Method for testing for disease caused by extracellular vesicle, and diagnostic method

Abstract

The present invention provides a technology for testing for and diagnosing various diseases by supplementing and recovering extracellular vesicles (EVs) by a simple method and analyzing the characteristics of the recovered extracellular vesicles (EVs) rapidly and accurately.　This method for testing for disease comprises: a step (a) for supplementing and / or recovering a plurality of extracellular vesicles from a biological sample of a test subject; a step (b) for detecting the plurality of supplemented and / or recovered extracellular vesicles; and a step (c) for analyzing a pattern of the detected extracellular vesicles. Step (a) includes: a step (a1) for causing the extracellular vesicles to be adsorbed to a solid-phase carrier containing a base material and an ion exchanger and having the ability to adsorb extracellular vesicles; a step (a2) for labeling the extracellular vesicles adsorbed to the solid-phase carrier; and a step (a3) for washing the labeled extracellular vesicles while in the state of being adsorbed to the solid-phase carrier.

Description

Method for testing and diagnosing diseases using extracellular vesicles

[0001] The present invention relates to a method for testing and diagnosing diseases using extracellular vesicles.

[0002] Extracellular vesicles (EVs) are a collective term for lipid bilayer-enclosed particles that are released from cells and lack a nucleus (they cannot replicate). Recent research has revealed that EVs are released by almost all cells, including not only animals such as humans, but also plants, fungi, and bacteria.

[0003] Extracellular vesicles contain nucleic acids (DNA, mRNA, miRNA), proteins, lipids, various metabolic products, etc. Extracellular vesicles derived from animal cells are classified into exosomes, apoptotic vesicles, and microvesicles based on their production mechanism, and are known to play an important role in intercellular communication. Not only EVs released from tissue cells in various organs in the body, but also EVs derived from intestinal bacteria and food are known to be taken up by the body and delivered to distant organs via the bloodstream, where they function in vivo.

[0004] Recently, it has been thought that EVs may be the main active ingredients in functional foods, fermented foods, and probiotic preparations. It has also been revealed that the effects of cells used in treatments, such as mesenchymal stem cells (MSCs), can be substituted by the EVs they produce. As a result, advances in research on EVs have led to increased momentum in EV drug discovery, with over 100 formulations using EVs as active ingredients already undergoing clinical trials in the United States. EVs are considered to have great potential as targets for future product development and drug discovery.

[0005] In recent years, progress has been made in the development of methods for testing and diagnosing various diseases, particularly cancer, using EVs. For example, specific cancers can be detected by analyzing the specific molecules (nucleic acids (DNA, mRNA, miRNA), proteins, lipids, various metabolites, etc.) contained in exosomes secreted by cancer cells, and various test kits are already commercially available. Other research is also being conducted on the possibility of measuring PD-L1(+) EVs to predict the effectiveness of immune checkpoint inhibitors (Non-Patent Document 1) and the early diagnosis of neurodegenerative diseases using EV testing (Non-Patent Document 2).

[0006] Current technology requires that EVs be isolated, collected, or concentrated before they can be analyzed. Ultracentrifugation, for example, is a widely used method for collecting EVs. However, because ultracentrifugation separates or fractionates substances based on sedimentation velocity, it also contains other particles (e.g., endosomes) with similar properties and concentrations, as well as high-mass proteins such as IgM. This makes it difficult to selectively capture and collect specific EVs. Furthermore, the high pressures applied by ultrahigh-speed centrifugation raise concerns about EV deformation and functional impairment.

[0007] Another method for effectively capturing, quantifying, and characterizing EVs is immunoprecipitation, which uses antibodies (e.g., EV markers such as CD9, CD63, and CD81) that specifically bind to specific proteins (antigens) on the surface of EVs. Because immunoprecipitation is based on an antigen-antibody reaction, it is possible to recover highly purified EVs of interest from a mixture. For example, a sample containing EVs is mixed with magnetic beads pre-conjugated with antibodies, allowing the EVs to bind to the antibodies, forming an EV-antibody-magnetic bead complex. The magnetic beads are then collected using a magnet, and finally, the EVs are eluted and recovered. However, recovery of EVs that do not possess specific proteins (antigens) on their surface is difficult, and the EVs that can be captured are limited to those that can be recognized by the antibody. Furthermore, this method is technically complex and laborious, requiring a certain level of expertise, and EVs may be damaged during processing.

[0008] Furthermore, quantification and characterization of EVs require a combination of techniques such as electron microscopy, nanoparticle tracking analysis, immunoprecipitation, and high-resolution flow cytometry (Non-Patent Document 3), all of which require time-consuming procedures. Therefore, a simple and efficient method for analyzing EVs, along with the aforementioned EV capture techniques, is highly desirable.

[0009] Flow cytometry, in particular, can rapidly obtain information on individual EVs, making it a potentially very effective tool for quantification and characterization of EVs. Cell flow cytometry analysis can generally be performed without the need for sample purification and / or concentration. However, conventional flow cytometry-based EV analysis often requires purification to remove unbound reagents, such as fluorescent antibodies used to label EVs, as well as reagents added prior to staining, such as anticoagulants and cell culture medium additives (e.g., phenol red). These purification steps require time, effort, and expense, and can result in sample loss.

[0010] https: / / crisp-bio.blog.jp / archives / 11218358.htmlFront Mol Neurosci. 2020 Mar 19:13:38. doi: 10.3389 / fnmol.2020.00038. eCollection 2020J Extracell Vesicles. 2023 Feb;12(2):e12299. doi: 10.1002 / jEV2.12299

[0011] Therefore, an object of the present invention is to provide a technology for testing and diagnosing various diseases by capturing and recovering extracellular vesicles (EVs) in a simple manner and analyzing the characteristics of the recovered extracellular vesicles (EVs) quickly and with high accuracy.

[0012] In light of the above objectives, the inventors have discovered that EVs can be analyzed quickly and easily with high sensitivity and accuracy by capturing EVs on a substrate coated with an ion exchanger, labeling the EVs with substances such as antibodies that specifically bind to specific proteins or glycans on the EV surface while the EVs are still adsorbed to the ion exchanger, detaching the labeled EVs from the ion exchanger and recovering them, and detecting the labeled EVs using methods such as flow cytometry. They have thus completed the present invention, discovering that EVs can be analyzed quickly and easily with high sensitivity and accuracy, and that various diseases can be tested and diagnosed from the analysis patterns.

[0013] That is, the method for testing for diseases caused by extracellular vesicles of the present invention comprises: (a) capturing and / or recovering a plurality of extracellular vesicles from a biological sample of a subject; (b) detecting the captured and / or recovered plurality of extracellular vesicles; and (c) analyzing the pattern of the detected extracellular vesicles, wherein the step (a) comprises: (a1) adsorbing the extracellular vesicles to a solid phase carrier comprising a substrate and an ion exchanger and having the ability to adsorb the extracellular vesicles; (a2) labeling the extracellular vesicles adsorbed to the solid phase carrier; and (a3) ​​washing the labeled extracellular vesicles while they are still adsorbed to the solid phase carrier.

[0014] The step (a2) of labeling the extracellular vesicles is preferably carried out using an extracellular vesicle detection substance.

[0015] The extracellular vesicle detection substance is preferably at least one selected from the group consisting of an antibody, a lectin, a membrane-reactive substance, and a nucleic acid-reactive substance.

[0016] Step (b) is preferably carried out by analyzing the captured and / or recovered extracellular vesicles by immunoassay.

[0017] Step (a) may include, after step (a3), step (a4) of detaching the washed extracellular vesicles from the solid phase carrier.

[0018] Step (b) is preferably carried out by analyzing the captured and / or recovered extracellular vesicles by flow cytometry.

[0019] The ion exchanger is preferably a weak anion exchanger having a secondary and / or tertiary amino group as a partial structure.

[0020] The ion exchanger is preferably a monovalent or polyvalent residue derived from polyethyleneimine.

[0021] The polyethyleneimine is preferably a branched high molecular weight polyethyleneimine.

[0022] The ion exchanger and the substrate are preferably bonded to each other by a covalent bond with or without a linking moiety.

[0023] The ion exchanger and the substrate are bonded via a linking moiety, and the linking moiety preferably contains a silicon-carbon bond.

[0024] The substrate is preferably made of glass, plastic or metal oxide.

[0025] The substrate is preferably in the form of a plate or particles.

[0026] The substrate is preferably a glass slide or a well plate.

[0027] The method for testing a disease using extracellular vesicles of the present invention is particularly preferably used when the disease is cancer, dementia, a metabolic disease, an inflammatory disease, a circulatory system disease, a digestive system disease, a neurodegenerative disease, a genetic disease, an infectious disease, or aging.

[0028] The method for diagnosing a disease caused by extracellular vesicles of the present invention comprises, in addition to the method for testing a disease caused by extracellular vesicles, a step of determining the disease of the subject based on the pattern of the extracellular vesicles analyzed in step (c).The diagnostic method of the present invention also includes a method for assisting in the diagnosis of a disease and a method for determining a disease.

[0029] The method for diagnosing a disease using extracellular vesicles, the method for assisting in the diagnosis of a disease, and the method for determining a disease of the present invention are particularly preferably used when the disease is cancer, dementia, a metabolic disease, an inflammatory disease, a cardiovascular disease, a digestive disease, a neurodegenerative disease, a genetic disease, an infectious disease, or aging.

[0030] The disease test kit of the present invention can carry out the disease test method using extracellular vesicles.

[0031] The disease test kit of the present invention can be used for diagnosing, assisting in diagnosis, and determining diseases.

[0032] The disease testing method of the present invention uses an ion exchanger to capture extracellular vesicles (EVs), labels the EVs with substances such as antibodies while they are still trapped on the ion exchanger, and then washes away impurities (such as unbound detection substances). This allows for the capture and recovery of EVs at low cost and with less effort than methods that use antibodies (immunoprecipitation), without requiring a special purification step. EVs labeled while adsorbed to the ion exchanger can be subjected to a subsequent detection step either while still adsorbed to the ion exchanger or after being recovered from the ion exchanger, allowing for simple detection of EVs.

[0033] Furthermore, the method of the present invention can comprehensively capture EVs that do not have specific antigens, making it possible to collect and analyze such EVs, and thus enabling the easy testing and diagnosis of various diseases.

[0034] Conventional testing and diagnostic methods using EVs have mainly focused on analyzing miRNAs and other proteins contained within EVs, but the method of the present invention stains EV surface markers and analyzes them using flow cytometry, etc., making it easier and less costly to perform than conventional methods.

[0035]

[0033] Figure 1 is a graph showing the results of flow cytometry analysis of EVs from plasma samples of human healthy subjects and human breast cancer patients, which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against human EpCAM, and analyzed using flow cytometry.

[0034] Figure 1 is a graph showing the results of flow cytometry analysis of EVs from plasma samples of human healthy subjects and human breast cancer patients, which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against human GPNMB, and analyzed using flow cytometry.

[0035] Figure 1 is a graph showing the results of flow cytometry analysis of EVs from plasma samples of human healthy subjects and human gastric cancer patients, which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against human EpCAM, and analyzed using flow cytometry.

[0036] Figure 1 is a graph showing the results of flow cytometry analysis of EVs from plasma samples of human healthy subjects and human gastric cancer patients, which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against human CA19-9, and analyzed using flow cytometry.

[0023] Figure 1 shows graphs illustrating the results of flow cytometry analysis of EVs from plasma samples of healthy humans and human gastric cancer patients, which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against human CD54 (ICAM-1), and analyzed using flow cytometry.

[0024] Figure 1 shows graphs illustrating the results of flow cytometry analysis of EVs from plasma samples of healthy humans and human gastric cancer patients, which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against human HER2, and analyzed using flow cytometry.

[0025] Figure 1 shows graphs illustrating the results of flow cytometry analysis of EVs from plasma samples of healthy humans and human Alzheimer's disease patients (AD patients), which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against human NCAM, and analyzed using flow cytometry.

[0026] Figure 1 shows graphs illustrating the results of flow cytometry analysis of EVs from plasma samples of healthy humans and human Alzheimer's disease patients (AD patients), which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against amyloid β, and analyzed using flow cytometry. 1 is a graph showing the results of flow cytometry analysis of EVs from plasma samples of healthy humans and Alzheimer's disease (AD) patients, which were immobilized on a solid support using the method of the present invention and stained with a fluorescently labeled antibody against human ICAM-1.FIG. 1 is a graph showing the results of flow cytometry analysis of EVs from plasma samples of healthy human subjects and human Alzheimer's disease patients (AD patients), which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against aggregated amyloid β (647-Aβ(aggregate)), and analyzed using flow cytometry. FIG. 2 is a graph showing the results of flow cytometry analysis of EVs from plasma samples of healthy human subjects and human Alzheimer's disease patients (AD patients), which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against human ICAM-1, and analyzed using flow cytometry. FIG. 3 is a graph showing the results of flow cytometry analysis of EVs from plasma samples of healthy human subjects and human Alzheimer's disease patients (AD patients), which were immobilized on a solid phase carrier by the method of the present invention, stained with a fluorescently labeled antibody against GPNMB.

[0036] The present invention is described in detail below, but is not limited thereto. The following descriptions of solid phase carriers, kits, detection methods, separation / purification methods, testing / diagnosis methods, immobilization methods, etc. can be mutually referenced and incorporated.

[0037] The method for testing and diagnosing diseases caused by extracellular vesicles of the present invention is characterized by the fact that extracellular vesicles can be detected by a simple operation by efficiently capturing extracellular vesicles using a solid phase carrier comprising a substrate and an ion exchanger and extracting them as needed. Before describing the testing and diagnosing methods of the present invention, extracellular vesicles and solid phase carriers will first be described.

[0038] [1] Extracellular vesicles. In this application, the term "extracellular vesicles (EVs)" is used in the sense commonly used in the art, referring to, for example, lipid bilayer-enclosed particles without a nucleus (non-replicating) that are released from cells. Extracellular vesicles derived from animal cells are primarily classified into exosomes, microvesicles, and apoptotic vesicles. Exosomes are endosomal membrane-derived vesicles formed during endocytosis, measuring approximately 50-200 nm in size, and are composed primarily of lipids, proteins, and nucleic acids. Microvesicles are vesicles with a wide size distribution ranging from 100-1,000 nm. They differ from exosomes in that they bud directly from the cell membrane and are secreted extracellularly, but it is difficult to completely distinguish them from exosomes. Apoptotic vesicles are particles budded from the membrane of cells undergoing apoptosis (cell death), measuring approximately 50-5,000 nm in size.

[0039] Extracellular vesicles have on their surface lipids, proteins, carbohydrates, etc. derived from the cell membrane of the cell from which they were secreted, and contain nucleic acids such as mRNA and miRNA and proteins inside, thus containing information derived from the cell that released them. The extracellular vesicles targeted by the present invention may be extracellular vesicles derived from any living cell, and include extracellular vesicles derived from animals (including humans), plants, fungi, microorganisms (including bacteria, yeast, etc.), etc.

[0040] [2] Solid-phase carriers are composite materials comprising a substrate and an ion exchanger. The inclusion of the ion exchanger allows the solid-phase carrier to adsorb extracellular vesicles and, if necessary, release them.

[0041] The ion exchanger is immobilized on the substrate by, for example, directly or indirectly bonding to a part or all of the surface of the substrate. The bonding mode between the substrate and the ion exchanger is not particularly limited, and the bonding may be by a covalent bond or by an attractive interaction other than a covalent bond. The ion exchanger can be directly bonded to the substrate by a modification reaction. Alternatively, the ion exchanger can be indirectly bonded to the substrate by a multi-step modification reaction. The bonding mode is preferably a covalent bond.

[0042] When an ion exchanger is indirectly bonded to a substrate by a multi-step modification reaction, the other modified portion between the substrate and the ion exchanger is called a linking part (also called a linker, spacer, etc.), and in this case, the ion exchanger is said to be indirectly bonded to the surface of the substrate via the linking part.

[0043] For example, by using a silane coupling agent (e.g., 3-hydroxypropyltriethoxysilane, 3-aminopropyltriethoxysilane, etc.) having a functional group at the end for introducing an ion exchanger, the functional group can be covalently linked to the surface of glass (substrate). By reacting diethylaminoethyl chloride hydrochloride with the functional group thus introduced onto the glass surface, a glass surface containing diethylaminoethyl can be obtained. Similarly, by reacting the functional group with glutaraldehyde and 2-diethylaminoethylamine as crosslinkers, a glass surface containing diethylaminoethyl can be obtained. In this case, the diethylaminoethyl (DEAE) portion is referred to as the ion exchanger, and the portion closer to the glass is referred to as the linking portion.

[0044] A commercially available slide glass coated with a functional group such as an amino group may also be used.

[0045] (1) Ion Exchanger Ion exchangers are classified into strong anion exchangers, weak anion exchangers, strong cation exchangers, and weak cation exchangers. As the ion exchangers used in the present invention, strong anion exchangers and weak anion exchangers are preferred, and weak anion exchangers are more preferred.

[0046] The strong anion exchanger refers to an ion exchanger that retains cationicity even at high pH, ​​and has a partial structure such as quaternary ammonium, N,N-dialkylimidazolium, N-alkylpyridinium, etc. Specific examples include partial structures such as tetraalkylammonium (e.g., trimethylammonioethyl, trimethylammoniopropyl, triethylammonioethyl, triethylammoniopropyl, etc.), 1-alkylpyridinium (e.g., 1-methylpyridinium-4-yl, 1-ethylpyridinium-3-yl, etc.), 1,3-dialkylimidazolium (e.g., 1,3-dimethylimidazolium-4-yl, etc.).

[0047] A weak anion exchanger refers to an ion exchanger that loses cationicity at high pH and has a monovalent or polyvalent residue partial structure derived from primary, secondary, or tertiary amines, imidazole, monoalkylimidazole, N-unsubstituted pyridine, or the like. Specific examples include partial structures such as secondary amino groups (e.g., methylaminoethyl, ethylaminoethyl, methylaminopropyl, etc.), tertiary amino groups (e.g., dimethylaminoethyl, dimethylaminopropyl, diethylaminoethyl, diethylaminopropyl, pyridyl, 1-methylimidazolyl, 1-ethylimidazolyl, etc.), and monovalent or polyvalent residue partial structures derived from polyethyleneimine. In the present invention, diethylaminoethyl (DEAE) and monovalent or polyvalent residues derived from polyethyleneimine are preferably used. In this specification, a monovalent or polyvalent residue derived from polyethyleneimine may be referred to as PEI.

[0048] Any type of polyethyleneimine can be used, for example, unbranched linear low-molecular-weight polyethyleneimine, branched low-molecular-weight polyethyleneimine, unbranched linear polyethyleneimine, branched high-molecular-weight polyethyleneimine, etc., and among these, branched high-molecular-weight polyethyleneimine is preferably used. Since the selectivity of the extracellular vesicles to be adsorbed varies depending on the type of polyethyleneimine, an appropriate type is selected depending on the purpose.

[0049] A strong cation exchanger refers to an ion exchanger that retains anionicity even at low pH and has a partial structure such as a strongly acidic group (partial structure) such as a sulfonic acid group (sulfo group) or a phosphonic acid group. Specific examples of such partial structures include a sulfophenyl group and a sulfoalkyl group (e.g., a sulfomethyl group, a sulfoethyl group, a sulfopropyl group, etc.).

[0050] A weak cation exchanger is an ion exchanger that loses its anionicity at low pH and has a weakly acidic group (partial structure) such as a carboxylic acid group (carboxyl group).Specific examples include carboxyalkyl groups (e.g., carboxymethyl, carboxyethyl, etc.).

[0051] (2) Substrate: A substrate is a material capable of supporting an ion exchanger. As described above, an ion exchanger is immobilized by directly or indirectly binding to a portion or all of the surface of the substrate to form a solid phase carrier. Substrates that can be used in the present invention are not particularly limited, and examples include instruments, tools, materials, and the like commonly used in the fields of biological detection, quantification, or separation / purification. Specific examples include glass slides, cover glasses, well plates, microplates, dishes, flasks, resin substrates for column packings, and separation beads (e.g., magnetic beads).

[0052] The substrate material is not particularly limited, and any substrate material (including instruments, tools, materials, etc.) commonly used in the fields of biological detection methods, quantification methods, or separation / purification methods can be used. Examples of substrate materials include glass, silicon, plastic, metal, ceramic, semiconductor, resin, polysaccharide, paper, cloth, etc. In the present invention, glass, silicon, plastic, ceramic, or resin can be particularly preferably used.

[0053] The shape of the substrate is not particularly limited, and substrates (including instruments, tools, materials, etc.) commonly used in the fields of biological detection methods, quantification methods, or separation / purification methods can be used. Examples of substrate shapes include plate-like, particulate, thread-like, net-like, fibrous, membrane-like, dish-like, and cylindrical shapes. Among these, plate-like or particulate (bead-like) substrates are preferred for ease of handling. The thickness of the plate-like substrate and the size of the particulate substrate are not particularly limited and may be appropriately set within a range that is preferable for ease of handling. These substrates may be porous. As the plate-like substrate, slide glasses are preferred, and well plates are particularly preferred. Furthermore, those that have been treated with a water-repellent finish (such as highly water-repellent printed slide glasses) are preferably used.

[0054] A particulate solid phase carrier may be prepared by immobilizing an ion exchanger on the surface of a particulate substrate. Such a particulate solid phase carrier can be used as a column packing material by filling it in a column.

[0055] The substrate forming the column packing may be either inorganic or organic, and examples of inorganic materials include silicon, titanium, zinc, aluminum, etc., while examples of organic materials include polysaccharides such as agarose, cellulose, chitin, chitosan, amylose, heparin, hyaluronic acid, pectin, polyacrylamide, polyacrylic acid, polystyrene, polyvinyl alcohol, polymethacrylic acid, polyacrylic acid, polymethacrylic acid esters, polyacrylic acid esters, and derivatives thereof. The particulate substrate forming the column packing may be porous or non-porous particles.

[0056] (3) Adsorption Ability: The ion exchanger constituting the solid phase carrier is responsible for the adsorption of extracellular vesicles. The ion exchanger imparts the solid phase carrier with the ability to adsorb extracellular vesicles. In other words, the solid phase carrier containing the ion exchanger has the ability to adsorb extracellular vesicles.

[0057] Ion exchangers have different adsorption capacities for various extracellular vesicles, and by selecting an ion exchanger appropriate for the purpose, the type of extracellular vesicles that can be adsorbed by the solid phase carrier can be selected. For example, when diethylaminoethyl (DEAE) is used as the ion exchanger, the solid phase carrier can adsorb any extracellular vesicles, regardless of their origin (e.g., human, plant, or bacterial).

[0058] [3] Method for testing for diseases using extracellular vesicles The method for testing for diseases in a subject using extracellular vesicles of the present invention comprises the steps of: (a) capturing and / or recovering a plurality of extracellular vesicles from a biological sample of the subject; (b) detecting the captured and / or recovered plurality of extracellular vesicles; and (c) analyzing the pattern of the detected extracellular vesicles, wherein the step (a) comprises the steps of: (a1) adsorbing the extracellular vesicles to a solid phase carrier comprising a substrate and an ion exchanger and capable of adsorbing the extracellular vesicles; (a2) labeling the extracellular vesicles adsorbed to the solid phase carrier; and (a3) ​​washing the labeled extracellular vesicles while still adsorbed to the solid phase carrier. These steps are described in detail below.

[0059] (a) Step of capturing and / or recovering a plurality of extracellular vesicles from a biological sample of a subject [Step (a)] Step (a) of capturing and / or recovering a plurality of extracellular vesicles from a biological sample of a subject includes a step (a1) of adsorbing the extracellular vesicles to a solid phase carrier containing a substrate and an ion exchanger and capable of adsorbing the extracellular vesicles, a step (a2) of labeling the extracellular vesicles adsorbed to the solid phase carrier, and a step (a3) ​​of washing the labeled extracellular vesicles while they are still adsorbed to the solid phase carrier. If necessary, after step (a3), a step (a4) of detaching the washed extracellular vesicles from the solid phase carrier may be included.

[0060] (a1) Step of adsorbing extracellular vesicles [Step (a1)] Extracellular vesicles are adsorbed onto a solid phase carrier that includes a substrate and an ion exchanger and has the ability to adsorb extracellular vesicles. In the present application, this step may also be simply referred to as the adsorption step.

[0061] In the adsorption step, the extracellular vesicles to be detected are immobilized on a solid phase by adsorbing them to the solid phase. This step allows the target extracellular vesicles to be efficiently immobilized on the solid phase. Furthermore, the target extracellular vesicles can be efficiently labeled by labeling the extracellular vesicles while they are still adsorbed on the solid phase (see the labeling step described below). The extracellular vesicles to be adsorbed on the solid phase are usually provided as a solution containing the extracellular vesicles (also referred to as a liquid sample). By contacting this liquid sample with the solid phase, the target extracellular vesicles are adsorbed to the solid phase.

[0062] The liquid sample may be, for example, a solution containing purified (including partially purified) extracellular vesicles, or a solution containing unpurified extracellular vesicles. Examples of unpurified liquid samples include plasma, serum, urine, saliva, tears, cerebrospinal fluid, bronchoalveolar lavage fluid, plant juice, fruit juice, cell culture supernatant, and fungal or bacterial culture supernatant.

[0063] Although not particularly limited, the liquid sample solution is preferably adjusted to be isotonic with body fluids, so that the extracellular vesicles are not deformed by osmotic pressure when the liquid sample comes into contact with the solid phase carrier.

[0064] It is preferable to adsorb extracellular vesicles to the solid support by contacting the liquid sample with the solid support. When the substrate is a solid support that is a variety of detection plates such as slide glasses, cover glasses, well plates, and microplates, or a cell culture vessel such as a dish or flask, the liquid sample is placed on the surface of the solid support and allowed to stand for a predetermined period of time, thereby adsorbing and immobilizing the extracellular vesicles to the solid support. Even if the target is, for example, extracellular vesicles that tend to float, immobilization can be easily achieved without the need for centrifugal force using a centrifuge or the like. The adsorption reaction is preferably carried out at a temperature ranging from 0 to 60°C and allowed to stand for at least 2 minutes, more preferably at least 3 minutes. The reaction temperature and reaction time may be adjusted appropriately depending on the sample and support used. The reaction temperature is preferably 1 to 37°C. The upper limit of the reaction time is not particularly limited, but from the viewpoint of work efficiency, it is preferably within 10 hours, and more preferably within 1 hour.

[0065] After the extracellular vesicles are adsorbed onto the solid support, the solid support may be washed with a washing solution, if necessary. For example, by washing the solid support with a washing solution under conditions that prevent the extracellular vesicles from being released from the solid support, unintended impurities can be removed more efficiently. As the washing solution, a solution that has little effect on the extracellular vesicles, such as PBS (phosphate buffered saline), can be preferably used. Washing may be performed using multiple washing solutions.

[0066] After EV adsorption, blocking may be effective in reducing nonspecific staining. Blocking agents include those using proteins such as serum, bovine serum albumin (BSA), and skim milk. Treatment with these blocking agents may be performed as needed.

[0067] (a2) Step of labeling extracellular vesicles adsorbed to a solid phase carrier [Step (a2)] In the adsorption step, extracellular vesicles are adsorbed to a solid phase carrier, and after blocking if necessary, the extracellular vesicles are labeled with an extracellular vesicle detection substance (hereinafter also referred to as a detection substance) while still adsorbed to the solid phase carrier. Note that in the present application, this step may also be simply referred to as a labeling step.

[0068] In the present invention, extracellular vesicles are indirectly detected by labeling them with a detection substance and detecting the label in step (b). Any detection substance commonly used in the art can be used without particular limitations. Typically, the detection substance is appropriately selected depending on the type of detection method and the type of extracellular vesicles to be detected. Examples of detection substances include antibodies [e.g., (FITC-labeled) anti-CD9 antibody, (rhodamine-labeled) anti-CD63 antibody], lectins, membrane-reactive substances [e.g., (rhodamine-labeled) polymyxin B, (FITC-labeled) vancomycin, DiI dye], nucleic acid-reactive substances [e.g., SYTO], and cytofluorescent stains [e.g., CFDA-SE]. These detection substances can specifically label the target extracellular vesicles by recognizing and binding to markers (proteins, sugar chains, etc.) on the surface of the target extracellular vesicles or nucleic acids within the extracellular vesicles. A single detection substance can be used, or two or more can be used in combination.

[0069] It is preferable that the detection substance be directly or indirectly bound to a signal substance capable of generating a detectable signal. Alternatively, the detection substance itself may be a substance that generates a signal. Examples of detection substances to which a signal substance is directly bound include reagents such as antibodies covalently bound to fluorescent dyes and nucleic acid binding molecules. Examples of detection substances to which a signal substance is indirectly bound include reagents such as secondary antibodies covalently bound to signal substances (note that in the present invention, antibodies (primary antibodies) before being captured by secondary antibodies are also referred to as detection substances). When using antibodies, labeling can be achieved by reacting them at 4 to 37°C for about 30 minutes.

[0070] Examples of signal substances include fluorescent substances and radioisotopes, which are substances that generate signals by themselves, as well as enzymes (e.g., alkaline phosphatase, peroxidase, β-galactosidase, luciferase) that generate signals by catalyzing the reaction of other substances. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of radioisotopes include 125I, 14C, and 32P.

[0071] (a3) Step of washing the labeled extracellular vesicles while they are still adsorbed to the solid phase carrier [Step (a3)] After labeling the extracellular vesicles with the detection substance, the solid phase carrier is washed with a washing solution while the extracellular vesicles are still adsorbed to the solid phase carrier. Note that in the present application, this step may also be simply referred to as the washing step. By washing the solid phase carrier with a washing solution under conditions that do not cause the extracellular vesicles to be released from the solid phase carrier, unintended impurities (such as unbound detection substance) can be efficiently removed.

[0072] As a washing solution, a solution that has little effect on extracellular vesicles, such as phosphate buffered saline (PBS), can be preferably used. Washing may be performed using multiple washing solutions, for example, after washing with PBS, further washing may be performed with ion-exchanged water, distilled water, etc.

[0073] For example, when using a 96-well plate, the supernatant is removed, followed by the addition of PBS and repeated suction. In the case of a flat glass slide, the slide can be washed by immersing it in a container filled with ion-exchanged water and shaking it.

[0074] (a4) Step of detaching labeled extracellular vesicles from the solid phase carrier [Step (a4)] After washing the labeled extracellular vesicles while they are still adsorbed to the solid phase carrier, the target extracellular vesicles adsorbed to the solid phase carrier are detached. Note that in the present application, this step may also be referred to simply as the detachment step. This operation allows extracellular vesicles with fewer impurities than in the initial liquid sample to be extracted in a state labeled with the detection substance, thereby obtaining a solution containing a high concentration of labeled extracellular vesicles. Note that if the labeled extracellular vesicles are analyzed while still adsorbed to the solid phase carrier, this detachment step can be omitted.

[0075] Extracellular vesicles can be released from the solid support by increasing the ionic strength (salt concentration) of the buffer, changing the pH, scraping, or other methods. Methods for increasing the ionic strength include elution with a high-concentration salt solution (e.g., 1M NaCl solution). When elution is performed by changing the pH, the eluent should be appropriately selected depending on the type of ion exchanger that makes up the solid support. When an anion exchanger is used as the ion exchanger, adsorbed extracellular vesicles can be released by using an alkaline eluent. Conversely, when a cation exchanger is used as the ion exchanger, adsorbed extracellular vesicles can be released by using an acidic eluent.

[0076] In addition, EVs can be detached from the solid support by scraping them off the solid surface using a cell scraper, which is used to detach adherent cells.

[0077] (b) Step of detecting the captured and / or recovered extracellular vesicles [Step (b)] The captured and / or recovered extracellular vesicles, i.e., the labeled extracellular vesicles released from the solid phase carrier (or remaining adsorbed), can be indirectly detected by detecting the signal generated from the labeled detection substance (a signal generated from a signal substance directly or indirectly bound to the detection substance) using various techniques. When the signal substance is an enzyme that generates a signal by catalyzing the reaction of another substance, the signal can be detected using a substrate for the enzyme. Examples of measurement techniques for detecting signals include flow cytometry.

[0078] Furthermore, for example, when using an immunoassay method, it is possible to analyze labeled extracellular vesicles while they are still adsorbed to the solid phase without being released from the solid phase.

[0079] (1) Flow cytometry is an experimental technique based on the detection of fluorescence, which can simultaneously analyze various properties of suspended objects (cells, extracellular vesicles, etc.) such as the number of objects and the amount of protein expressed by each object. Below, we will explain the specific method using extracellular vesicles as the object.

[0080] In flow cytometry, a suspension of extracellular vesicles stained with fluorescent dyes is passed through a thin stream, and the line of extracellular vesicles is adjusted so that they pass through a light source one after another. The scattering of the light source and the fluorescence emitted by each extracellular vesicle are measured at various wavelengths as they pass through. By using a combination of fluorescent molecules with different excitation and emission wavelengths, multiple parameters can be read simultaneously from a single extracellular vesicle. Fluorescent molecules that bind to specific components of extracellular vesicles can be used, or fluorescently labeled antibodies that recognize specific proteins, glycans, etc. can be used.

[0081] Flow cytometry can obtain quantitative information at the single extracellular vesicle level (e.g., the number of specific types of extracellular vesicles, the amount of specific proteins or glycans expressed on the membrane surface of those extracellular vesicles, and the amount of specific proteins or nucleic acids expressed within the extracellular vesicles by permeabilizing the membrane). It can also analyze the correlation between these multiple pieces of information for each single extracellular vesicle. Flow cytometry is particularly effective for identifying and quantifying the types of extracellular vesicles, taking advantage of the fact that different types of extracellular vesicles express different proteins on the cell surface. Antibodies specifically recognize and bind to specific proteins, and extracellular vesicles can be labeled with fluorescent dyes to distinguish them from other types of extracellular vesicles. This process is called phenotyping.

[0082] Phenotypic analysis uses antibodies labeled with fluorescent dyes. By labeling antibodies targeting different proteins with different fluorescent dyes, they can be analyzed simultaneously using a flow cytometer. Analysis using multiple antibodies simultaneously, also known as multiplex analysis, allows for simultaneous analysis using two or more antibodies. For example, in immunophenotypic analysis, depending on the antibodies used, it is possible to simultaneously identify multiple types of immune cell-derived extracellular vesicles, such as T cell-derived extracellular vesicles, B cell-derived extracellular vesicles, and monocyte-derived extracellular vesicles, from a single sample.

[0083] Flow cytometry is a suitable method for phenotypic analysis because it is very fast and can quantitatively analyze thousands or tens of thousands of extracellular vesicles in a short period of time. Another advantage of phenotypic analysis by flow cytometry is that extracellular vesicles sorted by phenotype can be used for analysis. Therefore, it is possible to analyze the function of specific extracellular vesicle populations after analysis depending on the analysis results.

[0084] (2) Immunoassay method When using an immunoassay method (e.g., ELISA), extracellular vesicles can be detected while still adsorbed to a solid phase. In this case, a detection reagent containing an appropriate detection substance (e.g., a fluorescently labeled antibody, an enzyme-labeled antibody, or a substrate for the enzyme) is selected and used according to the immunoassay method.

[0085] When detecting extracellular vesicles by immunoassay (e.g., ELISA), various extracellular vesicles can be quantified by binding a specific antibody to the target extracellular vesicles and detecting the intensity of fluorescence directly or indirectly bound to the antibody or the enzymatic reaction of a labeled enzyme using an appropriate substrate. Quantification by immunoassay is advantageous in that it can be performed relatively easily.

[0086] An example of the quantification (quantification step) of extracellular vesicles immobilized on a solid-phase carrier (the substrate is a detection plate such as a well plate or microplate) using an immunoassay method is shown below. First, extracellular vesicles are adsorbed and immobilized on the solid-phase carrier. If necessary, the solid-phase carrier is blocked with a blocking solution, and then an antibody specific to the extracellular vesicles of interest (e.g., an antibody specific to a protein on the surface of the extracellular vesicles of interest) is contacted with the solid-phase carrier to label the extracellular vesicles. After removing excess antibody using a washing solution, if necessary (if the antibody is not labeled), an enzyme-labeled secondary antibody is bound to the antibody. After that, excess antibody is removed using a washing solution, and then a substrate for the labeling enzyme is contacted with the solid-phase carrier to cause an enzymatic reaction. The product of the enzymatic reaction is detected and quantified, allowing the amount of extracellular vesicles of interest to be quantified.

[0087] Among immunoassay methods, the use of ELISA (Enzyme-Linked Immunosorbent Assay) is particularly preferred. ELISA is a method in which a target antigen or antibody contained in a sample solution is captured by a specific antibody or antigen and then detected and quantified using an enzymatic reaction.

[0088] (c) Step of analyzing the pattern of detected extracellular vesicles [Step (c)] The pattern of extracellular vesicles detected in step (b) is analyzed to examine the subject for a specific disease. The pattern is analyzed by comparing it with known patterns of extracellular vesicles specific to a specific disease to determine whether the subject has extracellular vesicles caused by a specific disease. If a subject has extracellular vesicles caused by a specific disease, it can be said that the subject may be suffering from the specific disease.

[0089] Extracellular vesicles released from diseased cells are thought to contain the same marker molecules as the original cells. For example, extracellular vesicles derived from cancer cells frequently express tumor markers used in cancer screening (e.g., CEA, CA19-9, CA125) and molecules whose expression is elevated in cancer (e.g., EpCAM and ICAM-1). Furthermore, amyloid beta molecules, which are thought to be related to the etiology of Alzheimer's disease, are known to be present on extracellular vesicles derived from brain neurons. Therefore, extracellular vesicles in a subject's body fluids (e.g., blood, urine, saliva) can be stained with fluorescently labeled antibodies that recognize these disease-related molecules and analyzed using a flow cytometer. The resulting patterns can be compared with those of patients with the disease, allowing for disease diagnosis in the subject.

[0090] Specifically, the present inventors have confirmed that the expression of EpCAM and GPNMB in extracellular vesicles in body fluids is increased in breast cancer patients, and that the expression of EpCAM and ICAM-1 is increased in gastric cancer patients (see Examples 1 and 2). Furthermore, the present inventors have confirmed that the expression of NCAM, Amyloid β, aggregated Amyloid β, ICAM-1, and GPNMB in extracellular vesicles in body fluids is increased in Alzheimer's disease patients (see Examples 3 and 4). Based on these results, the expression patterns of EpCAM and GPNMB in extracellular vesicles in body fluids can be compared with those of patients with each disease, or, if necessary, healthy individuals, to test for the disease in a subject.

[0091] Senescent cells also release specific extracellular vesicles, so analyzing extracellular vesicles derived from senescent cells in body fluids can be used to examine the state of aging in the body. Furthermore, because bacteria also release extracellular vesicles, analyzing extracellular vesicles derived from intestinal bacteria in body fluids can be used to examine the intestinal environment, for example.

[0092] [4] Method for diagnosing diseases using extracellular vesicles: The disease of the subject is diagnosed based on the pattern of extracellular vesicles obtained in step (c) of the above-mentioned method for testing diseases using extracellular vesicles. Note that the diagnostic method of the present invention may be a method for assisting in the diagnosis of a disease or a method for diagnosing a disease.

[0093] For example, by examining the expression patterns in extracellular vesicles in body fluids of multiple molecules whose expression increases in cancer, it will be possible to diagnose cancer early, assist in the diagnosis, and identify the disease early. Furthermore, by analyzing the patterns of molecules that increase specifically in cancer type, it will be possible to determine which organ the cancer is in.

[0094] The present inventors have confirmed that the expression of EpCAM and GPNMB in extracellular vesicles in body fluids is increased in breast cancer patients, and that the expression of EpCAM and ICAM-1 is increased in gastric cancer patients (see Examples 1 and 2). By analyzing the expression of EpCAM and / or GPNMB in extracellular vesicles in a subject's body fluids, if the expression of EpCAM and / or GPNMB is higher than that of healthy individuals, it can be determined that there is a certain degree of likelihood that the subject has breast cancer. Furthermore, by analyzing the expression of EpCAM and / or GPNMB in extracellular vesicles in a subject's body fluids, if the expression of EpCAM and GPNMB is equivalent to or lower than that of healthy individuals, it can be determined that there is a low likelihood that the subject has breast cancer. Furthermore, by analyzing the expression of EpCAM and / or ICAM-1 in extracellular vesicles in a subject's body fluids, if the expression of EpCAM and / or ICAM-1 is higher than that of healthy individuals, it can be determined that there is a certain degree of likelihood that the subject has gastric cancer. In addition, the expression of EpCAM and / or ICAM-1 in extracellular vesicles in the subject's body fluids can be analyzed, and if the expression of EpCAM and ICAM-1 is equivalent to or lower than that of healthy individuals, it can be determined that the subject is unlikely to have gastric cancer.

[0095] In Alzheimer's disease, abnormal expression of amyloid beta is known to be observed even before the onset of mild cognitive impairment (MCI). Therefore, analyzing the patterns of amyloid beta-positive extracellular vesicles in body fluids may enable early diagnosis, support for diagnosis, and early identification of dementia.

[0096] Specifically, the present inventors have confirmed that the levels of NCAM, Amyloid β, aggregated Amyloid β, ICAM-1, and GPNMB in extracellular vesicles in body fluids are increased in patients with Alzheimer's disease (see Examples 3 and 4). By analyzing the expression of NCAM, Amyloid β, aggregated Amyloid β, ICAM-1, and / or GPNMB in extracellular vesicles in the body fluids of a subject, if the expression of any of NCAM, Amyloid β, aggregated Amyloid β, ICAM-1, and GPNMB is higher than that of healthy individuals, it can be determined that there is a certain degree of possibility that the subject is suffering from Alzheimer's disease. Furthermore, the expression of NCAM, Amyloid β, aggregated Amyloid β, ICAM-1 and / or GPNMB in extracellular vesicles in the body fluids of a subject can be analyzed, and if the expression of NCAM, Amyloid β, aggregated Amyloid β, ICAM-1 and GPNMB is equivalent to or lower than that of healthy individuals, it can be determined that the subject is unlikely to suffer from Alzheimer's disease.

[0097] By analyzing the expression of disease-specific molecules in extracellular vesicles, it will be possible to test for not only cancer and dementia, but also liver disease, kidney disease, neuropsychiatric disorders, metabolic diseases, respiratory diseases, circulatory system diseases, digestive system diseases, age-related diseases, and excessive alcohol intake.

[0098] Furthermore, analyzing bacterial extracellular vesicles in body fluids will enable testing, diagnosis, and assistance in the diagnosis and determination of bacterial infections.

[0099] [5] Test Kit The disease test kit of the present invention uses the disease test method using the extracellular vesicles of the present invention and is used to detect extracellular vesicles and analyze their patterns. The test kit of the present invention can be used for diagnosing diseases, assisting in the diagnosis of diseases, determining diseases, etc.

[0100] According to the present invention, extracellular vesicles can be captured and recovered in a simple manner and their properties can be analyzed with high accuracy, compared to conventional methods including ultracentrifugation or immunoprecipitation. This makes it possible to clarify the properties of extracellular vesicles quickly and at low cost, and is useful for the testing and diagnosis of various diseases.

[0101] The present invention will be described in detail below with reference to examples, but these are intended to provide a better understanding of the present invention and are not intended to limit the scope of the present invention in any way.

[0102] In this example, a slide glass on which an ion exchanger was immobilized (PEI-immobilized slide glass) and a well plate on which an ion exchanger was immobilized (PEI-immobilized well plate) were used as solid phase supports.

[0103] <Preparation of ion exchanger-immobilized slide glass> A slide glass with an ion exchanger immobilized thereon (PEI-immobilized slide glass) was prepared by preparing a slide glass pre-coated with amino groups, and then binding polyethyleneimine (PEI) to the amino groups on the slide glass using glutaraldehyde as a crosslinker, thereby immobilizing PEI as an ion exchanger. The details of the method for preparing the PEI-immobilized slide glass are shown below.

[0104] A 2-5% glutaraldehyde solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well of the amino-coated slide as a crosslinker, at a rate of 70 μL per well, and the slide was left standing at room temperature for at least 2 hours. The slide was then washed with ion-exchanged water to remove any unreacted crosslinker or by-products. After washing, 70 μL of a 2-3% polyethyleneimine solution (Nacalai) was added to each well and left standing at room temperature for at least 2 hours. The slide surface was then washed with ion-exchanged water to remove any unreacted ion exchanger or by-products, resulting in the preparation of PEI-immobilized slides. The slides were then stored with 0.1 mol / L borate buffer (70 μL per well) until use.

[0105] <Preparation of ion exchanger-immobilized well plate> An ion exchanger-immobilized well plate (PEI-immobilized well plate) was prepared in the same manner as the above-mentioned ion exchanger-immobilized slide glass, except that an amino group-coated 96-well plate was used instead of the amino group-coated slide glass.

[0106] In this example, a slide glass or well plate already coated with amino groups was prepared to produce the ion exchanger-immobilized slide glass and well plate. However, coating of amino groups on the slide glass or well plate can be performed using, for example, a silane coupling agent (e.g., 3-aminopropyltriethoxysilane).

[0107] (a) Step of capturing and / or recovering extracellular vesicles: This section describes the step of capturing and recovering extracellular vesicles using the prepared solid support (PEI-immobilized glass slide or PEI-immobilized well plate). This step further includes the steps of (a1) adsorbing extracellular vesicles to the solid support, (a2) labeling the extracellular vesicles adsorbed to the solid support, (a3) ​​washing the labeled extracellular vesicles while still adsorbed to the solid support, and (a4) detaching the washed extracellular vesicles from the solid support. Note that a PEI-immobilized glass slide was used as the solid support in Example 1, and a PEI-immobilized well plate was used in Examples 2 to 4. However, the same procedure can be used whether a PEI-immobilized glass slide or a PEI-immobilized well plate is used as the solid support.

[0108] (a1) Step of adsorbing extracellular vesicles to a solid support The following sample solutions containing extracellular vesicles (EVs) were prepared: ・Healthy human plasma: Obtained by drawing blood from a healthy individual. ・Human breast cancer patient plasma: Purchased from ProteoGenex. ・Human stomach cancer patient plasma: Purchased from ProteoGenex. ・Human Alzheimer's disease patient plasma: Purchased from CentralLink.

[0109] The adsorption and immobilization of EVs onto the solid support was carried out as follows. First, the solid support was washed with ion-exchanged water to remove unreacted ion exchangers and by-products. Next, 10-30 μL of the sample solution containing EVs was added to each well of the solid support and allowed to stand at room temperature for 15-30 minutes, allowing the EVs in the sample solution to be adsorbed and immobilized onto the solid support.

[0110] (a2) Step of labeling extracellular vesicles The EVs fixed on the solid support were fluorescently stained so that they could be observed with a flow cytometer. The following reagents were used for fluorescent staining. Alexa Fluor 488-labeled human CD54 (ICAM-1) antibody (mouse monoclonal (clone CD54); BioLegend) Alexa Fluor 647-labeled human CA19-9 antibody (mouse monoclonal (1031516); R&D) Alexa Fluor 647-labeled human EGFR antibody (mouse monoclonal (AY13); BioLegend) Alexa Fluor 488-labeled human EpCAM antibody (mouse monoclonal (9C4); BioLegend) Alexa Fluor 647-labeled human HER2 antibody (mouse monoclonal (24D2); BioLegend) Alexa Fluor 647-labeled human GPNMB antibody (mouse monoclonal (D-9); Santa Cruz) Alexa Fluor 488-labeled human CD56 (NCAM) antibody (mouse monoclonal (5.1H11); BioLegend) Alexa Fluor 647-labeled anti-β-Amyloid, 1-16 antibody (mouse monoclonal (6E10); BioLegend) Alexa Fluor 647-labeled anti-β-Amyloid, aggregated antibody (mouse monoclonal (A17171C); BioLegend)

[0111] Fluorescent staining of EVs was performed using the following procedure. First, EVs were adsorbed and immobilized on a solid support as described above, and then the supernatant of the sample solution was removed and the plate was washed with PBS. After washing, 20-30 μL of blocking solution (20-100% FBS-PBS) was added per well and the plate was left to stand at room temperature for 30 minutes for blocking. After removing the blocking solution, the plate was washed with PBS. After washing, 20-30 μL of a fluorescent staining reagent (nucleic acid staining reagent or fluorescently labeled antibody) diluted to an appropriate concentration was added and the plate was left to stand at room temperature for 30 minutes.

[0112] (a3) A step of washing the extracellular vesicles while they are still adsorbed to the solid phase carrier. After fluorescent staining, the reagent supernatant was removed, the solid phase carrier was washed with ion-exchanged water, and the water was removed by suction.

[0113] (a4) Step of detaching washed extracellular vesicles from the solid phase carrier: PBS was added to the washed solid phase carrier, and the fixed EVs were scraped off with a cell scraper to obtain a PBS suspension of EVs.

[0114] (b) A step for detecting extracellular vesicles, and (c) a step for analyzing their patterns. The resulting PBS suspension of EVs was collected in a tube and subjected to EV detection and pattern analysis using a flow cytometer. The results of the analysis are shown below.

[0115] Example 1: EVs from plasma samples of three healthy subjects and four breast cancer patients were immobilized on a solid support (PEI-immobilized glass slide) according to the method described above, stained with fluorescently labeled antibodies against human EpCAM and human GPNMB, and then analyzed using a flow cytometer. The results are shown in Figures 1-1 and 1-2.

[0116] Figure 1-1 shows the results of no staining (negative control) [(a): healthy human subject] and the results of staining with anti-human EpCAM antibody [(b) to (d): healthy human subjects, (e) to (h): human breast cancer patients], and Figure 1-2 shows the results of no staining (negative control) [(a): healthy human subject] and the results of staining with anti-human GPNMB antibody [(b) to (d): healthy human subjects, (e) to (h): human breast cancer patients]. In each graph, the horizontal axis shows the green or red fluorescence intensity, and the vertical axis shows the count number (frequency). The percentage values ​​shown in each graph represent the fluorescence intensity of 2 x 10 3 Percentage of counts above (2 x 10 3 (The sum of the above counts divided by the total counts.)

[0117] Figures 1-1 and 1-2 confirm that EpCAM and GPNMB, known to be highly expressed in cancer cells, are highly expressed in EVs derived from breast cancer patients compared to healthy controls. These results demonstrate that human plasma-derived extracellular vesicles can be adsorbed onto a solid support (PEI-immobilized slide glass), fluorescently stained, and analyzed using a flow cytometer. Furthermore, it is possible to stratify healthy individuals from cancer patients by selecting markers, which means that breast cancer prevalence testing is possible.

[0118] Example 2: EVs from plasma samples of two healthy subjects and four gastric cancer patients were immobilized on a solid support (PEI-immobilized well plate) according to the method described above, stained with fluorescently labeled antibodies against human EpCAM, human CA19-9, human CD54 (ICAM-1), and human HER2, and then analyzed using a flow cytometer. The results are shown in Figures 2-1 to 2-4.

[0119] Figure 2-1 shows the results of unstained (negative control) [(a): healthy human subject] and stained with anti-human EpCAM antibody [(b) and (c): healthy human subject, (d) to (g): human gastric cancer patient]. Figure 2-2 shows the results of unstained (negative control) [(a): healthy human subject] and stained with anti-CA19-9 antibody [(b) and (c): healthy human subject, (d) to (g): human gastric cancer patient]. Figure 2-3 shows the results of stained with anti-human ICAM-1 antibody [(b) and (c): healthy human subject, (d) to (g): human gastric cancer patient]. Figure 2-4 shows the results of stained with anti-human HER2 antibody [(b) and (c): healthy human subject, (d) to (g): human gastric cancer patient]. In each graph, the horizontal axis represents green or red fluorescence intensity, and the vertical axis represents counts (frequency). The percentage values ​​shown in each graph represent the fluorescence intensity of 2 × 10 3 Percentage of counts above (2 x 10 3 (The sum of the above counts divided by the total counts.)

[0120] Figures 2-1 to 2-4 confirm that EpCAM and ICAM-1, which are known to be highly expressed in cancer cells, are highly expressed in EVs derived from gastric cancer patients compared to healthy controls. These results demonstrate that human plasma-derived extracellular vesicles can be adsorbed onto a solid support (PEI-immobilized slide glass), fluorescently stained, and analyzed using a flow cytometer. Furthermore, it is possible to stratify healthy individuals from cancer patients by selecting markers, which means that gastric cancer prevalence testing is possible.

[0121] Furthermore, Figures 2-1 to 2-4 confirm that some patients expressed HER2 (all cases) and CA19-9 (2 out of 4 cases), which were not expressed in healthy subjects. These results confirmed that it is possible not only to stratify healthy subjects and cancer patients, but also to analyze the type of gastric cancer.

[0122] Example 3: EVs from plasma samples of three healthy subjects and three Alzheimer's disease (AD) patients were immobilized on a solid support (PEI-immobilized well plate) according to the method described above, stained with fluorescently labeled antibodies against human NCAM, amyloid β, and human ICAM-1, and then analyzed using a flow cytometer. The results are shown in Figures 3-1 to 3-3.

[0123] Figure 3-1 shows the results of unstained (negative control) [(a): healthy human subject] and the results of staining with anti-human NCAM antibody [(b) to (d): healthy human subject, (e) to (g): AD patient]. Figure 3-2 shows the results of unstained (negative control) [(a): healthy human subject] and the results of staining with anti-Amyloid β antibody [(b) to (d): healthy human subject, (e) to (g): AD patient]. Figure 3-3 shows the results of staining with anti-human ICAM-1 antibody [(b) to (d): healthy human subject, (e) to (g): AD patient]. In each graph, the horizontal axis shows green or red fluorescence intensity, and the vertical axis shows the number of counts (frequency). The percentage values ​​shown in each graph represent the fluorescence intensity of 2 x 10 3 Percentage of counts above (2 x 10 3 (The sum of the above counts divided by the total counts.)

[0124] Figures 3-1 to 3-3 confirm that EVs positive for NCAM, Amyloid β, and ICAM-1 are increased in EVs derived from Alzheimer's disease patients compared to healthy controls. These results demonstrate that selecting markers can differentiate between healthy controls and Alzheimer's disease patients, making it possible to test for Alzheimer's disease.

[0125] Example 4: EVs from plasma samples of three healthy subjects and three Alzheimer's disease (AD) patients were immobilized on a solid support (PEI-immobilized well plate) according to the method described above, stained with fluorescently labeled antibodies against aggregated amyloid β, human ICAM-1, and GPNMB, and then analyzed using a flow cytometer. The results are shown in Figures 4-1 to 4-3.

[0126] Figure 4-1 shows the results of no staining (negative control) [(a): healthy human subject] and the results of staining with anti-aggregating amyloid β antibody [(b) to (d): healthy human subject, (e) to (g): AD patient], Figure 4-2 shows the results of no staining (negative control) [(a): healthy human subject] and the results of staining with anti-human ICAM-1 antibody [(b) to (d): healthy human subject, (e) to (g): AD patient], and Figure 4-3 shows the results of staining with anti-human GPNMB antibody [(b) to (d): healthy human subject, (e) to (g): AD patient]. In each graph, the horizontal axis shows green or red fluorescence intensity, and the vertical axis shows the number of counts (frequency). The percentage values ​​shown in each graph represent the percentage of the fluorescence intensity when the fluorescence intensity is 10 3 Percentage of counts above 10 3 (The sum of the above counts divided by the total counts.)

[0127] Figures 4-1 to 4-3 confirm that EVs derived from Alzheimer's disease patients show a dramatic increase in aggregated amyloid β and GPNMB-positive EVs, which are hardly expressed in healthy individuals. Because aggregated amyloid β exhibits neurotoxicity, it is thought to be deeply involved in the pathology of Alzheimer's disease. This indicates that analyzing EV phenotypes may enable the construction of tests that correlate with the cause of Alzheimer's disease.

Claims

1. A method for testing for diseases in a subject, comprising the steps of: (a) capturing and / or recovering multiple extracellular vesicles from a biological sample from the subject; (b) detecting the captured and / or recovered multiple extracellular vesicles; and (c) analyzing the pattern of the detected extracellular vesicles, wherein step (a) comprises the steps of: (a1) adsorbing the extracellular vesicles to a solid phase carrier comprising a substrate and an ion exchanger and having the ability to adsorb the extracellular vesicles; (a2) labeling the extracellular vesicles adsorbed to the solid phase carrier; and (a3) ​​washing the labeled extracellular vesicles while they are still adsorbed to the solid phase carrier.

2. A method for testing for diseases caused by extracellular vesicles according to claim 1, characterized in that the step (a2) of labeling the extracellular vesicles is carried out using an extracellular vesicle detection substance.

3. The method for testing for diseases caused by extracellular vesicles according to claim 2, wherein the extracellular vesicle detection substance is at least one substance selected from the group consisting of antibodies, lectins, membrane-reactive substances, and nucleic acid-reactive substances.

4. A method for testing for diseases using extracellular vesicles according to claim 1, characterized in that step (b) is carried out by analyzing the captured and / or collected multiple extracellular vesicles by an immunoassay method.

5. A method for testing for diseases using extracellular vesicles according to claim 1, wherein step (a) comprises, after step (a3), step (a4) of detaching the washed extracellular vesicles from the solid phase carrier.

6. A method for testing for diseases caused by extracellular vesicles according to claim 5, wherein step (b) is carried out by analyzing the captured and / or collected multiple extracellular vesicles by flow cytometry.

7. A method for testing for diseases using extracellular vesicles according to claim 1, characterized in that the ion exchanger is a weak anion exchanger having secondary and / or tertiary amino groups as partial structures.

8. The method for testing for diseases using extracellular vesicles according to claim 7, wherein the ion exchanger is a monovalent residue or a polyvalent residue derived from polyethyleneimine.

9. The method for testing for diseases using extracellular vesicles according to claim 8, wherein the polyethyleneimine is a branched high-molecular-weight polyethyleneimine.

10. A method for testing for diseases using extracellular vesicles according to claim 7, characterized in that the ion exchanger and the substrate are covalently bonded via a linking moiety or without a linking moiety.

11. A method for testing for diseases using extracellular vesicles according to claim 10, wherein the ion exchanger and the substrate are bonded via a linking moiety, and the linking moiety contains a silicon-carbon bond.

12. The method for testing for diseases using extracellular vesicles according to claim 1, wherein the substrate is made of glass, plastic or metal oxide.

13. The method for testing for diseases using extracellular vesicles according to claim 1, wherein the substrate is plate-shaped or particulate.

14. The method for testing for diseases using extracellular vesicles according to claim 1, wherein the substrate is a slide glass or a well plate.

15. A method for testing for diseases using extracellular vesicles according to any one of claims 1 to 14, wherein the disease is cancer, dementia, metabolic disease, inflammatory disease, circulatory system disease, digestive system disease, neurodegenerative disease, genetic disease, infectious disease, or aging.

16. A method for assisting in the diagnosis of diseases using extracellular vesicles, comprising the method for testing for diseases using extracellular vesicles according to any one of claims 1 to 14, and further comprising the step of determining the disease of the subject based on the pattern of the extracellular vesicles analyzed in step (c).

17. The method for assisting in the diagnosis of a disease using extracellular vesicles according to claim 16, wherein the disease is cancer, dementia, a metabolic disease, an inflammatory disease, a cardiovascular disease, a digestive disease, a neurodegenerative disease, a genetic disease, an infectious disease, or aging.

18. A disease testing kit for carrying out the disease testing method using extracellular vesicles according to claim 1.

19. A test kit for the disease described in claim 18 for use in diagnosis.

Images