained in wells 241, and the flow of detecting exosomes 110 when magnetic beads 150 are not contained in wells 241. FIG. 11 shows an example of a method for detecting exosomes 110.

[0098] As shown in Figure 11, when magnetic beads 150 are placed in a well 241, first, the exosomes 110 are captured by the magnetic beads 150 to remove impurities 114 other than the exosomes 110 contained in the blood sample (STEP 1). At this time, the magnetic beads 150 having TIM4 (T cell immunoglobulin and mucin domain-containing protein 4, 115 in the figure) on their surface can be used to specifically capture the exosomes 110. TIM4 specifically binds to the phospholipid phosphatidylserine 116 present on the membrane surface of the exosomes 110.

[0099] Next, the magnetic beads 150 are magnetically collected and washed with a buffer or the like to remove the impurities 114 (STEP 2).

[0100] Thereafter, the magnetic beads 150 are removed from the exosomes 110 (STEP 3), and the exosomes 110 derived from the brain neurons are again captured using magnetic beads 150 having on their surface a brain neuron-derived exosome marker-specific binding substance 151 that specifically binds to a brain neuron-derived exosome biomarker (e.g., ganglioside GM1) (STEP 4). Note that when capturing the exosomes 110 derived from the brain neuron from a blood sample using the brain neuron-derived exosome marker-specific binding substance 151 from the beginning, capture may be difficult due to impurities 114. Therefore, it is preferable to first capture the exosomes 110 using TIM4 and then capture the exosomes 110 derived from the brain neuron using the brain neuron-derived exosome marker-specific binding substance 151.

[0101] One method for removing the magnetic beads 150 from the exosomes 110 is to add EDTA to the sample. Adding EDTA reduces the calcium ion concentration in the sample, thereby cleaving the binding of TIM4 to the exosomes 110.

[0102] Next, an amyloid β-specific binding substance 160 labeled with a nucleic acid fragment is bound to the amyloid β on the surface of the exosome 110 (STEP 4), and then excess amyloid β-specific binding substance 160 is removed (STEP 5), and the exosome 110 is placed in a well 241 (STEP 6). Finally, the fluorescence of the well 241 is detected (STEP 7), thereby detecting the exosome 110 derived from brain neurons and bound to amyloid β (ICA reaction).

[0103] In contrast, when magnetic beads 150 are not contained in wells 241, the process is the same as the above-described method up to STEP 2, but thereafter, amyloid β-specific binding substance 160 labeled with a nucleic acid fragment is bound to amyloid β 111 on the surface of exosomes 110, and brain neuron-derived exosome marker-specific binding substance 151 labeled with a nucleic acid fragment is bound to brain neuron-derived exosome marker 112 (STEP 3A). At this time, exosome marker-specific binding substance 120 labeled with a nucleic acid fragment (see FIG. 1) may also be bound to exosome marker 113 (see FIG. 1).

[0104] Next, the magnetic beads 150 are removed from the exosomes 110 (STEP 4A). The method for removing the magnetic beads 150 is as described above. Thereafter, the exosomes 110 are placed in the wells 241 (STEP 5A), and the fluorescence of the wells 241 is detected (STEP 6A), thereby detecting the exosomes 110 derived from brain neurons and bound to amyloid β (ICA reaction).

[0105] As described above, by removing the exosomes 110 from the solid phase 150 such as beads and placing them in the wells 241, it is possible to prevent the exosomes 110 from being unable to be placed in the wells 241 due to aggregation of the solid phases 150, which would result in a decrease in detection sensitivity.

[0106] In the above example, the exosomes 110 are captured by the magnetic beads 150, and then the brain neuron-derived exosome marker-specific binding substance 151 and the amyloid β-specific binding substance 160 are bound to the exosomes 110, and the subsequent ICA reaction is carried out in the well 241 with the magnetic beads 150 removed from the exosomes 110. However, the exosomes 110 may also be captured by the magnetic beads 150, and then the exosome marker-specific binding substance 120 and the amyloid β-specific binding substance 160 are bound to the exosomes 110, and the subsequent ICA reaction may be carried out in the well 241 with the magnetic beads 150 removed from the exosomes 110.

[0107] In the example shown in Figure 11, the amount of exosomes 110 derived from brain neurons and bound with amyloid beta was detected. However, by using a similar method, i.e., by removing the solid phase 150 from the exosomes 110 before placing them in the well 241, the amount of exosomes 110 derived from brain neurons may be detected regardless of whether amyloid beta is bound, or the amount of all exosomes 110 contained in the blood sample may be detected regardless of the origin of the exosomes.

[0108] Furthermore, when the solid phase 150 is removed from the exosomes 110, the proportion of exosomes 110 derived from brain nerve cells to which amyloid beta is bound may be calculated, for example, as follows.

[0109] 12 is a schematic explanatory diagram showing the pattern of fluorescence presence or absence due to the presence of brain neuron-derived exosome marker 112, amyloid β111, and exosome marker 113. In FIG. 12, the fluorescence when brain neuron-derived exosome marker 112, such as ganglioside GM1, is detected using a nucleic acid fragment labeled with a brain neuron-derived exosome marker-specific binding substance 151 is shown as symbol 112, the fluorescence when amyloid β111 is detected using a nucleic acid fragment labeled with an amyloid β-specific binding substance 160 is shown as symbol 111, and the fluorescence when exosome marker 113, such as CD9, is detected using a nucleic acid fragment labeled with an exosome marker-specific binding substance 120 is shown as symbol 113. The detection method using nucleic acid fragments is the detection method using the ICA reaction described above.

[0110] The amount of amyloid β111 can be calculated by adding up the number of fluorescence signals detected in each of the fluorescence detection patterns B, D, E, and G (B+D+E+G) as shown in Figure 12. The value R1, which normalizes the amount of amyloid β111 by the total amount of exosomes, can be calculated using the following formula (i): R1 = (B+D+E+G) / (C+E+F+G) (i)

[0111] Furthermore, the amount of exosomes derived from brain neurons and bound to amyloid β111 can be calculated by summing the number of fluorescence detected in each of the B and G fluorescence detection patterns (B + G). The value R2 obtained by normalizing the amount of exosomes derived from brain neurons and bound to amyloid β111 to the total amount of exosomes (in other words, the proportion of exosomes derived from brain neurons and bound to amyloid β) can be calculated using the following formula (ii): R2 = (B + G) / (C + E + F + G) (ii)

[0112] The amount of exosomes bound to amyloid β111 can be calculated by adding up the number of fluorescent molecules detected in each of the fluorescent detection patterns E and G (E + G). The value R3, which is the amount of exosomes bound to amyloid β111 normalized by the amount of exosomes derived from brain neurons, can be calculated using the following formula (iii): R3 = (E + G) / (A + B + C + G) (iii)

[0113] Furthermore, the amount of exosomes derived from brain neurons and bound to amyloid β111 is the number of fluorescence detected in the fluorescence detection pattern G. The value R4 (in other words, the proportion of exosomes derived from neurons and bound to amyloid β111) normalized to the total amount of exosomes can be calculated using the following formula (iv): R4 = G / (C + E + F + G) ... (iv)

[0114] Furthermore, the value R5 obtained by normalizing the amount of exosomes derived from brain neurons and bound to amyloid β111 (in other words, the proportion of exosomes derived from brain neurons bound to amyloid β) can be calculated using the following formula (v): R5 = G / (A + B + C + G) (v)

[0115] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0116] <Reagent Preparation> (Preparation of a Sample Containing Exosomes Derived from Brain and Neurons) The sample containing exosomes derived from brain and neuron cells bound to amyloid beta was prepared by stably introducing a plasmid containing the gene for isoform 751 (APP751) of human amyloid beta precursor protein (hereinafter also referred to as APP) into mouse Neuro 2A (N2a) cells and culturing the cells in Dulbecco's Modified Eagle medium for 24 hours. Ultracentrifugation was used to recover exosomes from the culture supernatant. Specifically, the culture supernatant was centrifuged stepwise at 2,000 x g for 10 minutes, 10,000 x g for 30 minutes, and 100,000 x g for 70 minutes to recover exosomes, which was used as a sample containing exosomes derived from brain and neuron cells.

[0117] (Preparation of CTB-immobilized magnetic beads) To immobilize cholera toxin subunit B (CTB, Fujifilm Wako Pure Chemical Industries, Ltd.) on magnetic beads, CTB was added to a solution of carboxyl group-modified magnetic beads (Magnosphere, LC300, JSR Corporation) and mixed to a total volume of 100 μL. The mixture was reacted for 30 minutes on a rotator, and then the condensing agent EDC was added and reacted for 3 hours to immobilize CTB on the carboxyl magnetic beads. After the reaction, unreacted CTB and reagents were removed by magnetic collection of the CTB-immobilized carboxyl magnetic beads using a magnetic stand and washing with PBS-T (PBS containing 0.1% Tween 20) three times to prepare CTB-immobilized carboxyl magnetic beads (CTB beads).

[0118] (Nucleic acids for modification with anti-amyloid β antibodies and nucleic acids for detection) The following nucleic acids were used as nucleic acids for modification with anti-amyloid β antibodies and for the nucleic acid detection reaction. DNA fragment 1 corresponds to the second nucleic acid fragment 161 described above, and DNA fragment 2 corresponds to the first nucleic acid fragment 121 described above. DNA fragment 1: Has the base sequence (IL-28c) shown in SEQ ID NO: 2. DNA fragment 2: Has the base sequence (IL-28t) shown in SEQ ID NO: 3. Flap probe 1: Has the base sequence shown in SEQ ID NO: 4. Flap probe 2: Has the base sequence shown in SEQ ID NO: 5. Invasion probe 1: Has the base sequence shown in SEQ ID NO: 6. Invasion probe 2: Has the base sequence shown in SEQ ID NO: 7.

[0119] (Preparation of nucleic acid-modified anti-amyloid β antibody) A DNA fragment (DNA fragment 1) having the base sequence shown in SEQ ID NO: 2 was conjugated to an anti-amyloid β monoclonal antibody (clone number: BAN50, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a nucleic acid-labeled anti-amyloid β antibody (an example of an amyloid β-specific binding substance). The antibody has an epitope consisting of the amino acid sequence shown in SEQ ID NO: 1. A commercially available kit (trade name "Protein-Oligo Conjugation Kit", manufactured by Sollink) was used for conjugation of DNA fragment 1. The nucleic acid-labeled anti-amyloid β antibody was prepared so that 5 to 20 times the amount of nucleic acid was modified relative to the antibody (one antibody molecule).

[0120] (Preparation of nucleic acid-modified anti-CD9 antibody) Similarly, DNA fragment 2 having the base sequence shown in SEQ ID NO: 3 was conjugated to the anti-CD9 antibody to prepare a nucleic acid-labeled anti-CD9 antibody (an example of an exosome marker-specific binding substance, clone number: 479608, model number: MAB5218, manufactured by R&D Systems). This antibody was produced using an NS0 mouse myeloma cell line, into which the full-length mouse CD9 gene had been introduced, as an antigen. A commercially available kit (trade name "Protein-Oligo Conjugation Kit", manufactured by Sollink) was used to conjugate DNA fragment 2.

[0121] (Preparation of nucleic acid detection reagent) In order to detect nucleic acid-modified antibodies by ICA reaction, an ICA reaction reagent was prepared as a nucleic acid detection reagent. The reagent composition of the ICA reaction reagent in this experimental example is as follows. The ICA reaction reagents in this experimental example were 0.1 μM flap probe 1 (SEQ ID NO: 4), 0.1 μM flap probe 2 (SEQ ID NO: 5), 1 μM invasion probe 1 (SEQ ID NO: 6), 1 μM invasion probe 2 (SEQ ID NO: 7), 2 μM FRET Cassette (SEQ ID NO: 8, Alexa488, manufactured by TOPPAN), 2 μM FRET Cassette (base sequence: 5′-RedmondRed-TCT-EclipseQuencher-TCGGCCTTTTGGCCGAGAGACTCCGCGTCCGT-3′: SEQ ID NO: 9), 10 mM MOPS (pH 7.9), 10 mM MgCl ２ and Flapend Nuclease. The above two types of FRET Cassettes correspond to the nucleic acid fragment 820 shown in Figure 10. The concentrations of these components in the ICA reaction reagent are the final concentrations in the ICA reaction reagent used in this experimental example.

[0122] Analysis of CD9 and amyloid beta in exosomes derived from N2a cells transfected with the APP gene. In this experiment, N2a cells transfected with the APP gene and N2a cells not transfected with the APP gene were cultured for 24 hours, and exosomes were recovered from the culture supernatant by ultracentrifugation. The components were separated by molecular weight using SDS-PAGE. 1 μg of exosomes was loaded onto each lane. Subsequently, Western blot analysis was performed using 6E10 (BioLegend) as an anti-amyloid beta antibody and BA2 (R&D Systems) as an anti-CD9 antibody to confirm whether the isolated exosomes contained CD9 and amyloid beta.

[0123] Figure 13 is an image of the Western blot analysis results showing the presence or absence of CD9 in exosomes contained in the culture supernatant of N2a cells transfected with the APP gene and N2a cells not transfected with the APP gene, respectively, cultured for 24 hours. Figure 14 is an image of the Western blot analysis results showing the presence or absence of amyloid beta in exosomes contained in the culture supernatant of N2a cells transfected with the APP gene and N2a cells not transfected with the APP gene, respectively, cultured for 24 hours. In Figures 13 and 14, lane "1" shows the analysis results of a sample derived from N2a cells not transfected with the APP gene, and lane "2" shows the analysis results of a sample derived from N2a cells transfected with the APP gene. The numerical values ​​shown in Figures 13 and 14 are in kDa (Daltons). The arrow in Figure 13 indicates the position of the CD9 band, and the arrow in Figure 14 indicates the position of the amyloid beta band.

[0124] As shown in Figures 13 and 14, in the sample of N2a cells into which the APP gene had not been introduced, a band indicating CD9 was confirmed, but no band indicating amyloid β was confirmed.

[0125] In contrast, in the sample of N2a cells into which the APP gene had been introduced, a band representing CD9 and a band representing amyloid β were clearly observed.

[0126] These results demonstrate that N2a cells transfected with the APP gene secrete exosomes bound to amyloid beta.

[0127] [Example 1: Measurement of the Percentage of Exosomes Bound with Amyloid β] In this experiment, the percentage of exosomes bound with amyloid β was measured among the exosomes obtained from the culture supernatant of N2a cells transfected with the APP gene. <Reaction of Exosomes, CTB-Immobilized Magnetic Beads, and Nucleic Acid-Modified Anti-Amyloid β Antibody and Nucleic Acid-Modified Anti-CD9 Antibody> CTB specifically binds to ganglioside GM1 present on the membrane surface of exosomes derived from brain neurons. Therefore, exosomes derived from brain neurons can be captured using CTB-immobilized magnetic beads. Specifically, as described below, brain neuron-derived exosomes were captured using CTB-immobilized magnetic beads, and the captured brain neuron-derived exosomes were bound to an amyloid β-specific binding substance and a CD9-specific binding substance to form a complex.

[0128] In the sample tube, 6 x 10 5 CTB-immobilized carboxyl magnetic beads (CTB beads), PBS adjusted to contain 0, 100, 300, or 600 ng of exosomes, nucleic acid-modified anti-amyloid beta antibody adjusted to 0.1 ng / mL, and nucleic acid-modified anti-CD9 antibody adjusted to 0.1 ng / mL were mixed to a total volume of 100 μL and reacted on a rotator at room temperature for 1 hour to form a complex. The exosomes used here were the above-mentioned exosomes obtained from the culture supernatant of N2a cells transfected with the APP gene. After the reaction, the obtained magnetic beads were magnetically collected using a magnetic stand, and washed five times by removing the supernatant and adding PBS containing 0.1% Tween (PBS-T). Finally, the supernatant was removed to obtain a complex. The obtained complex was diluted after washing to prepare each reaction mixture.

[0129] <Preparation of device> A substrate provided with many minute wells was made of COP (cycloolefin polymer), and a COP cover material was attached to it to prepare a device. ２ The total volume per well was 0.93 μL. The total number of wells used in the measurement was 1,000,000.

[0130] <Delivery of reaction mixture> 18 μL of each of the reaction mixtures described above was delivered to each well of the device, followed by delivery of 200 μL of FC-40 (Sigma) as a sealing liquid to seal each well.

[0131] <Nucleic Acid Detection Reaction> After each reaction mixture was delivered, the device was placed on a hot plate and reacted at 66°C for 25 minutes. This resulted in the recognition of the nucleic acid fragment modifying the anti-amyloid β antibody or anti-CD9 antibody by the flap probe and invasion probe, cleavage of the flap probe by FEN, binding of the released flap probe fragment to the FRET Cassette, and cleavage of the FRET Cassette by FEN, resulting in the emission of a fluorescent signal. When a nucleic acid-modified anti-amyloid β antibody labeled with DNA fragment 1 was present, RedmondRed fluorescence (red) was emitted, and when a nucleic acid-modified anti-CD9 antibody labeled with DNA fragment 2 was present, Alexa488 fluorescence (green) was emitted.

[0132] <Fluorescence Observation of Wells> After heating at 66°C for 25 minutes, fluorescent images of the fluorescent signals obtained by the nucleic acid detection reaction in each well in the device were taken using a fluorescent microscope (BZ-700, manufactured by KEYENCE) with a 4x objective lens and a GFP fluorescent filter. The exposure time was 3000 msec. The obtained fluorescent images of each reaction mixture are shown in Figures 15 and 16.

[0133] Fig. 15 is a fluorescence image of exosomes derived from brain neurons. Fig. 16 is a fluorescence image of exosomes derived from brain neurons bound to amyloid β. In Fig. 15 and Fig. 16, areas emitting green or red fluorescence are shown in bright colors.

[0134] FIG. 17 is a graph showing the relationship between the ratio of the number of fluorescent lights shown in FIGS. 15 and 16 to the number of magnetic beads and the amount of exosomes in the reaction mixture supplied to the device.

[0135] As shown in Figure 17, the red and green fluorescence intensities increased in proportion to the amount of exosomes in the reaction mixture supplied to the device. However, the ratio of the amount of exosomes bound to amyloid beta (based on the red fluorescence count, which is the amyloid beta detection result) to the total amount of exosomes (based on the green fluorescence count, which is the CD9 detection result) was approximately 50% regardless of whether the amount of exosomes in the reaction mixture supplied to the device was 100 ng, 300 ng, or 600 ng. For example, when the amount of exosomes in the reaction mixture supplied to the device was 100 ng, the ratio of the green fluorescence count, which represents the total amount of exosomes, to the number of beads was approximately 10%, while the ratio of the red fluorescence count, which represents the amount of exosomes bound to amyloid beta, to the number of beads was approximately 5%. Table 1 below shows the above ratios for exosome amounts of 100 ng, 300 ng, and 600 ng.

[0136]

[0137] These results demonstrate that the ratio of amyloid-β-bound exosomes to the total amount of exosomes in a sample can be accurately determined regardless of the exosome concentration in the reaction mixture. Note that it is not necessary to divide the fluorescence intensity, which represents the total amount of exosomes or the amount of amyloid-β-bound exosomes, by the number of beads.

[0138] Meanwhile, for the above nucleic acid-modified anti-CD9 antibody, the total exosome amount was measured using the following four types of antibodies or CTB instead of the anti-CD9 antibody with clone number 479608. The amount of exosomes bound to amyloid beta was not measured. Except for changing the anti-CD9 antibody, the total exosome amount (more specifically, the ratio of the number of fluorescent light to the number of magnetic beads) was measured in the same manner as in the above measurement example. Three exosome concentrations were prepared: 0 ng / mL, 0.1 ng / mL, and 0.5 ng / mL. 1. Anti-human CD9 antibody, rat monoclonal antibody (clone number: 77B, Fujifilm Wako Pure Chemical Industries, Ltd.) 2. Anti-human CD9 antibody, monoclonal antibody (clone number: 1K, Fujifilm Wako Pure Chemical Industries, Ltd.) 3. Anti-human CD63 antibody, monoclonal antibody (clone number: 3-13, Fujifilm Wako Pure Chemical Industries, Ltd.) 4. CTB (cholera toxin B subunit, Fujifilm Wako Pure Chemical Industries, Ltd.)

[0139] Figure 18 is a graph showing the results of detecting total exosome amounts when using the anti-human CD9 antibody clone number 77B. Figure 19 is a graph showing the results of detecting total exosome amounts when using the anti-human CD9 antibody clone number 1K. Figure 20 is a graph showing the results of detecting total exosome amounts when using the anti-human CD63 antibody clone number 3-13. Figure 21 is a graph showing the results of detecting total exosome amounts when using CTB. As shown in Figures 18 to 21, when the above four antibodies or CTB were used instead of the anti-CD9 antibody clone number 479608, no proportional relationship was observed between the ratio of fluorescence intensity to the number of magnetic beads and the exosome concentration. Therefore, it was revealed that the anti-CD9 antibody clone number 479608 is optimal for detecting the total exosome amount in a sample.

[0140] [Measurement Example 2 of the Proportion of Exosomes Bound with Amyloid β] Figure 22 is a schematic diagram showing the mixing of exosomes obtained from the culture supernatant of N2a cells transfected with the APP gene with N2a cells not transfected with the APP gene. In this experiment, exosomes obtained from the culture supernatant of N2a cells transfected with the APP gene were mixed with exosomes obtained from the culture supernatant of N2a cells not transfected with the APP gene. Then, for each mixing ratio, the number of green fluorescent spots indicating the total amount of exosomes and the number of red fluorescent spots indicating the amount of exosomes bound with amyloid β were measured. Then, for each mixing ratio, the ratio of the amount of exosomes bound with amyloid β to the total amount of exosomes was calculated. The measurement methods were the same as in Measurement Example 1 above. The total amount of exosomes was 600 ng.

[0141] Figure 23 is a graph showing the ratio of the number of green fluorescence signals, which indicates the total amount of exosomes, to the number of magnetic beads, and the ratio of the number of red fluorescence signals, which indicates the amount of exosomes bound to amyloid β.

[0142] Table 2 below shows the ratio of the amount of exosomes bound to amyloid beta to the total amount of exosomes for each mixing ratio of exosomes obtained from the culture supernatant of N2a cells into which the APP gene had been introduced to exosomes obtained from the culture supernatant of N2a cells into which the APP gene had not been introduced.

[0143]

[0144] As shown in Table 2, the higher the mixing ratio of exosomes obtained from the culture supernatant of N2a cells transfected with the APP gene, the higher the ratio of amyloid beta-bound exosomes to the total exosome amount. From the above, it became clear that even when exosomes obtained from different cells are mixed, the ratio of amyloid beta-bound exosomes to the total exosome amount in the sample can be accurately calculated.

[0145] [Detection of exosomes bound to Aβ protofibrils] In this experiment, exosomes bound to Aβ protofibrils were detected without containing magnetic beads in the wells. The reagents used in this experiment are listed below.

[0146] (Reagents) Magnetic particles for purification: Capture immobilized particles in MagCapture Isolation kit (FUJIFILM, Wako Pure Chemical Industries, Ltd.) Washing solution: Wash Buffer in MagCapture Isolation kit (FUJIFILM, Wako Pure Chemical Industries, Ltd.) Extraction solution: Elution buffer in MagCapture Isolation kit (FUJIFILM, Wako Pure Chemical Industries, Ltd.) Blocking agent: Blocker BSA in TBS (ThermoFisher) CTB (FUJIFILM, Wako Pure Chemical Industries, Ltd.) Exosome antibody: CD9 (Cosmo Bio Co., Ltd.) Aβ fibril antibody: Lecanemab (Eisai Biogen Co., Ltd.) Probe: Allele probe, ICA oligo Antibody-labeled oligos: IL28t, IL28c, MTHFR Fluorescent probes: Alexa488-BHQ, ATTO542-BHQ, ATTO643-BHQ Enzyme: FlapEndNuclease (FEN-1) Salts, Buffer: NaCl, MgCl2 (Wako Pure Chemical Industries, Ltd.), Tris-HCl (pH 8.5), Tween 20 (Sigma-Aldrich) Oil: FC-40 (Sigma-Aldrich) Plasma: EDTA plasma purified by centrifugation according to the MagCapture Isolation kit protocol (Apparatus) Magnetic stand (Takara Bio Inc.), hot plate (TAITEC), fluorescence microscope: BZ-710 (Keyence Corporation) (Devices) Micro-hole (well) COP chip (diameter of each well: 12.5 μm, depth: 15 μm), flow channel member (flow channel height: 30 μm), device member by laser welding

[0147] (Experimental Method) First, Capture-immobilized magnetic beads were mixed with EDTA plasma according to the MagCapture Isolation kit protocol and stirred at room temperature for 2 hours. The magnetic beads had TIM4 bound to their surface, making them capable of capturing exosomes.

[0148] Next, the magnetic beads that captured the exosomes were magnetically collected using a magnetic stand. After removing the supernatant, the wash buffer included in the MagCapture Isolation kit was added and the beads were washed by redispersion. Magnetic collection and washing were then repeated three times, and the elution buffer included in the MagCapture Isolation kit was added to the beads after the final wash. This buffer contained EDTA, which cleaved the bond between the magnetic beads containing TIM4 and the exosomes.

[0149] The resulting exosome extract was then collected to prepare an exosome solution. Capture-immobilized magnetic beads (magnetic beads having TIM4 on their surface) prepared according to the MagCapture Isolation kit protocol were blocked with a 5% BSA blocker and washed while being magnetically captured. The beads were then mixed with EV solution adjusted to various concentrations (0, 50, or 200 mL of plasma), 0.5 ng / mL nucleic acid (IL28t)-modified CTB, and 5 ng / mL nucleic acid-modified antibodies (IL28c-lecanemab (anti-Aβ protofibril antibody), MTHFR-CD9 (CD9 antibody)), and stirred at room temperature for 2 hours.

[0150] The magnetic beads that captured the exosomes were then magnetically collected using a magnetic stand. After removing the supernatant, the beads were washed by adding 0.1% Tween-supplemented wash buffer and redispersing. This buffer contained calcium, ensuring the capture of exosomes by TIM4. Magnetic collection and washing were repeated nine times, and after the final wash, the EDTA-containing elution buffer in the MagCapture Isolation kit was added to the beads to sever the bond between the TIM4-containing magnetic beads and the exosomes. After a stirring extraction reaction, the exosome extract was collected.

[0151] Next, the exosome extract and nucleic acid detection reagent were mixed, and the mixed solution was pumped into each well of the device. FC-40 was pumped twice to seal the chamber (well). The device was then heated on a hot plate at 66°C for 25 minutes to allow the ICA reaction to proceed.

[0152] Finally, after the device was cooled to room temperature, bright-field and fluorescent images were observed using a 4x objective lens under a microscope. The exposure times for each fluorescent light were as follows: Alexa 488: 2000 msec, ATTO 643: 2000 msec, and ATTO 542: 667 msec.

[0153] Figure 24 is an image showing the fluorescence counts representing the amount of Aβ protofibrils at each plasma concentration, Figure 25 is an image showing the fluorescence counts representing the amount of ganglioside GM1 at each plasma concentration, and Figure 26 is an image showing the fluorescence counts representing the amount of CD9 at each plasma concentration.

[0154] As shown in Figures 24 to 26, the fluorescence counts increased with increasing plasma concentration for Aβ protofibrils, ganglioside GM1, and CD9. These results demonstrate that Aβ protofibrils (polymers of amyloid beta), exosomes derived from brain neurons, and the amount of exosomes can be detected without placing magnetic beads, an example of a solid phase, in the wells.

[0155] According to the present invention, a technique can be provided for measuring the ratio of the amount of exosomes to which amyloid β is bound to the total amount of exosomes.

[0156] 100... Complex, 110... Exosome, 111... Amyloid beta, 112... Biomarker for brain nerve cell-derived exosomes, 113... Exosome marker, 114... Impurities, 115... TIM4, 116... Phospholipid phosphatidylserine, 120... Exosome marker-specific binding substance, 121... First nucleic acid fragment, 130... Invasive probe, 150... Solid phase, 151... Brain nerve cell-derived exosome marker-specific binding substance, 160... Amyloid beta-specific binding substance, 161... Second nucleic acid fragment, 200 , 500...fluidic device, 210...substrate, 220...lid member, 221...protrusion, 222...inlet port, 223...exhaust port, 230...flow path, 240...well array, 241...well, 242...sealed well (microcompartment), L210...reagent solution, L220...sealing liquid, 242R...well in which signal was detected, 510...wall member, 810...flap probe, 811...flap region (nucleic acid fragment), 821...second flap region (nucleic acid fragment), 820, 820'...nucleic acid fragment, F...fluorescent substance, Q...quencher.

Claims

1. A method for evaluating the level of amyloid beta accumulation in the brain, comprising the steps of: binding exosomes obtained from a blood sample to an exosome marker-specific binding substance that specifically binds to an exosome marker present on the surface of exosomes and is labeled with a first nucleic acid fragment; binding exosomes obtained from the blood sample to an amyloid beta-specific binding substance that specifically binds to amyloid beta and is labeled with a second nucleic acid fragment; detecting the amount of exosomes obtained from the blood sample using the first nucleic acid fragment; detecting the amount of exosomes bound with amyloid beta using the second nucleic acid fragment; and calculating the ratio of the amount of exosomes bound with amyloid beta to the amount of exosomes obtained from the blood sample.

2. A method for evaluating the level of amyloid beta accumulation in the brain, comprising the steps of: binding exosomes derived from brain neurons obtained from a blood sample to an exosome marker-specific binding substance that specifically binds to an exosome marker present on the surface of exosomes and is labeled with a first nucleic acid fragment; binding exosomes derived from brain neurons obtained from the blood sample to an amyloid beta-specific binding substance that specifically binds to amyloid beta and is labeled with a second nucleic acid fragment; detecting the amount of exosomes derived from brain neurons using the first nucleic acid fragment; detecting the amount of exosomes derived from brain neurons and bound to amyloid beta using the second nucleic acid fragment; and calculating the ratio of the amount of exosomes derived from brain neurons and bound to amyloid beta to the amount of exosomes derived from brain neurons obtained from the blood sample.

3. A method for evaluating the level of amyloid beta accumulation in the brain described in claim 2, further comprising a step of obtaining the exosomes derived from brain neurons from a blood sample prior to the step of binding the exosome marker-specific binding substance to the exosomes derived from brain neurons and the step of binding an amyloid beta-specific binding substance to the exosomes derived from brain neurons.

4. A method for evaluating the level of amyloid beta accumulation in the brain described in claim 3, wherein the step of obtaining the exosomes derived from brain neurons is carried out using a substance that specifically binds to ganglioside GM1, a marker for the exosomes derived from brain neurons.

5. The method for evaluating the level of amyloid-β accumulation in the brain according to claim 4, wherein the substance is cholera toxin subunit B (CTB).

6. A method for evaluating the level of amyloid beta accumulation in the brain according to any one of claims 1 to 5, wherein the exosome marker is any one of CD9, CD63, CD81, and phosphatidylserine.

7. A method for evaluating the accumulation level of amyloid β in the brain according to any one of claims 1 to 5, wherein the amyloid β is amyloid β monomer, amyloid β protofibril, or N3pG Aβ.

8. A method for evaluating the level of amyloid beta accumulation in the brain described in any one of claims 1 to 5, wherein the amyloid beta-specific binding substance is an antibody or antibody fragment that specifically binds to amyloid beta.

9. A method for evaluating the level of amyloid beta accumulation in the brain described in any one of claims 1 to 5, wherein the amyloid beta-specific binding substance is a therapeutic agent for Alzheimer's disease that specifically binds to amyloid beta.

10. A method for evaluating the level of amyloid beta accumulation in the brain according to any one of claims 1 to 5, wherein the step of detecting the amount of exosomes derived from brain neurons and the step of detecting the amount of exosomes derived from brain neurons and bound to amyloid beta are performed by invasive cleavage assay.

11. A method for evaluating the level of amyloid beta accumulation in the brain described in any one of claims 1 to 5, wherein the exosome marker-specific binding substance is an anti-CD9 antibody of clone number 479608.

12. The method for evaluating the level of amyloid beta accumulation in the brain according to claim 10, wherein the exosomes are captured with magnetic beads, and then the exosome marker-specific binding substance and the amyloid beta-specific binding substance are bound to the exosomes, and the subsequent invasive cleavage assay is performed in the well with the magnetic beads removed from the exosomes.

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