Method for recovering extracellular vesicles

Abstract

The present invention provides a method for recovering extracellular vesicles from a sample containing extracellular vesicles. More specifically, the present invention provides a method for recovering extracellular vesicles, comprising separating the extracellular vesicles from a sample containing extracellular vesicles in the presence of a polymer.

Description

Technical Field

[0001] The present invention relates to a method for recovering extracellular vesicles and the like.

Background Art

[0002] Extracellular vesicles (EV) are minute vesicles with a membrane structure that are secreted from various types of cells and are present in body fluids such as blood or cell culture media. Extracellular vesicles secreted outside the cell include exosomes, ectosomes, and apoptotic blebs. Since extracellular vesicles are a diverse population containing various substances that play functions such as cell - to - cell communication, they have been analyzed for purposes such as diagnosis and drug discovery. Therefore, there is a demand for the development of a method for recovering extracellular vesicles useful for such analysis. For example, Patent Document 1 describes a method for recovering extracellular vesicles using a chelating agent.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] If extracellular vesicles can be recovered from a sample containing extracellular vesicles with high efficiency, it is promising for applications such as diagnosis and drug discovery. Extracellular vesicles are mainly recovered by immunoprecipitation using an antibody against an extracellular vesicle marker or by ultracentrifugation. However, with such methods, extracellular vesicles are not necessarily recovered with high efficiency, and the recovered extracellular vesicles are non - specifically concentrated, resulting in variations in quality.

[0005] Therefore, an object of the present invention is to develop a novel method capable of recovering extracellular vesicles.

Means for Solving the Problem

[0006] As a result of intensive studies, the present inventors have found that extracellular vesicles can be recovered with high efficiency by separating extracellular vesicles from a sample containing extracellular vesicles in the presence of a predetermined polymer, and have thus completed the present invention.

[0007] That is, the present invention is as follows. 〔1〕A method for recovering extracellular vesicles, comprising separating extracellular vesicles from a sample containing extracellular vesicles in the presence of a polymer. 〔2〕The method according to 〔1〕, wherein the polymer has a value of 1.5 mPa·s or more as the viscosity in a 1 to 20 wt% aqueous solution at 20 to 30°C. 〔3〕The method according to 〔1〕 or 〔2〕, wherein the polymer is a cellulose derivative or a polyvinyl derivative having a carbonyl-containing hydrophilic group. 〔4〕The method according to 〔3〕, wherein the polymer is a cellulose derivative in which at least one hydrogen atom of a hydroxyl group is substituted with a carboxyalkyl or hydroxyalkyl, or a polyvinyl derivative in which at least one hydrogen atom is substituted with a lactam. 〔5〕The method according to 〔4〕, wherein the polymer is carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, or polyvinylpyrrolidone. 〔6〕The method according to any one of 〔1〕 to 〔5〕, wherein the polymer has a weight average molecular weight of 10 kDa or more. 〔7〕The method according to any one of 〔1〕 to 〔6〕, wherein the concentration of the polymer when separating extracellular vesicles from the sample containing extracellular vesicles is 0.01 to 10.00 wt%. 〔8〕The method according to any one of 〔1〕 to 〔7〕, further comprising combining the sample containing extracellular vesicles with a chelating agent. 〔9〕The method according to any one of 〔1〕 to 〔8〕, wherein the extracellular vesicles are exosomes. 〔10〕The method according to any one of 〔1〕 to 〔9〕, wherein the separation is performed by a separation method using an extracellular vesicle membrane-binding substance or by ultracentrifugation of the sample containing extracellular vesicles. The method of

[10] , wherein the extracellular vesicle membrane-binding substance is an antibody against a tetraspanin membrane protein or an antibody against an extracellular matrix metalloprotease inducer substance. The method of

[11] , wherein the extracellular vesicle membrane-binding substance is an antibody against CD9, CD63, CD81, or CD147. The method according to any one of [1] to

[12] , wherein the extracellular vesicle-containing sample is a blood sample, urine, or saliva. The method for analyzing extracellular vesicles, comprising: (1) Separating extracellular vesicles from an extracellular vesicle-containing sample in the presence of a polymer; and (2) Analyzing the separated extracellular vesicles. The method of

[14] , further comprising adding a chelating agent to the extracellular vesicle-containing sample. The method according to

[14] or

[15] , wherein proteins or nucleic acids in the separated extracellular vesicles are analyzed. A kit comprising a polymer and an extracellular vesicle membrane-binding substance. The kit of

[17] , further comprising a chelating agent.

Advantages of the Invention

[0008] According to the present invention, by using a predetermined polymer, extracellular vesicles can be recovered with higher efficiency and higher purity.

Brief Description of the Drawings

[0009]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0010] The present invention provides a method for recovering extracellular vesicles.

[0011] Extracellular vesicles are minute vesicles having a membrane structure, which are secreted from various types of cells. Examples of extracellular vesicles include exosomes, ectosomes, and apoptotic bodies. Preferably, the extracellular vesicles are exosomes. Extracellular vesicles can also be defined by their size. The size of extracellular vesicles is, for example, 30 to 1000 nm, preferably 50 to 300 nm, more preferably 80 to 200 nm. The size of extracellular vesicles can be measured, for example, by a method based on the Brownian motion of extracellular vesicles, a light scattering method, and an electrical resistance method. Preferably, the size of extracellular vesicles is measured by NanoSight (manufactured by Malvern Instruments).

[0012] The recovery method of the present invention includes the following steps: (1) Separating extracellular vesicles from an extracellular vesicle-containing sample in the presence of a polymer.

[0013] An extracellular vesicle-containing sample is any sample containing extracellular vesicles. Preferably, the extracellular vesicle-containing sample is a biological liquid sample. The extracellular vesicle-containing sample may be subjected to other treatments before being used in the method of the present invention. Such treatments include, for example, centrifugation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring.

[0014] In one embodiment, the extracellular vesicle-containing sample is a culture supernatant. The culture supernatant may be a cell culture supernatant or a tissue culture supernatant. Examples of the organism from which the cells or tissues to be cultured are derived include mammals (e.g., primates such as humans and monkeys; rodents such as mice, rats, and rabbits; domestic animals such as cows, pigs, and goats; and draft animals such as horses and sheep), birds (e.g., chickens), insects, microorganisms (e.g., bacteria), plants, and fish. Preferably, the organism is a mammal such as a human.

[0015] In another embodiment, the extracellular vesicle-containing sample is a body fluid. The body fluid is a body fluid derived from an organism as described above. Examples of the body fluid include blood samples (e.g., whole blood, serum, and plasma), urine, saliva, lymph fluid, tissue fluid, cerebrospinal fluid, ascites, sweat, semen, tears, mucus, milk, pleural fluid, bronchoalveolar lavage fluid, and amniotic fluid. Preferably, the body fluid is a blood sample, urine, or saliva. Examples of plasma include plasma containing heparin, citrate, sodium fluoride, ACD (acid-citrate-dextrose), or CPD (citrate phosphate dextrose). Generally, the recovery of extracellular vesicles is difficult in body fluids (e.g., blood, urine, saliva) in which a larger amount of proteins (e.g., albumin, lysozyme, lactoferrin, histatin, peroxidase, agglutinin, defensin, immunoglobulin) are mixed compared to the culture supernatant. On the other hand, according to the method of the present invention, the recovery amount of extracellular vesicles from the extracellular vesicle-containing sample increases, and extracellular vesicles can be recovered with high efficiency and high purity even from such body fluids.

[0016] The polymer used in the present invention is preferably a water-soluble polymer. The "water-soluble polymer" refers to a polymer having a solubility in water at 4 to 80 °C (preferably 4 to 37 °C) of 0.01% by weight or more. The solubility of the water-soluble polymer in water at 4 to 80 °C (preferably 4 to 37 °C) may preferably be 0.05% by weight or more, more preferably 0.1% by weight or more.

[0017] For the purpose of achieving the object of the present invention, the polymer may have a value of 1.5 mPa·s or more as the viscosity in a 1 to 20% by weight aqueous solution at 20 to 30 °C. The viscosity of the polymer under the above conditions is preferably 5 mPa·s or more, more preferably 10 mPa·s or more, even more preferably 20 mPa·s or more, still even more preferably 30 mPa·s or more, particularly preferably 35 mPa·s or more. The viscosity of the polymer under the above conditions may preferably be 30000 mPa·s or less, more preferably 20000 mPa·s or less, even more preferably 10000 mPa·s or less, still even more preferably 5000 mPa·s or less, particularly preferably 1000 mPa·s or less. More specifically, the viscosity of the polymer under the above conditions is preferably 5 to 30000 mPa·s, more preferably 10 to 20000 mPa·s, even more preferably 20 to 10000 mPa·s, still even more preferably 30 to 5000 mPa·s, particularly preferably 35 to 1000 mPa·s.

[0018] The polymer may have a value of 1.5 mPa·s or more as the viscosity at 30°C in a PBS solution prepared by dissolving the polymer in phosphate buffered saline (PBS) to 2% by weight in order to achieve the object of the present invention. As the viscosity of the polymer under the above conditions, it is preferably 5 mPa·s or more, more preferably 10 mPa·s or more, still more preferably 20 mPa·s or more, even still more preferably 30 mPa·s or more, and particularly preferably 35 mPa·s or more. As the viscosity of the polymer under the above conditions, it is preferably 30000 mPa·s or less, more preferably 20000 mPa·s or less, still more preferably 10000 mPa·s or less, even still more preferably 5000 mPa·s or less, and particularly preferably 1000 mPa·s or less. More specifically, as the viscosity of the polymer under the above conditions, it is preferably 5 to 30000 mPa·s, more preferably 10 to 20000 mPa·s, still more preferably 20 to 10000 mPa·s, even still more preferably 30 to 5000 mPa·s, and particularly preferably 35 to 1000 mPa·s.

[0019] The viscosity of the polymer can be measured, for example, by a method of detecting the viscous frictional torque of the liquid generated on the outer periphery of the rotor when the liquid sample is rotated by a rotating body (method using a rotational viscometer), or a method of freely dropping a falling weight in a measuring tube filled with the sample and measuring the falling time (method using a falling ball viscometer). Also, the viscosity of the polymer can be measured, for example, by a method of putting a vibrating body (viscosity sensor) into a liquid sample and vibrating it. When the vibrating body vibrates, the vibration amplitude of the sensor is suppressed and decreased due to the viscous resistance as the liquid viscosity increases. However, by overcoming this suppressing force and increasing the vibrator drive current to maintain a constant amplitude, the input current amount at this time can be measured (method using a vibrating viscometer). The viscosity of the polymer can be measured, for example, using a viscosity analyzer (e.g., Rheology Spectrometer SKR100, Yamato Scientific Co., Ltd.).

[0020] The polymer may be, for example, a cellulose derivative, a polyvinyl derivative having a hydrophilic group, or a polyether compound.

[0021] The cellulose derivative is a cellulose derivative in which at least one hydrogen atom of the hydroxyl group of cellulose is substituted with a hydrophilic group. Examples of the hydrophilic group in the cellulose derivative include carboxyalkyl (e.g., carboxy C 1~6 alkyl), hydroxyalkyl (e.g., hydroxy C 1~6 alkyl). The hydrophilic group in the cellulose derivative is preferably carboxyalkyl or hydroxyalkyl. Examples of the carboxyalkyl include carboxymethyl, carboxyethyl (1-carboxyethyl, 2-carboxyethyl), carboxypropyl (1-carboxypropyl, 2-carboxypropyl, 3-carboxypropyl), carboxyisopropyl (1-carboxy-2-methylethyl, 2-carboxy-2-methylethyl), carboxybutyl (1-carboxybutyl, 2-carboxybutyl, 3-carboxybutyl, 4-carboxybutyl), carboxyt-butyl, carboxypentyl (1-carboxypentyl, 2-carboxypentyl, 3-carboxypentyl, 4-carboxypentyl, 5-carboxypentyl), carboxyhexyl (1-carboxyhexyl, 2-carboxyhexyl, 3-carboxyhexyl, 4-carboxyhexyl, 5-carboxyhexyl, 6-carboxyhexyl). Examples of the hydroxyalkyl include hydroxymethyl, hydroxyethyl (1-hydroxyethyl, 2-hydroxyethyl), hydroxypropyl (1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl), hydroxyisopropyl (1-hydroxy-2-methylethyl, 2-hydroxy-2-methylethyl), hydroxybutyl (1-hydroxybutyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl), hydroxy t-butyl, hydroxypentyl (1-hydroxypentyl, 2-hydroxypentyl, 3-hydroxypentyl, 4-hydroxypentyl, 5-hydroxypentyl), hydroxyhexyl (1-hydroxyhexyl, 2-hydroxyhexyl, 3-hydroxyhexyl, 4-hydroxyhexyl, 5-hydroxyhexyl, 6-hydroxyhexyl). Specific examples of the cellulose derivative include carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), or hydroxypropyl methyl cellulose (HPMC). The cellulose derivative includes a nanocellulose derivative. The nanocellulose derivative is a derivative of nanocellulose described later.

[0022] The polyvinyl derivative having a hydrophilic group is a polyvinyl derivative in which at least one hydrogen atom is substituted with a hydrophilic group, and a polyvinyl derivative in which one hydrogen atom in the methylene unit is substituted with a hydrophilic group is preferable. Examples of the hydrophilic group of the polyvinyl derivative include a carbonyl-containing hydrophilic group, a carboxy-containing hydrophilic group, a nitrogen-containing hydrophilic group, and a ring (carbocyclic or heterocyclic)-containing hydrophilic group. The hydrophilic group of the polyvinyl derivative is preferably a carbonyl-containing hydrophilic group, a nitrogen-containing hydrophilic group, or a heterocyclic-containing hydrophilic group, and lactam (e.g., α-lactam, β-lactam, γ-lactam, δ-lactam, ε-lactam) is more preferable. Specific examples of the polyvinyl derivative having a hydrophilic group include polyvinyl pyrrolidone.

[0023] A polyether compound is a polymer containing an ether structure in the main chain of the repeating unit. Examples of the polyether compound include polyalkyleneoxy compounds (e.g., poly C 1~6 alkyleneoxy compounds). Examples of the polyalkyleneoxy compounds include polyethylene glycol and polypropylene glycol. The polyalkyleneoxy compound is preferably polyethylene glycol.

[0024] Nanocellulose is fibrous cellulose having a fiber width on the order of nanometers. The fiber width of the nanocellulose is, for example, 500 nm or less, preferably 200 nm or less, more preferably 100 nm or less, even more preferably 50 nm or less, still even more preferably 10 nm or less, particularly more preferably 5 nm or less.

[0025] Cellulose derivatives, polyvinyl derivatives having a hydrophilic group, and polyether compounds include their salts. Examples of the salts include salts of metals (e.g., monovalent metals such as lithium, sodium, potassium, rubidium, and cesium, and divalent metals such as calcium, magnesium, and zinc), and salts of inorganic bases (e.g., ammonia).

[0026] The polymer may have, for example, a weight average molecular weight of 10 kDa or more in order to achieve the object of the present invention. The weight average molecular weight of the polymer is preferably 12 kDa or more, more preferably 14 kDa or more, still more preferably 16 kDa or more, yet still more preferably 18 kDa or more, and particularly preferably 20 kDa or more. The weight average molecular weight of the polymer is preferably 5000 kDa or less, more preferably 3000 kDa or less, still more preferably 2000 kDa or less, yet still more preferably 1000 kDa or less, and particularly preferably 500 kDa or less. More specifically, the weight average molecular weight of the polymer is preferably 12 to 5000 kDa, more preferably 14 to 3000 kDa, still more preferably 16 to 2000 kDa, yet still more preferably 18 to 1000 kDa, and particularly preferably 20 to 500 kDa.

[0027] The concentration of the polymer in the extracellular vesicle separation step is not particularly limited as long as extracellular vesicles can be recovered with high efficiency compared to the case where the polymer is not included and the polymer can be dissolved in the solution used in the separation step. Such a concentration varies depending on the type of the polymer, but may be, for example, 0.01 to 10.00% by weight, preferably 0.05 to 7.50% by weight, and more preferably 0.10 to 5.00% by weight.

[0028] In the extracellular vesicle separation step, the extracellular vesicles are separated in the liquid phase from the extracellular vesicle-containing sample. That is, the extracellular vesicles are separated from the extracellular vesicle-containing sample by a separation method described later without coprecipitating with the polymer. Therefore, the separation step in the present invention is different from the separation by the coprecipitation method using a coprecipitating polymer (for example, polyethylene glycol precipitation). Therefore, the polymer used in the present invention is preferably a non-coprecipitating polymer.

[0029] In the recovery method of the present invention, since the polymer only needs to be present in the separation step, the recovery method of the present invention may include combining the extracellular vesicle-containing sample and the polymer. For example, after combining the extracellular vesicle-containing sample and the polymer, extracellular vesicles may be separated from the extracellular vesicle-containing sample. Also, for example, when using the separation method using the extracellular vesicle membrane-binding substance described later, the polymer may be added in advance to the solution containing the extracellular vesicle membrane-binding substance, and then the extracellular vesicle-containing sample may be added thereto.

[0030] The recovery method of the present invention may further include combining the extracellular vesicle-containing sample and a chelating agent. In this case, in the recovery method of the present invention, since extracellular vesicles may be separated from the extracellular vesicle-containing sample in the presence of the polymer and the chelating agent, for example, the polymer and the chelating agent may be added to the extracellular vesicle-containing sample simultaneously, or the chelating agent may be added after adding the polymer to the extracellular vesicle-containing sample, or the polymer may be added after adding the chelating agent to the extracellular vesicle-containing sample. Also, for example, when using the separation method using the extracellular vesicle membrane-binding substance described later, the polymer and the chelating agent may be added in advance to the solution containing the extracellular vesicle membrane-binding substance, and then the extracellular vesicle-containing sample may be added thereto.

[0031] The chelating agent is a compound or a salt thereof having a coordination moiety capable of forming a coordination bond with a metal ion. The number of coordination moieties is preferably 2 or more, more preferably 3 or more (e.g., 3 or 6). Examples of the coordination atom as the coordination moiety include an oxygen atom, a phosphorus atom, a nitrogen atom, a sulfur atom, and a chlorine atom. The coordination atom is preferably an oxygen atom or a phosphorus atom, more preferably an oxygen atom. Examples of the coordination group as the coordination moiety include groups having the above-mentioned coordination atoms. The coordination group is preferably a carboxylic acid group or a phosphate group, more preferably a carboxylic acid group.

[0032] Examples of the chelating agent include oxalic acid, hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetra(methylenephosphonic acid) (EDTMP), glycol ether diamine tetraacetic acid (EGTA), and salts thereof. Examples of the salts include metal salts (e.g., monovalent metal salts such as sodium salt and potassium salt, and divalent metal salts such as calcium salt and magnesium salt), inorganic salts (e.g., halide salts such as fluoride, chloride, bromide, and iodide salts, and ammonium salts), organic salts (e.g., ammonium salts substituted with an alkyl group), and acid addition salts (e.g., salts with inorganic acids such as sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, and phosphoric acid, and salts with organic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, and trifluoroacetic acid). In the present invention, a mixture of two or more (e.g., two, three, four, five) chelating agents may be used for separating the extracellular vesicle-containing sample.

[0033] The chelating agent probably shows an effect on the high-purity recovery of extracellular vesicles by suppressing the adsorption of contaminants to the extracellular vesicles (International Publication No. 2018 / 070479). Therefore, separating extracellular vesicles from an extracellular vesicle-containing sample in the presence of a polymer and a chelating agent provides a synergistic effect on improving the recovery efficiency of extracellular vesicles. Since any of the above-mentioned chelating agents shows an effect on the high-purity recovery of extracellular vesicles, a synergistic effect of the same quality is provided by combining with a polymer.

[0034] The concentration of the chelating agent in the extracellular vesicle separation step is not particularly limited as long as it can suppress the adsorption of contaminants to the extracellular vesicles and the chelating agent can be dissolved in the solution used in the separation step. Such a concentration varies depending on the type of chelating agent, but for example, it is 1 mM to 200 mM. Preferably, the concentration of the chelating agent may be 10 mM or more, 15 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, or 50 mM or more. Such a concentration also varies depending on the type of chelating agent, but it may be 200 mM or less, 180 mM or less, 160 mM or less, 140 mM or less, 120 mM or less, or 100 mM or less.

[0035] Separation of extracellular vesicles from an extracellular vesicle-containing sample (step (1)) in the presence of a polymer can be carried out, for example, by a separation method using an extracellular vesicle membrane-binding substance or by ultracentrifugation. Separation using an extracellular vesicle membrane-binding substance may be carried out during the process of an analytical method (e.g., immunoassay described below). When separating extracellular vesicles using an extracellular vesicle membrane-binding substance, the extracellular vesicle membrane-binding substance is mixed with the extracellular vesicle-containing sample to bind the extracellular vesicle membrane-binding substance to the extracellular vesicles, and then the extracellular vesicles bound with the extracellular vesicle membrane-binding substance are separated from the sample to recover the extracellular vesicles. Also, when separating extracellular vesicles by ultracentrifugation, the extracellular vesicles in the extracellular vesicle-containing sample are precipitated by ultracentrifugation, and then the supernatant is discarded to recover the extracellular vesicles. Further, when separating extracellular vesicles during the process of an analytical method, the extracellular vesicles can be separated along with the removal of the solution or the washing of the solid phase in the analytical method (e.g., immunoassay such as ELISA). Specifically, in an immunoassay, the extracellular vesicles can be separated along with the binding of the extracellular vesicles to an antibody and the removal of the solution containing the extracellular vesicle-containing sample and / or the washing of the solid phase. The separation is preferably isolation or purification. Therefore, the recovery method of the present invention can also be used as an isolation or purification method.

[0036] In the recovery method of the present invention, the extracellular vesicle membrane-binding substance used is a substance having an affinity for an extracellular vesicle marker. Examples of extracellular vesicle markers include tetraspanin membrane proteins (extracellular vesicle membrane-specific four-pass transmembrane proteins, such as CD9, CD63, CD81), extracellular matrix metalloprotease inducer (CD147), carcinoembryonic antigen (CEA), heat shock protein (HSP) 70, HSP90, major histocompatibility complex (MHC) I, tumor susceptibility gene (TSG) 101, lysosome-associated membrane protein (LAMP) 1, intercellular adhesion molecule (ICAM)-1, integrin, ceramide, cholesterol, phosphatidylserine, ALIX, Annexins, Caveolin-I, Flotillin-I, Rab proteins, EpCAM, etc. The extracellular vesicle marker is preferably a tetraspanin membrane protein or an extracellular matrix metalloprotease inducer. Also, the extracellular vesicle marker is preferably CD9, CD63, CD81, or CD147. Examples of extracellular vesicle membrane-binding substances include antibodies (such as monoclonal antibodies and polyclonal antibodies) and their antigen-binding fragments, aptamers, phosphatidylserine-binding proteins, and ceramide-binding proteins. The antigen-binding fragment may be any antibody fragment that maintains binding to the target extracellular vesicle marker, and examples include Fab, Fab’, F(ab’)2, scFv, etc. The extracellular vesicle membrane-binding substance is preferably an antibody or its antigen-binding fragment, more preferably a monoclonal antibody or its antigen-binding fragment. Also, the extracellular vesicle membrane-binding substances used for recovering extracellular vesicles may be used alone or in combination of multiple ones.

[0037] The extracellular vesicle membrane-binding substance may be bound to a solid phase for facilitating the separation of extracellular vesicles. As the solid phase, for example, sepharose beads, agarose beads, magnetic beads, or plastic plates can be used. Immobilization of the extracellular vesicle membrane-binding substance on the solid phase can be carried out by a conventional method well-known to those skilled in the art.

[0038] In the recovery method of the present invention, when extracellular vesicles are recovered by a separation method using an extracellular vesicle membrane-binding substance, the polymer only needs to be present when at least the extracellular vesicle membrane-binding substance and the extracellular vesicle-containing sample are mixed. For example, when using magnetic beads, in the presence of the polymer, the extracellular vesicle membrane-binding substance bound to the magnetic beads and the extracellular vesicle-containing sample are mixed to bind the extracellular vesicle membrane-binding substance to the extracellular vesicles in the extracellular vesicle-containing sample. Then, the magnetic beads are magnetically collected using a permanent magnet or an electromagnet, etc., and subsequently, the supernatant containing the components not bound to the magnetic beads is discarded, whereby the extracellular vesicles bound to the magnetic beads can be separated from the extracellular vesicle-containing sample. As the method for magnetically collecting the magnetic beads, methods well-known to those skilled in the art can be used. Further, for example, when using Sepharose beads or agarose beads, in the presence of the polymer, the extracellular vesicle membrane-binding substance bound to these beads and the extracellular vesicle-containing sample are mixed to bind the extracellular vesicle membrane-binding substance to the extracellular vesicles in the extracellular vesicle-containing sample. Then, the beads are precipitated by centrifugation, and the supernatant containing the components not bound to the beads is discarded, whereby the extracellular vesicles bound to the beads can be separated from the extracellular vesicle-containing sample. As the method for precipitating the beads by centrifugation, methods well-known to those skilled in the art can be used.

[0039] When separating extracellular vesicles from a sample containing extracellular vesicles by a separation method using an extracellular vesicle membrane-binding substance, the temperature at which the extracellular vesicle membrane-binding substance and the sample containing extracellular vesicles are mixed may be any temperature as long as the mixture of the extracellular vesicle membrane-binding substance and the sample containing extracellular vesicles exists in a liquid state, and may be 0 to 100°C. Such a temperature may be, for example, 4°C or higher, preferably 15°C or higher, more preferably 35°C or higher, and even more preferably 40°C or higher. Such a temperature may also be, for example, 80°C or lower, preferably 70°C or lower, more preferably 60°C or lower, and even more preferably 50°C or lower. More specifically, such a temperature may be, for example, 4 to 80°C, preferably 15 to 70°C, more preferably 35 to 60°C, and even more preferably 40 to 50°C. The time for binding the extracellular vesicle membrane-binding substance to the extracellular vesicles is not particularly limited as long as it is sufficient for the extracellular vesicle membrane-binding substance to bind to the extracellular vesicles, and may be, for example, 1 minute or longer, 5 minutes or longer, 10 minutes or longer, or 20 minutes or longer. Such a time may also be 24 hours or shorter, 18 hours or shorter, 8 hours or shorter, 4 hours or shorter, 2 hours or shorter, or 1 hour or shorter.

[0040] In the recovery method of the present invention, when recovering extracellular vesicles by ultracentrifugation, the polymer only needs to be present when the sample containing extracellular vesicles is subjected to ultracentrifugation. For example, when ultracentrifugation is performed multiple times, at least one ultracentrifugation may be performed in the presence of the polymer. Ultracentrifugation can be performed using an ultracentrifuge. The gravity applied in ultracentrifugation is, for example, 10,000×g to 200,000×g, and preferably may be 70,000×g to 150,000×g. The time of ultracentrifugation is, for example, 0.5 to 24 hours, and preferably 1 to 5 hours. The temperature in ultracentrifugation is, for example, 4 to 30°C. Ultracentrifugation may be performed once or multiple times (e.g., 2 times, 3 times).

[0041] The present invention also provides a method for analyzing extracellular vesicles.

[0042] The analysis method of the present invention includes the following: (1) In the presence of a polymer, separating extracellular vesicles from a sample containing extracellular vesicles; and (2) Analyzing the separated extracellular vesicles.

[0043] Step (1) in the analysis method of the present invention can be carried out in the same manner as step (1) in the recovery method of the present invention.

[0044] In step (2), the separated extracellular vesicles are analyzed. The objects to be analyzed include, for example, components contained in extracellular vesicles (e.g., components contained inside extracellular vesicles, membrane components of extracellular vesicles, components present on the membrane surface of extracellular vesicles), and extracellular vesicles themselves (particles).

[0045] When the components contained in extracellular vesicles are analyzed, the analysis is the detection or quantification of the components. Such analysis is also the analysis of one or more components. Examples of the components to be analyzed include proteins, nucleic acids (e.g., RNA, DNA), sugars, lipids, amino acids, vitamins, polyamines, and peptides. By separating extracellular vesicles from a sample containing extracellular vesicles in the presence of a polymer, the recovery amount of extracellular vesicles can be increased. Therefore, according to the analysis method of the present invention, components such as proteins and nucleic acids in extracellular vesicles can be analyzed with high precision.

[0046] The analysis of the components can be carried out by any method known in the art. When the component to be analyzed is a protein, examples of analysis methods include immunoassay and mass spectrometry. Examples of immunoassays include direct competition method, indirect competition method, and sandwich method. Also, such immunoassays include chemiluminescent immunoassay (CLIA) [e.g., chemiluminescent enzyme immunoassay (CLEIA)], turbidimetric immunoassay (TIA), enzyme immunoassay (EIA) (e.g., direct competition ELISA, indirect competition ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination reaction method, fluorescence immunoassay (FIA), immunochromatography method, Western blotting, immunostaining, fluorescence activated cell sorting (FACS). When multiple components are analyzed, proteome analysis may be performed. When the component to be analyzed is a nucleic acid, examples of analysis methods include hybridization method using a probe, reverse transcription (RT) reaction using reverse transcriptase, gene amplification method using primers (e.g., 2, 3, or 4 primers) (e.g., PCR methods such as quantitative PCR, RT-PCR), sequencing, and mass spectrometry. When the component to be analyzed is a component other than protein and nucleic acid, examples of analysis methods include immunoassay and mass spectrometry. When multiple components are analyzed, metabolome analysis may be performed.

[0047] The analysis method of the present invention can be used for detecting markers contained in extracellular vesicles. Examples of markers contained in extracellular vesicles include markers that serve as indicators of the presence of extracellular vesicles, and markers that serve as indicators of diseases such as cancer (e.g., diagnostic markers, disease risk assessment markers). Examples of markers that serve as indicators of the presence of extracellular vesicles include tetraspanin membrane proteins (extracellular vesicle membrane-specific four-transmembrane proteins, e.g., CD9, CD63, CD81), extracellular matrix metalloprotease inducer (CD147), carcinoembryonic antigen (CEA), heat shock protein (HSP) 70, HSP90, major histocompatibility complex (MHC) I, tumor susceptibility gene (TSG) 101, lysosome-associated membrane protein (LAMP) 1, intercellular adhesion molecule (ICAM)-1, integrin, ceramide, cholesterol, phosphatidylserine, ALIX, Annexins, Caveolin-I, Flotillin-I, Rab proteins, EpCAM, and nucleic acids encoding these proteins (e.g., DNA, RNA). Examples of markers that serve as indicators of diseases include proteins that specifically exist in extracellular vesicles derived from organisms suffering from diseases such as cancer or extracellular vesicles secreted from abnormal cells such as cancer cells (hereinafter referred to as "abnormal proteins". E.g., mutant proteins, foreign proteins). Examples of mutant proteins include fusion proteins such as EML4-ALK. EML4-ALK has variants such as variant 1, 2, 3a, 3b, 4, 5a, 5b, 6, etc. Examples of foreign proteins include virus-derived proteins. Examples of markers that serve as indicators of diseases also include the presence or absence of expression of nucleic acids encoding these proteins or changes in their amounts, as well as the presence or absence of mutations such as SNPs, haplotypes, translocations, methylation of nucleic acids, and the types of variants.

[0048] Analysis of extracellular vesicles themselves (particles) can be performed by devices such as particle analyzers, electron microscopes, flow cytometers, etc. In this case, the number of particles of extracellular vesicles, the size and shape of the particles, and their distribution can be analyzed.

[0049] Extracellular vesicles have been reported to be involved in various diseases such as cancer (International Publication No. WO2014 / 003053; International Publication No. WO2014 / 152622; Taylor et al., Gynecologic Oncol, 100 (2008) pp13-21). Therefore, the recovery method and analysis method of the present invention are useful, for example, for diagnosis and drug discovery based on extracellular vesicles. For example, detecting EML4-ALK, which is a cancer marker such as lung cancer, by the analysis method of the present invention can be useful as an index for determining the administration of ALK tyrosine kinase inhibitors such as crizotinib and alectinib to cancer patients such as lung cancer patients.

[0050] In another embodiment, the present invention provides a method for analyzing extracellular vesicles, comprising: (1) separating extracellular vesicles from a sample containing extracellular vesicles using an extracellular vesicle membrane-binding substance; and (2) analyzing a marker that serves as an indicator of a disease such as cancer contained in the separated extracellular vesicles.

[0051] Examples of the extracellular vesicle membrane-binding substance in this embodiment include substances having an affinity for the above-described extracellular vesicle markers. The extracellular vesicle markers are preferably tetraspanin membrane proteins and extracellular matrix metalloprotease-inducing substances. The extracellular vesicle markers are preferably CD9, CD63, CD81, or CD147, more preferably CD9 or CD63. Examples of the extracellular vesicle membrane-binding substance include the above-described antibodies (e.g., monoclonal antibodies, polyclonal antibodies) and antigen-binding fragments thereof, aptamers, phosphatidylserine-binding proteins, and ceramide-binding proteins.

[0052] Examples of markers that indicate diseases such as cancer include the presence or absence of the expression of the above-described abnormal proteins or changes in their amounts. Examples of abnormal proteins include fusion proteins such as EML4-ALK. Examples of markers that indicate diseases also include the presence or absence of the expression of nucleic acids encoding these proteins or changes in their amounts, as well as mutations such as SNPs, haplotypes, translocations, the presence or absence of methylation of nucleic acids, and the types of variants.

[0053] The present invention also provides a kit containing a polymer and an extracellular vesicle membrane-binding substance as described above. The kit of the present invention may further contain a chelating agent. The kit of the present invention is useful, for example, for the convenient implementation of the recovery method and analysis method of the present invention.

Examples

[0054] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

[0055] (Example 1) The effects of three commercially available sodium CMC salts (hereinafter simply referred to as "CMC"; CAS No. 9004-32-4, Nacalai Tesque #07326-95 (average molecular weight unknown), Sigma-Aldrich #C5678 (average molecular weight 90 kDa), #C4888 (average molecular weight 250 kDa)) on EV recovery were examined. EV recovery was performed using an anti-CD9 antibody. After centrifuging normal human serum at 20,000×g for 15 minutes at 4°C, 200 μL of serum was diluted with 200 μL of PBS (2.9 mM NaH2PO4, 9.0 mM Na2HPO4, 137 mM NaCl), EDTA / EGTA-PBS (PBS containing EDTA and EGTA such that the final concentration after dilution of serum was 50 mM) (ED / EG), or CMC-PBS with a final concentration of 0.2 to 2.5 wt% (PBS in which CMC was dissolved such that the final concentration after dilution of serum was 0.2 to 2.5 wt%). Magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) fixed with anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After rotating the reaction overnight at 4°C, the magnetic beads were washed 3 times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare a sample for Western blotting. In the sample for Western blotting, the sample containing exosomes was treated with SDS, whereby the exosomes were disrupted and the marker protein of exosomes (e.g., CD9) was released into the sample solution. The immunoprecipitation efficiency was analyzed by Western blotting using biotinylated anti-CD9 antibody (manufactured in-house) (Figure 1). In all 4 types of CMC, an effect of improving the EV recovery efficiency was recognized as compared with the PBS-diluted sample. The physical property values of each CMC used in the examples are summarized in Table 1 below.

[0056]

Table 1

[0057] (Example 2) The effects of the concentration of CMC (Sigma-Aldrich #C4888) (final concentration of 0.06 wt% to 1.0 wt%) and reaction temperature (4°C, 37°C) on EV recovery were examined. After centrifuging normal human serum at 20,000×g for 15 minutes at 4°C, 200 μL of the serum was diluted with 200 μL of PBS or CMC-PBS (PBS in which CMC was dissolved so that the final concentration after serum dilution was 0.06 - 1.0 wt%). Magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) immobilized with anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After rotating the reaction overnight at 4°C or for 1 hour at 37°C, the magnetic beads were washed 3 times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare a sample for Western blotting. The recovery efficiency of EVs was analyzed by Western blotting using biotinylated anti-CD9 antibody (Figure 2). At reaction temperatures of 4°C and 37°C, in the range of 0.25 wt% - 1 wt% final concentration of CMC, an effect of improving the EV recovery efficiency was recognized compared to the PBS-diluted sample.

[0058] (Example 3) The effect of CMC (Sigma-Aldrich #C4888) on EV recovery was examined by nanoparticle tracking analysis (NanoSight LM10, Cantam Design). After centrifuging 200 μL of healthy human serum at 20,000×g for 15 minutes at 4°C, the supernatant was diluted with 200 μL of PBS, EDTA / EGTA-PBS (PBS containing EDTA and EGTA so that the final concentration of each after serum dilution is 50 mM) (ED / EG), or CMC-PBS (PBS in which CMC was dissolved so that the final concentration after serum dilution is 0.5% by weight) (CMC), and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) fixed with anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After rotating and reacting at 37°C for 30 minutes, the magnetic beads were washed 3 times with PBS-T, reacted with 40 μL of BRUB (Britton & Robinson Universal Buffer) (pH 2.6) for 5 minutes, and then neutralized with 20 μL of 1M Tris-HCl (pH 8.0) to release extracellular vesicles from the antibody magnetic bead particles. After quantifying the total protein concentration by Qubit protein assay (Life Technologies), 450 μL of PBS was added and the particle number was analyzed by NanoSight (Figure 3). An increase in the total number of recovered EV particles and the number of particles per total protein was observed under the condition of dilution with CMC, indicating that extracellular vesicles were recovered with high purity.

[0059] (Example 4) The effect of CMC (Sigma-Aldrich #C4888) on EV recovery was examined in plasma containing serum and five anticoagulants (heparin, EDTA, citrate, ACD (acid-citrate-dextrose), CPD (citrate phosphate dextrose)). 200 μL of normal human serum and normal human plasma containing an anticoagulant were centrifuged at 20,000×g for 15 minutes at 4°C. Then, 200 μL of PBS, EDTA / EGTA-PBS (PBS containing EDTA and EGTA such that the final concentration of each after serum or plasma dilution is 50 mM) (ED / EG), CMC-PBS (PBS in which CMC was dissolved such that the final concentration after serum or plasma dilution is 0.5 wt%) (CMC), or EDTA / EGTA / CMC-PBS (PBS containing EDTA, EGTA, and CMC such that the respective final concentrations after serum or plasma dilution are 37.5 mM / 37.5 mM / 0.5 wt%) (ED / EG / C) was added to the supernatant, and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) immobilized with an anti-CD9 antibody (self-made) were added to a concentration of 0.26 mg / mL. After a rotary reaction at 37°C for 1 hour, the magnetic beads were washed 3 times with PBS-T and diluted with sample buffer (BIO-RAD) to obtain a sample for Western blotting. The immunoprecipitation efficiency was analyzed by Western blotting with a biotinylated anti-CD9 antibody (Figure 4). Regardless of the type of anticoagulant, an effect of improving the EV recovery efficiency by CMC was also observed in plasma specimens. Furthermore, the EV recovery efficiency was further improved by combining CMC with a chelating agent.

[0060] (Example 5) The effect of CMC (Sigma-Aldrich #C4888) on the recovery of EVs using antibodies against two types of tetraspanin membrane proteins other than CD9 (CD63 and CD81) and extracellular matrix metalloprotease inducer (CD147) was examined. After centrifuging 200 μL of normal human serum at 20,000×g for 15 minutes at 4°C, 200 μL of PBS, EDTA / EGTA-PBS (each final concentration of 50 mM after serum dilution) (ED / EG), CMC-PBS (final concentration of 0.5 wt% after serum dilution) (CMC), or EDTA / EGTA / CMC-PBS (each final concentration of 37.5 mM / 37.5 mM / 0.5 wt% after serum dilution) (ED / EG / C) was added to the supernatant, and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) immobilized with each of the antibodies anti-CD63 antibody (8A12: Cosmo Bio), anti-CD81 antibody (M38: Abcam), and anti-CD147 antibody (MEM-M6 / 1: Abcam) were added to a concentration of 0.26 mg / mL. After rotating the reaction overnight at 4°C, the magnetic beads were washed 3 times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare a sample for Western blotting. The EV recovery efficiency was analyzed by Western blotting using an anti-CD63 antibody (self-made), an anti-CD81 antibody (12C4: Cosmo Bio), and a biotinylated anti-CD9 antibody (self-made) (FIGS. 5A and 5B). The effect of improving the EV recovery efficiency by CMC was also observed when antibodies against CD63, CD81, and CD147 were used. In addition, the EV recovery efficiency was further improved by combining CMC with a chelating agent.

[0061] (Example 6) In two types of body fluids (urine and saliva), the effect of CMC (Sigma-Aldrich #C4888) on EV recovery was examined. 200 μL of normal human urine and saliva (two samples each, designated as "#1" and "#2") were centrifuged at 15,000×g for 15 minutes at 4°C, filtered through a 0.22 μm filter, and then diluted with 200 μL of PBS, EDTA / EGTA-PBS (each final concentration of 50 mM after urine or saliva dilution) (ED / EG), CMC-PBS (final concentration of 0.5 wt% after urine or saliva dilution) (CMC), or EDTA / EGTA / CMC-PBS (each final concentration of 37.5 mM / 37.5 mM / 0.5 wt% after urine or saliva dilution) (ED / EG / C). Magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) immobilized with anti-CD9 antibody (self-made) were added to a concentration of 0.26 mg / mL. After rotating the reaction at 37°C for 1 hour, the magnetic beads were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. The immunoprecipitation efficiency was analyzed by Western blotting with biotinylated anti-CD9 antibody (Figure 6). The effect of improving the EV recovery efficiency by CMC was observed not only in serum and plasma but also in urine and saliva.

[0062] (Example 7) The effects of the cellulose derivatives shown in Table 2 below (each final concentration of 0.13 wt% to 4.0 wt%) on EV recovery were examined. After centrifuging normal human serum at 20,000×g for 15 minutes at 4°C, 200 μL of the serum was diluted with 200 μL of PBS, CMC-PBS (final concentration of 0.5% by weight after serum dilution) (CMC), or a cellulose derivative dissolved in PBS (each final concentration after serum dilution of 0.13 - 4.0% by weight). Magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) immobilized with an anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After rotating the reaction at 37°C for 1 hour, the magnetic beads were washed 3 times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare a sample for Western blotting. The recovery efficiency of EVs was analyzed by Western blotting with a biotinylated anti-CD9 antibody (Figs. 7A - C). An effect of improving the EV recovery efficiency was also observed for the three types of cellulose derivatives other than CMC, as compared with the PBS-diluted sample (HEC in the range of 0.13 - 2.0% by weight, HPC in the range of 0.25 - 4.0% by weight, and HPMC in the range of 0.25 - 2.0% by weight).

[0063]

Table 2

[0064] (Example 8) The effects of polyvinylpyrrolidone (final concentrations of 1%, 2%, and 4% by weight) shown in Table 3 below on EV recovery were examined. After centrifuging normal human serum at 20,000×g for 15 minutes at 4°C, 200 μL of the serum was diluted with 200 μL of PBS or polyvinylpyrrolidone dissolved in PBS (each final concentration after serum dilution was 1.0 - 4.0 wt%). Magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) fixed with anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After rotating and reacting at 37°C for 1 hour, the magnetic beads were washed 3 times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare a sample for Western blotting. The recovery efficiency of EVs was analyzed by Western blotting using biotinylated anti-CD9 antibody (Figure 8). In the range of 1.0 wt% to 4.0 wt% of polyvinylpyrrolidone, an effect of improving the EV recovery efficiency was observed compared to the PBS-diluted sample.

[0065]

Table 3

[0066] (Example 9) Regarding CMC (Sigma-Aldrich #C4888), the effect on EV recovery at each reaction temperature from 35°C to 60°C was examined. After centrifuging normal human serum at 20,000×g for 15 minutes at 4°C, 100 μL of the serum was diluted with 100 μL of PBS or CMC-PBS (final concentration 0.5% by weight after serum dilution), and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) immobilized with anti-CD9 antibody (manufactured in-house), anti-CD63 antibody (8A12: Cosmo Bio), or anti-CD81 antibody (12C4: Cosmo Bio) were added to a concentration of 0.26 mg / mL. After reacting for 5 minutes at each reaction temperature, the magnetic beads were washed 3 times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. The recovery efficiency of EVs was analyzed by Western blotting using biotinylated anti-CD9 antibody (manufactured in-house), anti-CD63 antibody (manufactured in-house), and anti-CD81 antibody (12C4: Cosmo Bio) (Figs. 9A to 9C). The effect of improving the EV recovery efficiency was recognized at each reaction temperature from 35°C to 60°C by adding CMC. Furthermore, a higher effect of improving the EV recovery efficiency by adding CMC was recognized under high-temperature conditions of 40°C or higher. Also, the effect of improving the EV recovery efficiency by CMC was recognized even in short-time reactions.

[0067] (Example 10) The viscosities of the cellulose derivatives used in Example 7 and polyvinylpyrrolidone used in Example 8 in PBS solutions were measured. Specifically, each PBS solution prepared by dissolving each cellulose derivative or polyvinylpyrrolidone in PBS to a concentration of 2% by weight was measured using a viscosity analyzer rheology spectrometer SKR100 (Yamato Scientific Co., Ltd.) at 30°C for a measurement time of 60 seconds, and the average of the measurement results at each rotation speed of 200 rpm, 400 rpm, 600 rpm, and 800 rpm was determined (Table 4).

[0068] [Table 4]

[0069] (Example 11) The effects of immunoprecipitation using antibodies against CMC (Sigma-Aldrich #C4888) and tetraspanin membrane proteins (CD9 and CD63) on EV recovery and the detection of marker (EML4-ALK fusion gene) RNA from EVs were examined. The culture supernatant of human lung cancer cells H2228 cultured in serum-free medium for 3 days was used as a sample. The culture supernatant was centrifuged at 2,000×g for 5 minutes at 4°C, filtered through a 0.22 μm filter (Millipore), and then concentrated 100-fold using Amicon Ultra-15 (Millipore). The concentrate was diluted with an equal volume of PBS, EDTA / EGTA-PBS (final concentration of each after dilution of the concentrate: 50 mM) (ED / EG), CMC-PBS (final concentration after dilution of the concentrate: 0.5 wt%) (CMC), or EDTA / EGTA / CMC-PBS (final concentrations of each after dilution of the concentrate: 37.5 mM / 37.5 mM / 0.5 wt%) (ED / EG / C), and Dynabeads M-280 tosylactivated (Life Technologies) immobilized with each of the antibodies, anti-CD9 antibody (self-made) and anti-CD63 antibody (self-made), was added to a concentration of 0.26 mg / mL each. After rotating the reaction overnight at 4°C, it was washed 3 times with PBS-T, and total RNA was purified using the miRNeasy micro kit (QIAGEN). cDNA was prepared from the purified total RNA using the SuperScript(™)IV First-Strand Synthesis System (Thermo Fisher Scientific), and EML4-ALK mRNA was quantified using Droplet Digital PCR (BioRad) (Figure 10). Primers and fluorescent probes with the sequences shown in Table 5 below were used for the detection of EML4-ALK mRNA. As the fluorescent probe, a double quencher probe having a fluorescent substance HEX at the 5'-end, a ZEN quencher inside the probe, and an Iowa Black(®) quencher (IABkFQ) at the 3'-end was used. By using the primers and fluorescent probes with the sequences shown in Table 5, variants 3a and 3b of the EML4-ALK fusion gene can be detected.

[0070]

Table 5

[0071] EML4-ALK contained in EVs recovered by immunoprecipitation was detected. By using CMC, the detected amount of EML4-ALK mRNA increased, and an effect of improving the EV recovery efficiency was observed. Furthermore, by adding a chelating agent, the detected amount of EML4-ALK mRNA further increased, and an effect of further improving the EV recovery efficiency was observed.

Claims

1. A method for recovering extracellular vesicles, comprising separating extracellular vesicles from a sample containing extracellular vesicles in a single-phase aqueous solution containing a polymer, wherein the polymer comprises a cellulose derivative, polyvinylpyrrolidone, or a salt thereof, the separation is performed by a separation method using an extracellular vesicle membrane-binding substance, and the extracellular vesicle membrane-binding substance comprises (i) an antibody against CD9, CD63, CD81 or CD147, or (ii) a fragment of the antibody having an affinity for CD9, CD63, CD81 or CD147. A method for recovering extracellular vesicles.

2. The method according to claim 1, wherein the polymer has a value of 1.5 mPa·s or more as the viscosity in a 1 to 20% by weight aqueous solution at 20 to 30°C.

3. The method according to claim 1 or 2, wherein the cellulose derivative is a cellulose derivative in which at least one hydrogen atom of a hydroxyl group is substituted with a carboxyalkyl or hydroxyalkyl.

4. The method according to claim 1 or 2, wherein the cellulose derivative is carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, or hydroxypropyl methyl cellulose.

5. The method according to any one of claims 1 to 4, wherein the polymer has a weight average molecular weight of 10 kDa or more.

6. The method according to any one of claims 1 to 5, wherein the concentration of the polymer when separating extracellular vesicles from a sample containing extracellular vesicles is 0.01 to 10.00% by weight.

7. The method according to any one of claims 1 to 6, further comprising combining the extracellular vesicle-containing sample with a chelating agent.

8. The method according to any one of claims 1 to 7, wherein the extracellular vesicles are exosomes.

9. The method according to any one of claims 1 to 8, wherein the extracellular vesicle-containing sample is a blood sample, urine, or saliva.

10. The following: (1) Separating extracellular vesicles from a sample containing extracellular vesicles in a single-phase aqueous solution containing a polymer; and (2) Analyzing the separated extracellular vesicles, wherein the polymer comprises a cellulose derivative, polyvinylpyrrolidone, or a salt thereof, the separation is performed by a separation method using an extracellular vesicle membrane-binding substance, and the extracellular vesicle membrane-binding substance comprises (i) an antibody against CD9, CD63, CD81 or CD147, or (ii) a fragment of the antibody having an affinity for CD9, CD63, CD81 or CD147. Method for analyzing extracellular vesicles.

11. The method according to claim 10, further comprising adding a chelating agent to the extracellular vesicle-containing sample.

12. The method according to claim 10 or 11, wherein the protein or nucleic acid in the isolated extracellular vesicles is analyzed.

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