Method for detecting intracellular proteins in extracellular vesicles and extracellular vesicle membrane permeabilizing agent

Abstract

The present invention addresses the problem of providing: a method for simply and accurately detecting a specific protein contained in extracellular vesicles; and a membrane permeabilization treatment agent that can be used in the method. A method for detecting a specific protein contained in extracellular vesicles, the method comprising (A) a step for capturing extracellular vesicles using a carrier capable of binding to an extracellular vesicle-specific marker which exists on the surface of extracellular vesicles, (B) a step for performing a membrane permeabilization treatment on extracellular vesicles captured by the carrier using a membrane permeabilization treatment agent, and (C) a step for introducing a reagent capable of detecting a specific protein contained in extracellular vesicles, into the extracellular vesicles subjected to the membrane permeabilization treatment, wherein the step (B) is carried out such that the specific protein is prevented from leaking off the extracellular vesicles, and the reagent can be introduced into the extracellular vesicles.

Description

Technical Field

[0001] The present invention relates to a method for simply and accurately detecting a specific protein contained inside extracellular vesicles and a membrane permeation treatment agent that can be used in the method.

Background Art

[0002] It is known that body fluids such as blood and cell culture media contain extracellular vesicles secreted from cells contained in the body fluid or culture medium. In recent years, the functions of such extracellular vesicles as mediators of intercellular communication in vivo and their relevance to diseases such as cancer and physiological phenomena have been reported, and research aimed at elucidating physiological functions and applying them to disease tests has been underway.

[0003] Extracellular vesicles represented by exosomes and apoptotic bodies are colloidal particles covered with a lipid bilayer membrane, and detection methods for extracellular vesicles targeting proteins on the membrane surface have been reported (Patent Document 1).

[0004] As a method for detecting a specific protein contained inside extracellular vesicles, a method has been reported in which the membrane is permeated with a surfactant containing sodium deoxycholate and lauroyl sarcosinate, and the protein leaked from inside the extracellular vesicles is detected with a mass spectrometer (Patent Document 2). In addition, a method has been reported in which extracellular vesicles are concentrated with magnetic particles, the membrane of the extracellular vesicles is solubilized, and then a specific protein leaked from inside the extracellular vesicles is detected by Western blotting (Patent Document 3). However, the detection methods described in these documents are complicated, and since detection is performed after leakage to the outside of the extracellular vesicles, high-precision detection is also difficult.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] An object of the present invention is to provide a method for simply and accurately detecting a specific protein contained inside extracellular vesicles, and a membrane permeation treatment agent that can be used in the method. [Means for Solving the Problems]

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that by using a specific membrane permeation treatment agent, a reagent capable of detecting a specific protein can be introduced into extracellular vesicles without leaking the specific protein contained inside the extracellular vesicles to the outside of the extracellular vesicles, and thus have reached the present invention.

[0008] That is, the present invention can be exemplified as follows. [1] A method for detecting a specific protein contained inside extracellular vesicles, comprising: (A) a step of capturing extracellular vesicles using a carrier capable of binding to an extracellular vesicle-specific marker present on the surface of the extracellular vesicles; (B) a step of subjecting the extracellular vesicles captured by the carrier to membrane permeation treatment using a membrane permeation treatment agent; and (C) a step of introducing a reagent capable of detecting a specific protein contained inside the extracellular vesicles into the membrane-permeated extracellular vesicles and wherein the step (B) is carried out so as not to leak the specific protein to the outside of the extracellular vesicles and to enable introduction of the reagent into the inside of the extracellular vesicles. [2] The method according to any one of the above [1], further comprising a step of removing impurities after the step (A) and / or after the step (B). [3] The method further includes a step of detecting the specific protein using the introduced reagent after the step (C). [4] The method, wherein the membrane permeation treatment agent is a surfactant and / or an organic solvent. [5] The method, wherein the surfactant is an anionic surfactant, a cationic surfactant, or a nonionic surfactant. [6] The method, wherein the surfactant is deoxycholate, glycocholate, SDS, saponin, Triton X-100, or CTAB. [7] The method, wherein the surfactant is any one of the following (a) to (f). (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycocholate (c) 0.01 to 0.1% (w / v) SDS (d) 0.1 to 2% (w / v) saponin (e) 0.005 to 0.5% (w / v) Triton X-100 (f) 0.002 to 0.2% (w / v) CTAB [8] The method, wherein the surfactant is any one of the following (a) to (c). (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycocholate (c) 0.01 to 0.1% (w / v) SDS [9] The method, wherein the organic solvent is ethanol and / or acetone.

[10] The method, wherein the organic solvent is the following (a) or (b). (a) 20 to 60% (v / v) ethanol (b) 20 to 70% (v / v) acetone

[11] The method, wherein the carrier is magnetic particles.

[12] An extracellular vesicle membrane permeabilization agent, comprising a surfactant and / or an organic solvent, characterized in that it can perform membrane permeabilization treatment on extracellular vesicles so as not to leak specific proteins contained inside the extracellular vesicles to the outside of the extracellular vesicles and to introduce a reagent capable of detecting the specific proteins contained inside the extracellular vesicles into the inside of the extracellular vesicles.

[13] The membrane permeabilization agent, wherein the surfactant is an anionic surfactant, a cationic surfactant, or a nonionic surfactant.

[14] The membrane permeabilization agent, wherein the surfactant is one or more components selected from the group consisting of deoxycholate, glycocholate, SDS, saponin, Triton X-100, and CTAB.

[15] The membrane permeabilization agent, wherein the surfactant is any one of the following (a) to (f). (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycocholate (c) 0.01 to 0.1% (w / v) SDS (d) 0.1 to 2% (w / v) saponin (e) 0.005 to 0.5% (w / v) Triton X-100 (f) 0.002 to 0.2% (w / v) CTAB

[16] The membrane permeabilization agent, wherein the surfactant is any one of the following (a) to (c). (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycocholate (c) 0.01 to 0.1% (w / v) SDS

[17] The membrane permeabilization agent, wherein the organic solvent is ethanol and / or acetone.

[18] The membrane permeabilization agent, wherein the organic solvent is the following (a) or (b). (a) 20~60% (v / v) ethanol (b) 20~70% (v / v) acetone

[19] A kit for detecting a specific protein contained inside extracellular vesicles, comprising magnetic particles capable of binding to an extracellular vesicle-specific marker present on the surface of extracellular vesicles, the membrane permeabilization agent, and a reagent capable of detecting a specific protein contained inside extracellular vesicles.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0010] Hereinafter, the present invention will be described in detail.

[0011] <1> Method of the Present Invention The method of the present invention is a method for detecting a specific protein contained inside extracellular vesicles.

[0012] The method of the present invention comprises a step of subjecting extracellular vesicles to membrane permeabilization treatment using a membrane permeabilization agent (hereinafter, also simply referred to as "membrane permeabilization step"), and a step of introducing a reagent capable of detecting a specific protein contained inside extracellular vesicles into the extracellular vesicles subjected to membrane permeabilization treatment (hereinafter, also simply referred to as "introduction step") and may include.

[0013] The extracellular vesicle-specific marker present on the surface of extracellular vesicles will be hereinafter also simply referred to as "vesicle-specific marker". A reagent capable of detecting a specific protein will be hereinafter also simply referred to as "detection reagent".

[0014] The term "extracellular vesicles" refers to vesicles with a diameter of 1 nm to 1 μm that are covered with lipids and are released from cells, either actively or passively. Examples of extracellular vesicles include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, and apoptotic vesicles (Nature Reviews, 9, 2009, 581-593). It has been generally reported that extracellular vesicles are composed of lipids and proteins with compositions different from those of cells (BioScience, 65, 2015, 783-797).

[0015] The origin of extracellular vesicles is not particularly limited. Examples of the origin of extracellular vesicles include body fluids, bacterial suspensions, culture media or culture supernatants after cell culture, and tissue cell lysates. Among these, body fluids and culture supernatants after cell culture are preferred as the origin of extracellular vesicles. Examples of body fluids include blood components such as whole blood, serum, plasma, blood components, various blood cells, blood clots, and platelets, as well as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymph, bone marrow fluid, tissue fluid, saliva, gastric juice, synovial fluid, pleural effusion, bile, ascites, and amniotic fluid. Among body fluids, blood components are preferred. Body fluids such as blood components may be treated with anticoagulants such as citrate, heparin, and EDTA.

[0016] The method of the present invention may include a step of capturing extracellular vesicles (hereinafter, also simply referred to as the "capturing step") before the membrane permeation step. "Capturing of extracellular vesicles" may be used interchangeably with "recovery of extracellular vesicles". The capturing step can be carried out, for example, using a carrier capable of binding to an extracellular vesicle-specific marker present on the surface of extracellular vesicles. That is, the extracellular vesicles to be subjected to the membrane permeation step may be, for example, extracellular vesicles captured by the carrier.

[0017] That is, specifically, the method of the present invention is, for example, a step of capturing extracellular vesicles using a carrier capable of binding to an extracellular vesicle-specific marker present on the surface of extracellular vesicles, and a step of subjecting the extracellular vesicles captured by the carrier to membrane permeation using a membrane permeation treatment agent. A step of introducing a reagent capable of detecting a specific protein contained inside extracellular vesicles into the membrane-permeated extracellular vesicles may be included.

[0018] The carrier used in the capture step can be obtained, for example, by immobilizing a substance capable of specifically binding to a vesicle-specific marker on a substrate.

[0019] The substrate is not particularly limited as long as it is a water-insoluble substance. The substrate may be hydrophilic or hydrophobic. Specifically, as the substrate, for example, polysaccharides such as agarose-based, dextran-based, chitosan-based, cellulose-based, etc., polyacrylamide-based, polyvinyl alcohol-based, polyvinyl pyrrolidone-based, polyacrylonitrile-based, styrene-divinylbenzene copolymer, polystyrene-based, acrylate-based, methacrylate-based, polyethylene-based, polypropylene-based, polytetrafluoroethylene-based, ethylene-vinyl acetate copolymer-based, polyamide-based, polycarbonate-based, polyvinylidene fluoride-based, polyvinyl formal-based, polyarylate-based, polyethersulfone-based, etc. organic synthetic polymers, glass-based, titanium-based, activated carbon-based, various ceramic-based such as alumina, silica, hydroxyapatite, metal-based inorganic substances such as iron oxide, gold, etc., known materials commonly used in this technical field can be used without special limitation. Also, the shape of the substrate is not particularly limited. Specifically, as the substrate, for example, known shapes such as particulate, fibrous, hollow fiber, membrane, flat plate, etc. can be used. Among the shapes of the substrate, in particular, in terms of a large surface area, the ability to bind to target cells uniformly and efficiently, the difficulty of physically damaging the cells, the difficulty of causing damage, and the ease of obtaining a uniform carrier, the particulate shape is preferred. The shape of the particles is not particularly limited. Among the particles, in particular, in terms of easy handling, the difficulty of physically damaging the extracellular vesicles, the difficulty of causing damage to the particles, and the ease of obtaining uniform particles, spherical (including not only true spheres but also almost spherical) particles are preferred. Furthermore, as the particles, magnetic particles are particularly preferred in terms of being able to be recovered simply, in a short time, and with high precision by applying an external magnetic field. Examples of the magnetic particles include particles manufactured by incorporating a magnetic substance.

[0020] The vesicle-specific marker is not particularly limited as long as it is a substance that specifically exists on the surface of extracellular vesicles. Examples of vesicle-specific markers include substances that exist on the surface of extracellular vesicles and have antigenicity, and substances that exist on the surface of extracellular vesicles and have recognition ability for specific receptors. Specifically, examples of vesicle-specific markers include proteins, sugar chains, nucleic acids, and lipids. For example, when the extracellular vesicle is an exosome, examples of substances that exist on the surface of the extracellular vesicle and have antigenicity include tetraspanins such as CD9, CD63, and CD81; antigen presentation-related proteins such as MHC (Major Histocompatibility Complex) I and MHCII; adhesion molecules such as integrin, ICAM-1 (InterCellular Adhesion Molecule 1), and EpCAM (Epithelial Cell Adhesion Molecule); cytokines / cytokine receptors such as EGFR (Epidermal Growth Factor Receptor) vIII and TGF (Transforming Growth Factor)-β, and enzymes. Also, for example, when the extracellular vesicle is an exosome, an example of a substance that exists on the surface of the extracellular vesicle and has recognition ability for a specific receptor can be a lipid such as phosphatidylserine.

[0021] Examples of substances capable of specifically binding to extracellular vesicle-specific markers include antibodies against proteins specifically present on the surface of extracellular vesicles, lectins capable of specifically binding to sugar chains specifically present on the surface of extracellular vesicles, aptamers capable of binding to proteins or nucleic acids specifically present on the surface of extracellular vesicles, and receptors for lipids specifically present on the surface of extracellular vesicles. Among these, antibodies against proteins specifically present on the surface of extracellular vesicles are particularly preferred as substances capable of specifically binding to extracellular vesicle-specific markers. When a substance capable of specifically binding to a membrane surface marker derived from a specific organ or specific cells that are the source of extracellular vesicles is used as a substance capable of specifically binding to an extracellular vesicle-specific marker, extracellular vesicles released from the specific organ or specific cells can be specifically captured. Examples of methods for immobilizing a substance capable of specifically binding to an extracellular vesicle-specific marker to a substrate include covalent bonding, electrostatic interaction, hydrophobic interaction, and coordination bonding. Among these, covalent bonding, which is a strong modification method, is particularly preferred as a method for immobilizing a substance capable of specifically binding to an extracellular vesicle-specific marker to a substrate.

[0022] The membrane permeation step is a step of subjecting extracellular vesicles to membrane permeation treatment using a membrane permeation treatment agent. "Membrane permeation treatment of extracellular vesicles" may mean a treatment for enhancing the membrane permeability of extracellular vesicles, and specifically may mean a treatment for enhancing the membrane permeability of extracellular vesicles to a detection reagent.

[0023] The membrane permeation step is carried out such that specific proteins contained inside the extracellular vesicles do not leak outside the extracellular vesicles and a detection reagent can be introduced into the inside of the extracellular vesicles. Specifically, in the membrane permeation step, a membrane permeation pore having a size that allows a detection reagent to be introduced into the inside of the extracellular vesicles may be formed without allowing specific proteins contained inside the extracellular vesicles to leak outside the extracellular vesicles. If the size of the membrane permeation pore is small, the detection reagent cannot enter the inside of the extracellular vesicles. If the size of the membrane permeation pore is large, the proteins present inside the extracellular vesicles leak out. In either case, it becomes difficult to accurately detect the proteins present inside the extracellular vesicles. "Specific proteins do not leak outside the extracellular vesicles" means that specific proteins contained inside the extracellular vesicles remain inside the vesicles in an amount that can be accurately detected with a detection reagent. Specifically, "specific proteins do not leak outside the extracellular vesicles" may mean, for example, that the amount (e.g., number of molecules) of specific proteins remaining inside the extracellular vesicles after the membrane permeation step is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the amount (e.g., number of molecules) of specific proteins contained inside the extracellular vesicles before the membrane permeation step.

[0024] The membrane permeation treatment agent is not particularly limited as long as it can carry out the membrane permeation step such that specific proteins contained inside the extracellular vesicles do not leak outside the extracellular vesicles and a detection reagent can be introduced into the inside of the extracellular vesicles. Specifically, the membrane permeation treatment agent may be one that can form a membrane permeation pore having a size that allows a detection reagent to be introduced into the inside of the extracellular vesicles without allowing specific proteins contained inside the extracellular vesicles to leak outside the vesicles. As the membrane permeation treatment agent, one kind of component may be used, or two or more kinds of components may be used in combination.

[0025] Examples of the membrane permeation treatment agent include surfactants and organic solvents. As the membrane permeation treatment agent, a surfactant is preferred. In particular, at least a surfactant may be used as the membrane permeation treatment agent, and more particularly, a surfactant and an organic solvent may be used in combination.

[0026] Examples of surfactants include cationic surfactants, nonionic surfactants, and anionic surfactants. Examples of cationic surfactants include didecyldimethylammonium bromide (DDAB), cetyltrimethylammonium bromide (CTAB), cetylpyridinium bromide (CPB), dodecyltrimethylammonium chloride (DOTAC), sodium perfluorononanoate (SPFN), and hexadecyltrimethylammonium bromide (HDTMA). Examples of nonionic surfactants include Triton X-100 (trade name) and saponin. Examples of anionic surfactants include alkyl sulfate salts (such as SDS (sodium dodecyl sulfate)), bile acids or bile salts (such as cholate salts such as sodium cholate, deoxycholate salts such as sodium deoxycholate, glycocholate salts such as sodium glycocholate, chenodeoxycholate salts such as sodium chenodeoxycholate, lithocholate salts such as sodium lithocholate, glycolithocholate salts such as sodium glycolithocholate, taurolithocholate salts such as sodium taurolithocholate, etc.), sodium lauryl sulfosuccinate, sodium α-olefin sulfonate, sodium polyoxyethylene lauryl ether sulfate, sodium polyoxyethylene lauryl ether phosphate, and sodium lauryl benzene sulfonate. Among the surfactants, anionic surfactants are preferred in terms of accurately detecting specific proteins inside extracellular vesicles. Among the anionic surfactants, bile acids or bile salts are preferred in terms of having no protein-denaturing ability. Examples of surfactants particularly include deoxycholate salts (such as sodium deoxycholate), glycocholate salts (such as sodium glycocholate), SDS, saponin, Triton X-100, and CTAB. More particularly, examples of surfactants include deoxycholate salts (such as sodium deoxycholate), glycocholate salts (such as sodium glycocholate), and SDS.

[0027] Examples of organic solvents include alcohols such as ethanol and methanol, and acetone. Particularly, examples of organic solvents include ethanol and acetone.

[0028] The concentration of the membrane permeation treatment agent can be appropriately set according to various conditions such as the type of the membrane permeation treatment agent and the amount of extracellular vesicles to be subjected to membrane permeation treatment. The membrane permeation treatment agent may be used, for example, in the form of an aqueous solution containing the membrane permeation treatment agent at a predetermined concentration. The aqueous solution containing the membrane permeation treatment agent can be prepared, for example, by diluting the membrane permeation treatment agent with an aqueous medium such as water or an aqueous buffer. Unless otherwise specified, the "concentration of the membrane permeation treatment agent" may mean the concentration of the membrane permeation treatment agent in contact with the extracellular vesicles.

[0029] When a surfactant is used as the membrane permeation treatment agent, the concentration of the surfactant can be appropriately set according to various conditions such as the critical micelle concentration of the surfactant and the amount of extracellular vesicles to be subjected to membrane permeation treatment. The concentration of the surfactant may be, for example, 0.002% (w / v) or more, 0.005% (w / v) or more, 0.01% (w / v) or more, 0.02% (w / v) or more, 0.05% (w / v) or more, or 0.1% (w / v) or more, and may also be 2% (w / v) or less, 1% (w / v) or less, 0.5% (w / v) or less, 0.2% (w / v) or less, or 0.1% (w / v) or less, and may be any non-contradictory combination thereof.

[0030] Specifically, the concentration of the surfactant may be any of the following (a) to (f). That is, the surfactant may be, for example, any of the following (a) to (f). Specifically, the surfactant may be, for example, an aqueous solution containing any of the following (a) to (f). (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycocholate (c) 0.01 to 0.1% (w / v) SDS (d) 0.1 to 2% (w / v) saponin (e) 0.005 to 0.5% (w / v) Triton X-100 (f) 0.002 to 0.2% (w / v) CTAB

[0031] As the concentration of the surfactant, more particularly, the following (a) to (c) may be mentioned. That is, the surfactant may be, for example, any one of the following (a) to (c). Specifically, the surfactant may be, for example, an aqueous solution containing any one of the following (a) to (c). (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycolate (c) 0.01 to 0.1% (w / v) SDS

[0032] When an organic solvent is used as the membrane permeation treatment agent and particles are used as the carrier, the organic solvent may reduce the dispersion stability in the particle suspension. When aggregation of particles occurs due to a decrease in dispersion stability, the ability to detect specific proteins contained inside the extracellular vesicles is significantly reduced. On the other hand, if the added concentration of the organic solvent is low, the effect of membrane permeation cannot be fully exerted, so the ability to detect specific proteins contained inside the extracellular vesicles is not improved. Therefore, when using an organic solvent as the membrane permeation treatment agent, it is preferable to use the organic solvent at a concentration at which particle aggregation does not occur and the effect of membrane permeation is exerted. The concentration of the organic solvent may be, for example, 5% (v / v) or more, 10% (v / v) or more, 20% (v / v) or more, or 30% (v / v) or more, and may also be 90% (v / v) or less, 80% (v / v) or less, 70% (v / v) or less, 60% (v / v) or less, or 50% (v / v) or less, or a combination thereof.

[0033] When ethanol is used as the organic solvent, the concentration of ethanol may be, for example, 5% (v / v) or more, 10% (v / v) or more, 20% (v / v) or more, or 30% (v / v) or more, and may also be 70% (v / v) or less, 60% (v / v) or less, or 50% (v / v) or less, or a combination thereof. Specifically, the concentration of ethanol is preferably, for example, 5 to 70% (v / v), more preferably 20 to 60% (v / v), and even more preferably 30 to 50% (v / v). When acetone is used as the organic solvent, the concentration of acetone may be, for example, 5% (v / v) or more, 10% (v / v) or more, 20% (v / v) or more, or 30% (v / v) or more, and may also be 90% (v / v) or less, 80% (v / v) or less, or 70% (v / v) or less, or a combination thereof. Specifically, the concentration of acetone is preferably, for example, 5 to 80% (v / v), more preferably 20 to 70% (v / v), and even more preferably 30 to 70% (v / v).

[0034] Examples of the concentration of the organic solvent particularly include the following (a) and (b). That is, the organic solvent may be, for example, the following (a) or (b). Specifically, the organic solvent may be, for example, an aqueous solution containing the following (a) or (b). (a) 20 to 60% (v / v) ethanol (b) 20 to 70% (v / v) acetone

[0035] The method of the present invention may further include a step of immobilizing extracellular vesicles (hereinafter, also simply referred to as the "immobilization step"). "Immobilizing extracellular vesicles" may mean, for example, immobilizing a specific protein contained inside the extracellular vesicles. The immobilization step can be carried out using a reagent for immobilizing extracellular vesicles (hereinafter, also simply referred to as the "immobilization treatment agent"). Examples of the immobilization treatment agent include the organic solvents described above, formaldehyde donor compounds such as formaldehyde and imidazolidinyl urea (compounds capable of releasing formaldehyde by undergoing hydrolysis), and aldehydes such as glutaraldehyde.

[0036] When the method of the present invention includes an immobilization step, the immobilization step may be carried out, for example, simultaneously with the membrane permeation step. As an example of carrying out the immobilization step and the membrane permeation step simultaneously, there is a mode in which a surfactant and an organic solvent are combined and allowed to act on extracellular vesicles. In other words, by using a surfactant and an organic solvent in combination as a membrane permeation treatment agent, the immobilization step and the membrane permeation step can be carried out simultaneously. Specifically, the surfactant and the organic solvent may be used in the form of a mixed solution containing the surfactant and the organic solvent. The concentrations of the surfactant and the organic solvent may be the concentrations exemplified above, respectively. The surfactant can function as a membrane permeation treatment agent. However, when the membrane permeation step is carried out using only a surfactant, if a high concentration of the surfactant is used, leakage of a specific protein contained inside the extracellular vesicles may occur, and thus accurate detection of the specific protein may be difficult. In addition to its function as a membrane permeation treatment agent, the organic solvent may also have a function as an immobilization treatment agent that insolubilizes proteins. However, when the immobilization step and the membrane permeation step are carried out using only an organic solvent, if a high concentration of the organic solvent is used, aggregation of the particles used as a carrier may occur, and thus accurate detection of the specific protein may be difficult. Also, when the immobilization step and the membrane permeation step are carried out using only an organic solvent, if a low concentration of the organic solvent is used, the membrane permeation step may be insufficient, and thus accurate detection of the specific protein may be difficult. On the other hand, when a mixed solution of a surfactant and an organic solvent is allowed to act on extracellular vesicles, the function of the organic solvent as an immobilization treatment agent causes insolubilization of a specific protein contained in the extracellular vesicles, so that leakage of the specific protein can be suppressed, and at the same time, the function of the organic solvent and the surfactant as a membrane permeation treatment agent causes the membrane permeation treatment of the extracellular vesicles to proceed.

[0037] The method of the present invention may further include a step of activating the antigen (hereinafter, also simply referred to as the "activation step"). The "activation treatment of the antigen" may mean, for example, a treatment for restoring the antigenicity of a specific protein. The antigenicity of a specific protein may be reduced, for example, by steric hindrance or masking of the antigenic determinant. The steric hindrance or masking of the antigenic determinant may occur, for example, when the immobilization step is carried out. Due to the reduction of the antigenicity of the specific protein, for example, the binding property between the specific protein and the detection reagent may be reduced. Therefore, by carrying out the activation step, for example, the reduced binding property between the specific protein and the detection reagent can be restored, and thus highly sensitive detection of the specific protein by the detection reagent may become possible. The activation step may be carried out before the introduction step is carried out. The activation step may be carried out, for example, after the immobilization step is carried out. Further, the activation step may be carried out, for example, when the immobilization step is not carried out. Examples of the activation treatment method include a heat treatment method and a proteolytic enzyme treatment method, which can be appropriately selected according to various conditions such as the type of the antigen. Examples of the heat treatment method include heating methods using microwave irradiation, an autoclave, a thermostatic bath, an electric pressure cooker, etc. Examples of the proteolytic enzyme treatment method include treatment with an enzyme capable of cleaving the amino acids of a protein such as pepsin, trypsin, chymotrypsin, proteinase K, etc.

[0038] The method of the present invention may further include a step of removing impurities (hereinafter, also simply referred to as the "removing step"). "Impurities" may mean substances not captured by the carrier. Examples of impurities include substances other than extracellular vesicles. In addition, the impurities may include extracellular vesicles not captured by the carrier. The removing step may be carried out, for example, after the capturing step and / or after the membrane permeation step. Carrying out the removing step is preferable in that a concentrated fraction of the extracellular vesicles captured by the carrier is provided to the introduction step and the detection step described later in a state where impurities have been removed. The removal of impurities can be carried out, for example, based on the difference in the physical or chemical properties between the carrier and the impurities. Examples of removal based on the difference in physical or chemical properties include removal based on specific gravity difference, sedimentation rate difference, particle size difference, and dielectric constant difference. When the carrier is magnetic particles, the carrier (magnetic particles) bound with extracellular vesicles and the impurities can be separated simply, in a short time, and with high precision by applying an external magnetic field, so that the removing step can be carried out simply, in a short time, and with high precision.

[0039] The introduction step is a step of introducing a detection reagent into the extracellular vesicles that have been subjected to membrane permeation treatment. The detection reagent may be introduced into the extracellular vesicles, for example, through the membrane permeation pores formed by carrying out the membrane permeation step. The detection reagent is not particularly limited as long as it can detect a specific protein contained inside the extracellular vesicles.

[0040] The specific protein is not particularly limited as long as it is a protein present inside extracellular vesicles (i.e., inside the lipid bilayer constituting the extracellular vesicles). Examples of the specific protein include internal proteins common to many cell-derived exosomes such as actin, tubulin, and GAPDH (GlycerAldehyde-3-Phosphate DeHydrogenase), sorting proteins of endosomes such as ESCRT (Endosomal Sorting Complex Required for Transport)-III, ALIX (Apoptosis-Linked gene 2-Interacting protein X), syntenin, and Tsg101, heat shock proteins such as HSP70 and HSP90, and proteins involved in membrane transport and fusion such as RAB and annexin.

[0041] In addition, a protein that is specifically expressed in a specific organ or specific cell that is the source of extracellular vesicle release and is contained inside the extracellular vesicles may be used as the specific protein. By detecting such a protein as the specific protein, extracellular vesicles secreted from a specific organ or specific cell can be accurately detected. Furthermore, when the capture step is carried out using a carrier immobilized with a substance capable of specifically binding to a membrane surface marker derived from a specific organ or specific cell that is the source of release, extracellular vesicles secreted from a specific organ or specific cell can be detected even more accurately.

[0042] Examples of reagents introduced in the introduction step include antibodies and aptamers that can specifically bind to a specific protein. Both antibodies and aptamers may be appropriately modified with a labeling substance. The labeling substance may be for direct detection or for indirect detection. Examples of the labeling substance include dyes, enzymes, and radioactive substances. Examples of the dye include fluorescent substances such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), and Alexa Fluor (trade name). Examples of the enzyme include alkaline phosphatase and horseradish peroxidase. For example, a dye such as a fluorescent dye may be used as the labeling substance and detected directly based on the dye, but it is preferable in that a specific protein can be detected with high sensitivity by using an enzyme as the labeling substance and adding a substrate that reacts with the enzyme to develop color for detection. The binding mode between the antibody or aptamer and the labeling substance is not particularly limited. The antibody or aptamer may or may not be directly bound to the labeling substance. For example, the antibody may be directly bound to the labeling substance by a chemical bond or the like, or may be indirectly bound to the labeling substance by binding to a secondary antibody (labeled secondary antibody) bound to the labeling substance. Also, for example, when an antibody is modified with biotin, the antibody may be indirectly labeled using an enzyme modified with avidin.

[0043] The introduced detection reagent can be used to detect a specific protein. That is, the method of the present invention may further include a step of detecting a specific protein using the introduced detection reagent (hereinafter, also simply referred to as the "detection step").

[0044] Detection can be carried out according to conventional methods. The detection method can be appropriately selected according to various conditions such as the type of detection reagent. For example, when the detection reagent is an antibody that can specifically bind to a specific protein, examples of the detection method include immunoassay methods, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), and luminescence immunoassay. Among these detection methods, the ELISA method is preferred in terms of its simplicity and high sensitivity for detecting antibodies. Examples of the ELISA method include the competitive method and the sandwich method.

[0045] Hereinafter, an example of the method of the present invention when the detection step is carried out by the sandwich ELISA method will be described with reference to FIG. 1.

[0046] (1) A suspension containing extracellular vesicles 10 and magnetic particles 20 immobilized with an antibody 30 against a vesicle-specific marker present on the membrane surface of the extracellular vesicles 10 are placed in a well 100 of a 96-well plate to capture the extracellular vesicles 10 by the magnetic particles 20 (capture step).

[0047] (2) After removing the extracellular vesicles 10 not captured by the magnetic particles 20, the extracellular vesicles captured on the particle surface are subjected to membrane permeabilization treatment using a membrane permeabilization treatment agent (membrane-permeabilized extracellular vesicles 11) (membrane permeabilization step).

[0048] (3) A blocking reagent is added to the membrane-permeabilized extracellular vesicles 11 (not shown).

[0049] (4) After removing the blocking reagent, a detection reagent containing an enzyme-labeled antibody that can specifically bind to a specific protein contained inside the extracellular vesicles is introduced into the membrane-permeabilized extracellular vesicles (labeled extracellular vesicles 12) (introduction step). By the membrane permeabilization step in (2), the enzyme-labeled antibody can be introduced into the membrane-permeabilized extracellular vesicles, while the specific protein contained inside the extracellular vesicles does not leak, so that the enzyme-labeled antibody can bind to the specific protein in a sufficient amount. In addition, the non-specific adsorption by the enzyme-labeled antibody and the like is also suppressed by the blocking reagent added in (3).

[0050] (5) Add substrate 40 to the labeled extracellular vesicles 12. An optically detectable product 41 is generated by the reaction 50 between the enzyme modified on the antibody bound to the specific protein contained inside the extracellular vesicles and the substrate 40. For example, when peroxidase is used as the labeled enzyme, TMB (tetramethylbenzidine) or the like may be used as the substrate 40.

[0051] (6) After adding a reagent to stop the enzyme reaction, bring the magnet 200 close to the bottom of the well 100 to attract the magnetic particles 20 to the bottom of the well, and then collect the solution (supernatant) containing the product 41 so as to substantially not contain the magnetic particles 20.

[0052] (7) Put the solution recovered in (6) into a well 100 without magnetic particles, and detect the amount of the specific protein contained inside the extracellular vesicles by measuring the absorbance of the product 41 with the detector 300 (detection step).

[0053] <2> Membrane permeation treatment agent of the present invention The membrane permeation treatment agent of the present invention is a membrane permeation treatment agent characterized in that it can perform membrane permeation treatment on extracellular vesicles so that a specific protein contained inside the extracellular vesicles does not leak outside the extracellular vesicles and a detection reagent can be introduced into the inside of the extracellular vesicles.

[0054] The membrane permeation treatment agent of the present invention contains a component that functions as a membrane permeation treatment agent (hereinafter, also simply referred to as "membrane permeation treatment component"). The membrane permeation treatment agent of the present invention may be composed of, for example, a membrane permeation treatment component, or may contain other components in addition to the membrane permeation treatment component. The membrane permeation treatment agent of the present invention may contain one kind of membrane permeation treatment component, or may contain two or more kinds of membrane permeation treatment components.

[0055] Regarding the membrane permeation treatment component (for example, type and concentration) contained in the membrane permeation treatment agent of the present invention, the description of the membrane permeation treatment agent in the method of the present invention can be applied mutatis mutandis.

[0056] That is, the membrane permeation treatment agent of the present invention may contain, for example, a surfactant and / or an organic solvent. The membrane permeation treatment agent of the present invention may particularly contain at least a surfactant, and more particularly may contain a surfactant and an organic solvent. As the surfactant, an anionic surfactant is preferable. As the surfactant, particularly, deoxycholate (sodium deoxycholate, etc.), glycollate (sodium glycollate, etc.), SDS, saponin, Triton X-100, CTAB can be mentioned. As the surfactant, more particularly, deoxycholate (sodium deoxycholate, etc.), glycollate (sodium glycollate, etc.), SDS can be mentioned. As the organic solvent, particularly, ethanol and acetone can be mentioned.

[0057] The surfactant contained in the membrane permeation treatment agent of the present invention may be any one of the following (a) to (f), for example. Specifically, the surfactant contained in the membrane permeation treatment agent of the present invention may be an aqueous solution containing any one of the following (a) to (f), for example. (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycollate (c) 0.01 to 0.1% (w / v) SDS (d) 0.1 to 2% (w / v) saponin (e) 0.005 to 0.5% (w / v) Triton X-100 (f) 0.002 to 0.2% (w / v) CTAB

[0058] The surfactant contained in the membrane permeation treatment agent of the present invention may be any one of the following (a) to (c), for example. Specifically, the surfactant contained in the membrane permeation treatment agent of the present invention may be an aqueous solution containing any one of the following (a) to (c), for example. (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycollate (c) 0.01 to 0.1% (w / v) SDS

[0059] The organic solvent contained in the film permeation treatment agent of the present invention may be, for example, the following (a) or (b). Specifically, the organic solvent may be, for example, an aqueous solution containing the following (a) or (b). (a) 20 - 60% (v / v) ethanol (b) 20 - 70% (v / v) acetone

[0060] The film permeation treatment agent of the present invention can be used, for example, to implement the method of the present invention. Specifically, it can be used to implement the film permeation step of the method of the present invention. That is, the film permeation treatment agent of the present invention can be used, for example, as the film permeation treatment agent in the method of the present invention. Specifically, it can be used as the film permeation treatment agent in the film permeation step of the method of the present invention.

[0061] <3>The detection kit of the present invention The detection kit of the present invention is a detection kit for a specific protein contained inside extracellular vesicles.

[0062] The detection kit of the present invention may contain a carrier capable of binding to an extracellular vesicle-specific marker present on the surface of extracellular vesicles. Regarding the carrier capable of binding to the extracellular vesicle-specific marker present on the surface of extracellular vesicles contained in the detection kit of the present invention, the description of the carrier capable of binding to the extracellular vesicle-specific marker present on the surface of extracellular vesicles in the method of the present invention can be applied mutatis mutandis. The carrier capable of binding to the extracellular vesicle-specific marker present on the surface of extracellular vesicles contained in the detection kit of the present invention may be, for example, magnetic particles.

[0063] The detection kit of the present invention may contain the film permeation treatment agent of the present invention. The film permeation treatment agent of the present invention is as described above.

[0064] The detection kit of the present invention may contain a detection reagent. Regarding the detection reagent contained in the detection kit of the present invention, the description of the detection reagent in the method of the present invention can be applied mutatis mutandis.

[0065] The detection kit of the present invention can be used, for example, to implement the method of the present invention.

Example

[0066] Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

[0067] Example 1 (1) Human fetal kidney cells (293T cells) were cultured at 37 °C in D-MEM (Dulbecco's Modified Eagle's Medium) containing 10% (v / v) FBS (fetal bovine serum) from which extracellular vesicles had been removed under a 5% CO2 environment until confluent. Then, the culture supernatant (lot number 1) was collected and centrifuged at 10,000×g for 30 minutes. The obtained supernatant was centrifuged at 100,000×g for 70 minutes, and the obtained pellet was suspended in PBS (Phosphate Buffered Saline) to obtain a concentrated suspension of extracellular vesicles released from 293T cells.

[0068] (2) To 40 μL of a 10% (w / v) suspension of magnetic particles (manufactured by JSR Corporation) with a particle diameter of 2 to 3 μm whose surface was modified with carboxy groups, an aqueous solution containing 0.5 mg of EDC (1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide) was added and stirred for 1 hour. After removing the reaction solution, 20 μL of an anti-CD9 antibody (manufactured by Frontier Institute) at 1 mg / mL was added and stirred for 3 hours. After removing the reaction solution, it was immersed in a 3% (w / v) BSA (Bovine serum albumin) aqueous solution to suppress non-specific adsorption, thereby obtaining an anti-CD9 antibody-conjugated magnetic particle suspension.

[0069] 10 μL of the suspension containing 2% (w / v) anti-CD9 antibody-conjugated magnetic particles obtained in (3)(2), 5 μL of the extracellular vesicle-concentrated suspension obtained in (1), and 90 μL of PBS containing 3% (w / v) BSA were mixed in the wells of a 96-well plate and stirred for 90 minutes. After stirring, a magnet was brought close to the bottom of the 96-well plate to accumulate the magnetic particles at the bottom of the plate, and then the supernatant was removed and washed three times with PBS.

[0070] (4) After removing the washing solution, an aqueous solution prepared by dissolving sodium deoxycholate, an anionic surfactant, in PBS at 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), 0.2% (w / v), or 0.5% (w / v) was used as a membrane permeation treatment agent, and 100 μL of each was added and stirred for 5 minutes. The supernatant was removed using a magnet as in (3) and washed three times with PBS.

[0071] (5) After removing the washing solution, a mixture of 90 μL of PBS containing 3% (w / v) BSA and 10 μL of FcR Blocking Reagent (manufactured by Miltenyi Biotec) was added as a blocking reagent and stirred for 10 minutes.

[0072] (6) After removing the blocking reagent, 100 μL of PBS containing 2.5 μg / mL biotinylated mouse-derived anti-human ALIX (Apoptosis-Linked gene 2-Interacting protein X, a protein present inside the extracellular vesicles released from 293T cells) antibody (manufactured by Santa Cruz) or 2.5 μg / mL biotinylated mouse control antibody (mouse Isotype Control IgG, manufactured by abcam) and 3% (w / v) BSA was added and stirred for 30 minutes. The supernatant was removed using a magnet as in (3) and washed three times with PBS.

[0073] (7) After removing the washing solution, 100 μL of PBS containing streptavidin conjugated with HRP (Horseradish Peroxidase) in a multivalent manner was added and stirred for 20 minutes. The supernatant was removed using a magnet as in (3) and washed three times with PBS.

[0074] (8) After removing the washing solution, 100 μL of a solution containing TMB (tetramethylbenzidine), which is a chromogenic substrate, was added and stirred for 20 minutes. After stirring, 100 μL of an acidic aqueous solution containing 1 M hydrochloric acid was added to stop the enzymatic reaction.

[0075] (9) 180 μL of the supernatant was collected using a magnet and transferred to a new 96-well plate, and then the absorbance at a wavelength of 450 nm was measured with a plate reader (manufactured by Tecan).

[0076] Comparative Example 1 In Example 1(4), except that 100 μL of PBS was added, the color development of TMB was measured in the same manner as in Example 1.

[0077] The results of Example 1 and Comparative Example 1 are summarized in Table 1. When PBS was added in the membrane permeation step (Comparative Example 1), membrane permeation of extracellular vesicles did not occur, and no difference in absorbance was confirmed between the anti-ALIX antibody and the mouse control antibody (anti-ALIX antibody: 0.04, mouse control antibody: 0.03). On the other hand, when PBS containing at least 0.05% (w / v) or more of sodium deoxycholate was added in the membrane permeation step (Example 1), a difference in absorbance was confirmed between the anti-ALIX antibody and the mouse control antibody. This indicates that by adding sodium deoxycholate, at least a part of the membrane of extracellular vesicles was solubilized, the anti-ALIX antibody entered the inside of the extracellular vesicles, and it was able to bind to ALIX present inside the extracellular vesicles and was obtained as a detection value (synonymous with the absorbance value in the examples of this specification). The mouse control antibody is not an IgG that specifically recognizes human antigens. The fact that the system with the addition of sodium deoxycholate (Example 1) also showed a low absorbance value similar to that of Comparative Example 1 (0.03 for both Example 1 and Comparative Example 1) means that no non-specific binding of mouse IgG occurred even after the membrane permeation treatment with sodium deoxycholate, and it can be said that the absorbance value obtained by adding the anti-ALIX antibody in Example 1 does not result from non-specific adsorption specific to mouse IgG and reflects the amount of the anti-ALIX antibody that recognized ALIX present inside the extracellular vesicles.

[0078] Although a high absorbance value was shown in the range of the content concentration of sodium deoxycholate from 0.05% (w / v) to 0.2% (w / v), the absorbance value decreased when it deviated from the said concentration range. At concentrations lower than 0.05% (w / v), the membrane permeation process was insufficient, so the antibody could not enter inside and the absorbance value was low. At concentrations higher than 0.2% (w / v), although the membrane permeation process itself was sufficient, the proteins including ALIX contained inside the extracellular vesicles also leaked outside the vesicles, and it was presumed that the absorbance value decreased because the amount of protein remaining inside the extracellular vesicles decreased.

[0079]

Table 1

[0080] Example 2 In Example 1(4), an aqueous solution prepared by dissolving Triton X-100 (trade name), a nonionic surfactant, in PBS to a concentration of 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.1% (w / v) or 0.5% (w / v) was used as the membrane permeation treatment agent, and the color development of TMB was measured in the same manner as in Example 1.

[0081] The results of Example 2 are shown in Table 2. Since the absorbance value with the mouse control antibody did not depend on the Triton X-100 concentration and showed almost the same value (from 0.03 to 0.04) as in Comparative Example 1 (Table 1), it can be seen that non-specific adsorption due to the membrane permeation process did not occur. On the other hand, when at least 0.01% (w / v) or more of Triton X-100 was added in the membrane permeation process, the absorbance value with the anti-ALIX antibody was significantly higher than that with the mouse control antibody, so it can be seen that nonionic surfactants can also permeate the membrane of extracellular vesicles. Although a high absorbance value was shown in the range of the content concentration of Triton X-100 from 0.01% (w / v) to 0.1% (w / v), the absorbance value decreased when it deviated from the said range.

[0082]

Table 2

[0083] Example 3 In Example 1(4), an aqueous solution prepared by dissolving saponin, a nonionic surfactant, in PBS at concentrations of 0.01% (w / v), 0.1% (w / v), 0.25% (w / v), 1% (w / v), 2% (w / v), or 10% (w / v) as a membrane permeation treatment agent was used, and the color development of TMB was measured in the same manner as in Example 1.

[0084] The results of Example 3 are shown in Table 3. Different from the cases of sodium deoxycholate (Example 1) and Triton X-100 (Example 2), the absorbance values with the anti-ALIX antibody increased depending on the saponin concentration added. Also, the absorbance values with the mouse control antibody increased depending on the concentration when the saponin concentration was 2% (w / v) or higher, indicating that non-specific binding of mouse IgG occurred. If it is at least in the range of 0.1% (w / v) to 1% (w / v) of the saponin concentration, the absorbance value with the anti-ALIX antibody is significantly higher than that with the mouse control antibody, and no increase in the absorbance value with the mouse control antibody due to non-specific binding is observed. Therefore, it can be determined that it can be used as the membrane permeation treatment agent in the present invention.

[0085]

Table 3

[0086] Example 4 In Example 1(1), the lot of the culture supernatant was changed (lot number 2), and an extracellular vesicle concentrated suspension released from 293T cells was obtained. In Example 1(4), except that an aqueous solution prepared by dissolving sodium glycolate, an anionic surfactant, in PBS at 0.01% (w / v), 0.05% (w / v), 0.08% (w / v), 0.1% (w / v), 0.2% (w / v), 0.3% (w / v), 0.4% (w / v), 0.5% (w / v), 0.6% (w / v), 0.8% (w / v), or 1% (w / v) was used as the membrane permeation treatment agent, the color development of TMB was measured in the same manner as in Example 1.

[0087] Comparative Example 2 In Example 1(1), the lot of the culture supernatant was changed (lot number 2), and an extracellular vesicle concentrated suspension released from 293T cells was obtained. In Example 1(4), except that 100 μL of PBS was added, the color development of TMB was measured in the same manner as in Example 1.

[0088] The results of Example 4 and Comparative Example 2 are summarized in Table 4. Since the absorbance value with the mouse control antibody is independent of the sodium glycolate concentration and shows a value equivalent to that of Comparative Example 2 (both 0.02), it can be seen that non-specific adsorption does not occur in the membrane permeation step. On the other hand, when at least 0.05% (w / v) or more of sodium glycolate is added in the membrane permeation step, the absorbance value with the anti-ALIX antibody is significantly higher than that with the mouse control antibody, indicating that sodium glycolate can also permeate the membrane of extracellular vesicles.

[0089] The content concentration of sodium glycolate showing a high absorbance value is from 0.05% (w / v) to 0.8% (w / v), which is higher than that of sodium deoxycholate (from 0.05% (w / v) to 0.2% (w / v)), which is the same anionic surfactant. This is presumably because the critical micelle concentration of sodium glycolate is higher than that of sodium deoxycholate (sodium deoxycholate: 5 mM, sodium glycolate: 13 mM).

[0090]

Table 4

[0091] Example 5 In Example 1(1), the lot of the culture supernatant was changed (lot number 2), and an extracellular vesicle concentrated suspension released from 293T cells was obtained. In Example 1(4), except that an aqueous solution prepared by dissolving SDS (sodium dodecyl sulfate), an anionic surfactant, in PBS at 0.01% (w / v), 0.02% (w / v), 0.1% (w / v), 0.2% (w / v), 0.5% (w / v), or 1% (w / v) was used as the membrane permeation treatment agent, the color development of TMB was measured in the same manner as in Example 1.

[0092] The results of Example 5 are shown in Table 5. Since the absorbance value with the mouse control antibody is independent of SDS and shows a value equivalent to that of Comparative Example 2 (Table 4) (both 0.02), it can be seen that non-specific adsorption due to the membrane permeation step does not occur. On the other hand, when 0.02% (w / v) of SDS is added in the membrane permeation step, since the absorbance value with the anti-ALIX antibody is significantly higher than the value with the mouse control antibody, it can be seen that SDS can also permeate the membrane of extracellular vesicles.

[0093] The SDS concentration showing a high absorbance value is only 0.02% (w / v), and when compared with sodium deoxycholate (0.05% (w / v) to 0.2% (w / v)) and sodium glycolate (0.05% (w / v) to 0.8% (w / v)), which are the same anionic surfactants, the optimal concentration range is narrow. This is presumably because the protein denaturing ability of SDS is higher than that of sodium deoxycholate and sodium glycolate.

[0094]

Table 5

[0095] Example 6 In Example 1(1), the extracellular vesicle concentrated suspension released from 293T cells was obtained by changing the lot of the culture supernatant (lot number 3), and in Example 1(4), except that cetyltrimethylammonium bromide (CTAB), a cationic surfactant, was dissolved in PBS to a concentration of 0.001% (w / v), 0.002% (w / v), 0.01% (w / v), 0.05% (w / v), or 0.2% (w / v) as a membrane permeabilizing agent, the color development of TMB was measured in the same manner as in Example 1.

[0096] Comparative Example 3 In Example 1(1), the extracellular vesicle concentrated suspension released from 293T cells was obtained by changing the lot of the culture supernatant (lot number 3), and in Example 1(4), except that 100 μL of PBS was added, the color development of TMB was measured in the same manner as in Example 1.

[0097] The results of Example 6 and Comparative Example 3 are summarized in Table 6. Since the absorbance value with the mouse control antibody is independent of CTAB and shows a value equivalent to that of Comparative Example 3 (both 0.02), it can be seen that non-specific adsorption does not occur in the membrane permeation step. On the other hand, when at least 0.002% (w / v) of CTAB is added in the membrane permeation step, the absorbance value with the anti-ALIX antibody is significantly higher than that with the mouse control antibody, indicating that the cationic surfactant can also permeate the membrane of extracellular vesicles. Although high absorbance values were shown in the range of CTAB content concentration from 0.002% (w / v) to 0.05% (w / v), the absorbance value decreased outside this range.

[0098] [Table 6]

[0099] Example 7 In Example 1(1), an extracellular vesicle concentrated suspension released from 293T cells was obtained by changing the lot of the culture supernatant (lot number 4), and in Example 1(4), except that an aqueous solution dissolved in PBS containing any one of the following (a) to (g) was used as the membrane permeation treatment agent, the color development of TMB was measured in the same manner as in Example 1. The addition concentrations in (a) to (f) are the optimal concentrations of each surfactant obtained from the results of Examples 1 to 6. (a) 0.02% (w / v) SDS (b) 0.5% (w / v) sodium glycolate (c) 0.075% (w / v) sodium deoxycholate (d) 0.01% (w / v) CTAB (e) 0.25% (w / v) saponin (f) 0.02% (w / v) Triton X-100 (g) 95% (v / v) ethanol

[0100] Comparative Example 4 In Example 1(1), an extracellular vesicle concentrated suspension released from 293T cells was obtained by changing the lot of the culture supernatant (lot number 4), and in Example 1(4), except that 100 μL of PBS was added, the color development of TMB was measured in the same manner as in Example 1.

[0101] Comparative Example 5 In Example 1(1), an extracellular vesicle concentrated suspension released from 293T cells was obtained by changing the lot of the culture supernatant (lot number 4), and in Example 1(4), except that after adding 100 μL of PBS, ultrasonic treatment was carried out for 5 minutes, the color development of TMB was measured in the same manner as in Example 1.

[0102] The results of Example 7 and Comparative Examples 4 and 5 are summarized in Table 7. Using the extracellular vesicle concentrated suspension of the same lot and performing the membrane permeation step at the optimal concentration of each surfactant, the absorbance values when using anionic surfactants ((a) SDS: 1.10, (b) sodium glycolate: 1.12, (c) sodium deoxycholate: 1.13) were higher than those of nonionic surfactants ((e) saponin: 0.77, (f) Triton X-100: 0.78) and cationic surfactants ((d) CTAB: 0.82). From this result, among the surfactants, anionic surfactants are more suitable as the membrane permeation treatment agent used in the present invention in terms of better membrane permeability of extracellular vesicles and / or the ability to suppress the leakage of proteins contained inside the extracellular vesicles after membrane permeation.

[0103] Even when using ethanol (g), which is an organic solvent, the absorbance value with the anti-ALIX antibody (0.28) is higher than that with the mouse control antibody (0.04) and also higher than the absorbance value with the anti-ALIX antibody in Comparative Example 4 (0.10), indicating that the membrane of extracellular vesicles can be permeated. However, the absorbance value with the anti-ALIX antibody was lower than the values (from 0.77 to 1.13) when a surfactant was added. This is presumably because the magnetic particles aggregated when ethanol was added, resulting in a decrease in the reaction efficiency after the membrane permeation step.

[0104] When ultrasonic treatment was performed instead of the membrane permeation step (Comparative Example 5), the absorbance value with the anti-ALIX antibody was higher than that in Comparative Example 4 (0.10) at 0.25, but lower than the values (from 0.77 to 1.13) when a surfactant was added. This is presumably because the membrane disruption of extracellular vesicles was not sufficient in the above treatment and / or the binding between extracellular vesicles and magnetic particles was disrupted in the above treatment.

[0105]

Table 7

[0106] Example 8 In Example 1(1), the lot of the culture supernatant was changed (lot number 4), and an extracellular vesicle concentrated suspension released from 293T cells was obtained. In Example 1(4), after removing the washing solution, 100 μL of PBS containing 1% (w / v) formaldehyde as an immobilizing agent was added, stirred for 5 minutes, the supernatant was removed using a magnet, and then 100 μL of PBS containing 0.075% (w / v) sodium deoxycholate as a membrane permeabilization treatment agent was added and stirred again for 5 minutes. The color development of TMB was measured in the same manner as in Example 1.

[0107] Example 9 In Example 1(1), the lot of the culture supernatant was changed (lot number 4), and an extracellular vesicle concentrated suspension released from 293T cells was obtained. In Example 1(4), 100 μL of an aqueous PBS solution in which formaldehyde as an immobilizing agent and sodium deoxycholate as a membrane permeabilization treatment agent were dissolved in PBS to have any of the concentrations from (a) to (f) was added. The color development of TMB was measured in the same manner as in Example 1. (a) 1% (w / v) formaldehyde + 0.05% (w / v) sodium deoxycholate (b) 1% (w / v) formaldehyde + 0.075% (w / v) sodium deoxycholate (c) 1% (w / v) formaldehyde + 0.1% (w / v) sodium deoxycholate (d) 1% (w / v) formaldehyde + 0.2% (w / v) sodium deoxycholate (e) 1% (w / v) formaldehyde + 0.5% (w / v) sodium deoxycholate (f) 4% (w / v) formaldehyde + 0.1% (w / v) sodium deoxycholate

[0108] Comparative Example 6 In Example 1(1), the lot of the culture supernatant was changed (lot number 4), and an extracellular vesicle concentrated suspension released from 293T cells was obtained. In Example 1(4), 100 μL of PBS containing 1% (w / v) or 4% (w / v) formaldehyde was added. The color development of TMB was measured in the same manner as in Example 1.

[0109] The results of Examples 8 and 9 and Comparative Examples 4 and 6 are summarized in Table 8. Even when the step of adding formaldehyde as an immobilizing agent (immobilization step) is added, the absorbance values with the anti-ALIX antibody (from 0.47 to 1.05) are all higher than the value with the mouse control antibody (0.04), indicating that it is possible to permeate the membrane of extracellular vesicles (Examples 8 and 9). Furthermore, by simultaneously performing the above immobilization step and the step of adding sodium deoxycholate as a membrane permeabilizing agent (membrane permeabilization step) (Example 9(b)), it can be seen that the absorbance value with the anti-ALIX antibody is improved compared to when the membrane permeabilization step is performed after the immobilization step (Example 8).

[0110] On the other hand, when only the immobilization step was performed (Comparative Example 6), the absorbance values with the anti-ALIX antibody were almost the same as when only PBS was added (that is, when neither the immobilization step nor the membrane permeabilization step was performed, Comparative Example 4) (Comparative Example 4: 0.10, Comparative Example 6: 0.10 to 0.11).

[0111] In Example 9, even when the concentration of sodium deoxycholate was increased to 0.5% (w / v), there was no decrease in the absorbance value generated in Example 1 (Table 1). This can be presumably because the addition of formaldehyde as an immobilizing agent suppressed the leakage of proteins contained inside extracellular vesicles. Also, when the formaldehyde concentration was set to 4% (w / v) without changing the concentration of sodium deoxycholate, a higher detection value was shown compared to when the formaldehyde concentration was 1% (w / v) (Example 9(c): 0.85, Example 9(f): 1.05). From this result as well, the inhibitory effect of formaldehyde as an immobilizing agent on protein leakage can be confirmed.

[0112]

Table 8

[0113] Example 10 The color development of TMB was measured in the same manner as in Example 1, except that in Example 1(1), the lot of the culture supernatant was changed (lot number 4) to obtain a concentrated suspension of extracellular vesicles released from 293T cells, and in Example 1(4), 100 μL of an aqueous solution in which the fixative and membrane permeabilizing agent, ethanol, and the membrane permeabilizing agent, sodium deoxycholate, were dissolved in PBS to give a concentration of any one of (a) to (h) was added. (a) 5% (v / v) ethanol + 0.1% (w / v) sodium deoxycholate (b) 10% (v / v) ethanol + 0.1% (w / v) sodium deoxycholate (c) 25% (v / v) ethanol + 0.1% (w / v) sodium deoxycholate (d) 50% (v / v) ethanol + 0.1% (w / v) sodium deoxycholate (e) 5% (v / v) ethanol (f) 10% (v / v) ethanol (g) 25% (v / v) ethanol (h) 50% (v / v) ethanol

[0114] The results of Example 10 are shown in Table 9. When 25% (v / v) ethanol or 50% (v / v) ethanol was combined with 0.1% (w / v) sodium deoxycholate (Examples 10(c) and (d)), the absorbance value was higher than that of sodium deoxycholate alone (Example 7(c), Table 7) (Example 7(c): 1.13, Example 10(c): 1.58, Example 10(d): 1.40). On the other hand, when 25% (v / v) ethanol alone (Example 10(g)) was used, the absorbance was 0.16, which did not show sufficient membrane permeation effect. From this, it is presumed that the insolubilization of protein by ethanol (immobilization step) and the membrane permeation by sodium deoxycholate (membrane permeation step) occurred simultaneously, which significantly suppressed the leakage of protein from inside the extracellular vesicles and resulted in a high detection value (absorbance value).

[0115] [Table 9]

[0116] Example 11 The color development of TMB was measured in the same manner as in Example 1, except that in Example 1(1), the lot of the culture supernatant was changed (lot number 5) to obtain a concentrated suspension of extracellular vesicles released from 293T cells, and in Example 1(4), 100 μL of an aqueous solution of 40% (v / v) ethanol alone, or 40% (v / v) ethanol and 0.0001% (w / v), 0.001% (w / v), 0.01% (w / v), 0.02% (w / v), 0.05% (w / v), 0.1% (w / v), 0.2% (w / v), or 0.5% (w / v) sodium deoxycholate, a fixative and membrane permeabilizing agent, dissolved in PBS, was added.

[0117] The results of Example 11 are shown in Table 10. In this Example, the absorbance values (0.46 to 0.72) for the anti-ALIX antibody were all higher than the value (0.04) for the mouse control antibody, indicating that the membrane permeation of the extracellular vesicles was performed appropriately. In addition, when 0.001% (w / v) to 0.2% (w / v) sodium deoxycholate was added to 40% (v / v) ethanol, the absorbance value for the anti-ALIX antibody was improved (0.64 to 0.72) compared with the fixation step and the membrane permeation step (0.46) when 40% (v / v) ethanol alone was used. It is presumed that when the concentration of sodium deoxycholate was higher than 0.2% (w / v), the insolubilization of the protein by ethanol was insufficient and the protein leaked from the inside of the extracellular vesicles.

[0118] As shown in Example 1 (Table 1), when the membrane permeation step was carried out with sodium deoxycholate alone, almost no membrane permeation of extracellular vesicles occurred at 0.01% (w / v) or less (absorbance value 0.05). On the other hand, in this example, when 40% (v / v) ethanol was further added, even with sodium deoxycholate at 0.01% (w / v) or less, the absorbance value with the anti-ALIX antibody improved (both 0.001% (w / v) and 0.01% (w / v) were 0.70) compared to when the immobilization step and the membrane permeation step were carried out with 40% (v / v) ethanol alone (0.46). From this, it can be seen that even in the concentration range of the surfactant where almost no effect of membrane permeation of extracellular vesicles is obtained, the addition of ethanol, which is an immobilizing agent and a membrane permeation treatment agent, may improve the detection value (absorbance value).

[0119]

Table 10

[0120] Example 12 In Example 1(1), the lot of the culture supernatant was changed (lot number 6) to obtain a concentrated suspension of extracellular vesicles released from 293T cells, and in Example 1(4), 100 μL of an aqueous solution dissolved in PBS containing 40% (v / v) ethanol, which is an immobilizing agent and a membrane permeation treatment agent, and any one of the surfactants (a) to (c), which is a membrane permeation treatment agent, were added. The color development of TMB was measured in the same manner as in Example 1. (a) 0.1% (w / v) sodium deoxycholate (b) 0.05% (w / v) Triton X-100 (c) 0.01% (w / v) CTAB

[0121] The results of Example 12 are shown in Table 11. By using sodium deoxycholate, an anionic surfactant, as the surfactant to be mixed with 40% (w / v) ethanol, the absorbance value with the anti-ALIX antibody was higher (sodium deoxycholate: 0.82) compared to when Triton X-100 (nonionic surfactant) or CTAB (cationic surfactant) was used (Triton X-100: 0.63, CTAB: 0.38).

[0122]

Table 11

[0123] Example 13 In Example 1(1), the lot of the culture supernatant was changed (lot number 7) to obtain a concentrated suspension of extracellular vesicles released from 293T cells. In Example 1(3), 0.5 μL of the concentrated suspension of extracellular vesicles was used. In Example 1(4), 100 μL of an aqueous solution in which ethanol, an immobilizing agent and a membrane permeabilizing agent, was dissolved in PBS to a concentration of any one of (a) to (g) was added. In Example 1(9), except that a plate reader manufactured by Corona Electric Co., Ltd. was used, the color development of TMB was measured in the same manner as in Example 1. (a) 20% (v / v) ethanol (b) 30% (v / v) ethanol (c) 40% (v / v) ethanol (d) 50% (v / v) ethanol (e) 60% (v / v) ethanol (f) 70% (v / v) ethanol (g) 95% (v / v) ethanol

[0124] Comparative Example 7 Except that PBS was used as the immobilizing agent and the membrane permeabilizing agent in Example 13, the color development of TMB was measured in the same manner as in Example 13.

[0125] The results of Example 13 and Comparative Example 7 are summarized in Table 12. By using ethanol, which is an organic solvent, the relative value of the absorbance of the anti-ALIX antibody against the mouse control antibody was higher (Example 13: 1.472 - 3.408) compared to the case without ethanol addition (Comparative Example 7: 1.470). In particular, the relative value was high in the range of 30% - 50% ethanol concentration (3.363 - 3.408). It is presumed that when the ethanol concentration is low, the membrane permeability of extracellular vesicles is insufficient, and when the ethanol concentration is high, the dispersion stability of magnetic particles decreases, resulting in the existence of an optimal concentration range.

[0126] [Table 12]

[0127] Example 14 In Example 1(1), the lot of the culture supernatant was changed (lot number 7) to obtain a concentrated suspension of extracellular vesicles released from 293T cells. In Example 1(3), 0.5 μL of the concentrated suspension of extracellular vesicles was used. In Example 1(4), 100 μL of an aqueous solution in which acetone, which is an immobilizing agent and a membrane permeation treatment agent, was dissolved in PBS to a concentration of any one of (a) to (f) was added. In Example 1(9), except that a plate reader manufactured by Corona Electric Co., Ltd. was used, the color development of TMB was measured in the same manner as in Example 1. (a) 20% (v / v) acetone (b) 30% (v / v) acetone (c) 40% (v / v) acetone (d) 60% (v / v) acetone (e) 70% (v / v) acetone (f) 99% (v / v) acetone

[0128] The results of Example 14 are shown in Table 13. By using 20% to 70% acetone, which is an organic solvent, the relative value of the absorbance of the anti-ALIX antibody against the mouse control antibody was higher (Example 14: 2.75 - 3.75) compared to the case without acetone addition (Comparative Example 7: 1.47). When 99% acetone was used, the 96-well plate melted and it became difficult to evaluate. In particular, the relative value was high (3.30 - 3.75) in the range of 30% to 70% acetone concentration. It is presumed that when the acetone concentration was low, the membrane permeation of extracellular vesicles was insufficient and the value decreased.

[0129]

Table 13

[0130] Example 15 In Example 1(1), the lot of the culture supernatant was changed (lot number 8) to obtain a concentrated suspension of extracellular vesicles released from 293T cells. In Example 1(3), 0.5 μL of the concentrated suspension of extracellular vesicles was used. In Example 1(4), 100 μL of an aqueous solution containing any one of the surfactants or organic solvents (a) to (e), which is an immobilizing agent and / or a membrane permeation treatment agent, was added. In Example 1(9), except that a plate reader manufactured by Corona Electric Co., Ltd. was used, the color development of TMB was measured in the same manner as in Example 1. The addition concentrations in (a) and (e) are the optimal concentrations of each surfactant or organic solvent obtained from the results of Examples 1 to 6 and 13, 14. (a) 0.075% (w / v) sodium deoxycholate (b) 0.02% (w / v) Triton X-100 (c) 0.01% (w / v) CTAB (d) 40% (v / v) ethanol (e) 40% (v / v) acetone

[0131] The results of Example 15 are shown in Table 14. By using an organic solvent, the relative absorbance value of the anti-ALIX antibody against the mouse control antibody was higher compared to surfactants (sodium deoxycholate: 2.72, Triton X-100: 2.59, CTAB: 1.49) (ethanol: 7.56, acetone: 4.10). Among the surfactants, the relative value of sodium deoxycholate, which is an anionic surfactant, was the highest, and among the organic solvents, the value of ethanol was high.

[0132]

Table 14

[0133] Example 16 As the amount of extracellular vesicle suspension in Example 15, 2 μL was used. As the antibody in Example 15, 2.5 μg / mL biotinylated mouse-derived anti-human HSP70 (Heat shock protein 70, a protein present inside extracellular vesicles released from 293T cells) antibody (manufactured by StressMarq Biosciences) or 2.5 μg / mL biotinylated mouse control antibody (Mouse Isotype Control IgG, manufactured by abcam) was used. Otherwise, the color development of TMB was measured in the same manner as in Example 15.

[0134] The results of Example 16 are shown in Table 15. Even when using an organic solvent, the relative absorbance value of the anti-ALIX antibody against the mouse control antibody hardly changed compared to surfactants (sodium deoxycholate: 2.46, Triton X-100: 1.40, CTAB: 1.66) (ethanol: 2.37, acetone: 1.95). Among the surfactants, the relative value of sodium deoxycholate, which is an anionic surfactant, was the highest, and among the organic solvents, the value of ethanol was high. From the measurement results for HSP70 and the measurement results for ALIX (Example 15), it can be seen that regardless of the type of specific protein to be measured, high sensitivity measurement can be achieved by using an anionic surfactant among surfactants and ethanol among organic solvents.

[0135]

Table 15

[0136] Comparative Example 8 (1) An extracellular vesicle suspension (lot number 7) similar to that of Example 13 was adjusted to 0.5 μL (prepared by adding 4.5 μL of PBS to a total of 5 μL) or 5 μL, and 1.25 μL of loading buffer (250 mM Tris-HCl (pH 6.8), 8% SDS, 0.1% bromophenol blue, 40% Glycerol, 100 mM DTT) was added, followed by heating at 95°C for 5 minutes to permeabilize the extracellular vesicles and solubilize and denature the proteins inside the vesicles.

[0137] (2) The sample prepared in (1) was electrophoresed (30 mA, 40 minutes) using a polyacrylamide gel (catalog number: 4561096, manufactured by Bio-Rad) to separate the proteins.

[0138] (3) The proteins separated by the gel in (2) were blotted onto a PVDF membrane (catalog number: 1704156, manufactured by Bio-Rad) using a Trans-Blot Turbo transfer system (manufactured by Bio-Rad).

[0139] (4) The membrane blotted in (3) was blocked using Blocking One (manufactured by Nacalai Tesque), and immersed in a solution containing Blocking One diluted 20-fold with TBST (Tris Buffered Saline containing 0.1% Tween 20) and 1 μg / mL anti-ALIX antibody at room temperature for 60 minutes.

[0140] (5) After washing the membrane in (4) with TBST, it was immersed in a solution containing Blocking One diluted 20-fold with TBST and HRP-conjugated goat anti-mouse IgG antibody (catalog number: 31430, manufactured by ThermoFisher) diluted 10,000-fold at room temperature for 60 minutes.

[0141] After washing the membranes of (6)(5) with TBST, they were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Catalog No.: 34580, manufactured by ThermoFisher), and then photographed to confirm the ALIX band.

[0142] The results of Comparative Example 8 are shown in Figure 2. When the amount of the extracellular vesicle suspension was 0.5 μL, especially when the ethanol concentration was from 30% to 50% in Example 13 (relative value of absorbance with the anti-ALIX antibody against the mouse control antibody: 3.363 - 3.408), ALIX inside the extracellular vesicles could be detected with high sensitivity. In contrast, in the Western blotting results of Comparative Example 16, a band derived from ALIX could be confirmed when the amount of the extracellular vesicle suspension was 5 μL, but the band could not be confirmed when the amount was 0.5 μL.

[0143] Even for an amount of extracellular vesicles that could not be detected by the method of Patent Document 3 in which detection is performed by Western blotting after leaking the proteins inside the extracellular vesicles, it was possible to detect with high sensitivity by performing detection without leaking the proteins inside the extracellular vesicles by this detection method.

[0144] Example 17 (1) In the same manner as in Example 1(1), a concentrated suspension of extracellular vesicles released from 293T cells was obtained by changing the lot of the culture supernatant (lot number 9).

[0145] (2) Using 0.5 μL of the concentrated extracellular vesicle suspension obtained in (1), except that 100 μL of an aqueous solution containing 40% (v / v) ethanol was used as the membrane permeabilization agent, the membrane permeabilization treatment of the extracellular vesicles was performed in the same manner as in Example 1(2) to (4).

[0146] (3) After removing the washing solution, 100 μL of the activation reagent from (a) to (c) was added and stirred for 10 minutes. After removing the supernatant using a magnet in the same manner as in Example 1(3), washing 3 times with PBS, and then performing blocking treatment, HRP-modified streptavidin was bound in the same manner as in Example 1(5) to (7). (a) PBS (control for the activation reagent) (b) Proteinase K (Catalog No.: 9034, manufactured by Takara) diluted 50-fold with PBS (c) HistoReveal (manufactured by abcam)

[0147] (4) After removing the washing solution, 150 μL of the substrate reaction solution of SuperSignal ELISA Pico Chemiluminescent Substrate (manufactured by ThermoFisher) as the chromogenic substrate was added, and the luminescence amount was measured with a plate reader (manufactured by Tecan) within 5 minutes.

[0148] Example 18 In Example 17(2), in the treatment with the immobilizing agent and the membrane permeabilizing agent, 100 μL of PBS containing 1% or 4% (w / v) formaldehyde was added, and after stirring for 5 minutes, the supernatant was removed using a magnet. Then, 100 μL of an aqueous solution containing 40% (v / v) ethanol, which is the immobilizing agent and the membrane permeabilizing agent, was added and stirred again for 5 minutes. In the activation treatment of Example 17(3), the luminescence amount was measured in the same manner as in Example 17, except that 100 μL of the activation reagent of PBS or Proteinase K diluted 50-fold with PBS was used.

[0149] The results of Examples 17 and 18 are summarized in Table 16. In Example 17, by adding the activation reagent, the relative value of the luminescence amount of the anti-ALIX antibody against the mouse control antibody was higher (Proteinase K: 6.36, HistoReveal: 7.28) compared to the case without addition (PBS: 2.22). From this result, it can be seen that the relative value is improved by adding the activation reagent, and the sensitivity is improved when using either the enzyme treatment agent of Proteinase K or HistoReveal containing trypsin. In particular, the luminescence amount in the mouse control antibody decreased by adding the activation reagent, indicating that the activation treatment contributes to the suppression of non-specific adsorption.

[0150] In Example 18, by increasing the amount of formaldehyde added, as compared to no formaldehyde added (Example 17: 6.36), the relative value was improved (1% formaldehyde: 7.57, 4% formaldehyde: 8.67) by performing the activation treatment. On the other hand, when the activation treatment was not performed, even if the amount of formaldehyde added was increased, the relative value only increased slightly (PBS: 2.22, 4% formaldehyde: 2.50). Formaldehyde can retain proteins inside extracellular vesicles by cross-linking proteins. However, on the other hand, the recognition site of the antibody by the cross-linking is masked, and sufficient reactivity is not exhibited. Since the activation treatment enables the antibody to regain its binding ability to the antigen again by cleaving the cross-linked portion, it is presumed that when the formaldehyde treatment and the activation treatment are combined, the luminescence amount in the anti-ALIX antibody increases and the detection sensitivity is improved.

[0151]

Table 16

[0152] Example 19 In Example 1(1), the lot of the culture supernatant was changed (lot number 7) to obtain a concentrated suspension of extracellular vesicles released from 293T cells, in Example 1(3), 0.5 μL of the concentrated suspension of extracellular vesicles was used, in Example 1(4), 100 μL of an aqueous solution dissolved in PBS to contain 0.075% (w / v) sodium deoxycholate, which is a membrane permeation treatment agent, was added, and in Example 1(9), except that a plate reader manufactured by Corona Electric Co., Ltd. was used, the color development of TMB was measured in the same manner as in Example 1.

[0153] Comparative Example 9 Except that PBS was used instead of the membrane permeation treatment agent in Example 19, the color development of TMB was measured in the same manner as in Example 19.

[0154] The results of Example 19 and Comparative Example 9 are shown in Table 17. Even when the extracellular vesicle suspension volume was 0.5 μL, the relative value of the absorbance of the anti-ALIX antibody against the mouse control antibody was higher in the membrane permeabilization treatment with sodium deoxycholate compared to PBS (Comparative Example 9: 1.21) (Example 19: 8.45). On the other hand, as described above, in the Western blotting results of Comparative Example 8, a band derived from ALIX could be confirmed when the extracellular vesicle suspension volume was 5 μL, but the band could not be confirmed when it was 0.5 μL.

[0155] Even for an amount of extracellular vesicles that cannot be detected by the method of Patent Document 3 in which detection is performed by Western blotting after leaking the proteins inside the extracellular vesicles, it was possible to detect with high sensitivity by detecting without leaking the proteins inside the extracellular vesicles by this detection.

[0156] [Table 17]

Industrial Applicability

[0157] According to the present invention, for example, the membrane permeabilization treatment of the vesicles can be carried out in order to simply and accurately detect a specific protein contained inside the extracellular vesicles. Further, according to the present invention, for example, a specific protein contained inside the extracellular vesicles can be simply and accurately detected. Therefore, the internal protein of a specific extracellular vesicle can be detected from among a plurality of types of extracellular vesicles. The method of the present invention can be expected not only for elucidating the physiological functions of extracellular vesicles but also for application to disease tests using body fluids.

Explanation of Signs

[0158] 100: Well 200: Magnet 300: Detector 10: Extracellular vesicle 11: Membrane-permeated extracellular vesicle 12: Labeled extracellular vesicle 20: Magnetic particle 30: Antibody immobilized on magnetic particles 40: Substrate 41: Product 50: Enzyme reaction

Claims

1. A method for detecting a specific protein contained inside extracellular vesicles, comprising: (A) a step of capturing extracellular vesicles using a carrier immobilized with a substance capable of binding to an extracellular vesicle-specific marker present on the surface of the extracellular vesicles; (B) a step of subjecting the extracellular vesicles captured on the carrier to membrane permeabilization treatment using a membrane permeabilization treatment agent; and (C) a step of introducing a reagent capable of detecting a specific protein contained inside the extracellular vesicles into the extracellular vesicles subjected to membrane permeabilization treatment wherein the step (B) is carried out so as not to leak the specific protein to the outside of the extracellular vesicles and to allow the reagent to be introduced into the inside of the extracellular vesicles; the membrane permeabilization treatment agent is a surfactant and / or an organic solvent; the surfactant is any one of the following (a) to (c): (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycocholate (c) 0.01 to 0.1% (w / v) SDS the organic solvent is any one of the following (a) or (b): (a) 20 to 60% (v / v) ethanol (b) 20 to 70% (v / v) acetone.

2. The method according to claim 1, further comprising a step of removing contaminants after the step (A) and / or after the step (B).

3. The method according to claim 1 or 2, further comprising a step of detecting the specific protein using the introduced reagent after the step (C).

4. The method according to any one of claims 1 to 3, wherein the carrier is magnetic particles.

5. A membrane permeabilization treatment agent for extracellular vesicles, comprising a surfactant and / or an organic solvent, characterized in that it can subject extracellular vesicles to membrane permeabilization treatment so as not to leak a specific protein contained inside the extracellular vesicles to the outside of the extracellular vesicles and to allow a reagent capable of detecting the specific protein contained inside the extracellular vesicles to be introduced into the inside of the extracellular vesicles, wherein the surfactant is any one of the following (a) to (c): (a) 0.05 to 0.5% (w / v) deoxycholate (b) 0.01 to 1% (w / v) glycocholate (c) 0.01 to 0.1% (w / v) SDS the organic solvent is any one of the following (a) or (b): (a) 20 to 60% (v / v) ethanol (b) 20 to 70% (v / v) acetone.

6. A kit for detecting a specific protein contained inside extracellular vesicles, comprising A kit comprising magnetic particles capable of binding to an extracellular vesicle-specific marker present on the surface of extracellular vesicles, the membrane permeation treatment agent according to claim 5, and a reagent capable of detecting a specific protein contained inside the extracellular vesicles.

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