Method for recovering extracellular vesicles

Abstract

To provide a novel method of recovering an extracellular vesicle from an extracellular vesicle-containing sample.SOLUTION: The present invention provides a method of recovering an extracellular vesicle that includes separating the extracellular vesicle from the extracellular vesicle-containing sample in the presence of a polymer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for recovering extracellular vesicles, etc. [Background technology]

[0002] Extracellular vesicles (EVs) are minute vesicles with a membrane structure secreted from various types of cells, and are present in body fluids such as blood or cell culture fluids. Extracellular vesicles secreted outside cells include exosomes, ectosomes, and apoptotic blebs. Extracellular vesicles are a diverse group containing various substances that perform functions such as intercellular signaling, and therefore are analyzed for the purpose of diagnosis, drug discovery, and the like. Therefore, there is a demand for the development of a method for recovering extracellular vesicles that is useful for such analyses. 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] International Publication No. 2018 / 070479 Summary of the Invention [Problem to be solved by the invention]

[0004] If extracellular vesicles could be efficiently collected from samples containing extracellular vesicles, it would be useful for applications in diagnosis, drug discovery, etc. Extracellular vesicles are mainly collected by immunoprecipitation using antibodies against extracellular vesicle markers or ultracentrifugation. However, these methods do not necessarily collect extracellular vesicles with high efficiency, and the quality of the collected extracellular vesicles varies due to non-specific concentration.

[0005] Therefore, the 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 extensive research, the inventors discovered that extracellular vesicles can be recovered with high efficiency by separating them from a sample containing extracellular vesicles in the presence of a specific polymer, and 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 viscosity of 1.5 mPa s or more 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 of a hydroxyl group 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] Any of the methods according to [1] to [6], wherein the concentration of the polymer when separating extracellular vesicles from an extracellular vesicle-containing sample is 0.01 to 10.00% by weight. [8] Any of the methods according to [1] to [7], further comprising combining the extracellular vesicle-containing sample with a chelating agent. [9] Any of the methods according to [1] to [8], wherein the extracellular vesicles are exosomes.

[10] Any of the methods according to [1] to [9], wherein the separation is carried out by a separation method using an extracellular vesicle membrane-binding substance or by ultracentrifugation of an extracellular vesicle-containing sample.

[11] The method according to

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

[12] The method according to

[11] , wherein the extracellular vesicle membrane-bound substance is an antibody against CD9, CD63, CD81, or CD147.

[13] Any of the methods according to [1] to

[12] , wherein the extracellular vesicle-containing sample is a blood sample, urine, or saliva.

[14] A method for analyzing extracellular vesicles, comprising: (1) isolating extracellular vesicles from an extracellular vesicle-containing sample in the presence of a polymer; and (2) To analyze isolated extracellular vesicles.

[15] The method of

[14] , further comprising adding a chelating agent to the sample containing extracellular vesicles.

[16] The method of

[14] or

[15] , wherein proteins or nucleic acids in the separated extracellular vesicles are analyzed.

[17] A kit comprising a polymer and an extracellular vesicle membrane-binding substance.

[18] The kit according to

[17] , further comprising a chelating agent. Effect of the Invention

[0008] According to the present invention, by using a specific polymer, it is possible to recover extracellular vesicles more efficiently and with higher purity. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation using anti-CD9 antibody of serum specimens diluted with PBS, EDTA / EGTA-PBS ("ED / EG"), or various concentrations of CMC-PBS in Example 1. [Diagram 2] FIG. 2 shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation with anti-CD9 antibody at 4° C. overnight or at 37° C. for 1 hour of serum specimens diluted with PBS or various concentrations of CMC-PBS in Example 2. [Diagram 3] FIG. 3 shows the results of particle count measurement by nanoparticle tracking analysis of samples obtained by immunoprecipitation using an anti-CD9 antibody from serum specimens diluted with PBS, EDTA / EGTA-PBS ("ED / EG"), or CMC-PBS in Example 3. [Figure 4] FIG. 4 shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation using anti-CD9 antibody of specimens in Example 4, which were serum and plasma containing five types of anticoagulants (heparin, EDTA, citrate, ACD (acid-citrate-dextrose), and CPD (citrate phosphate dextrose)) diluted with PBS, CMC-PBS, EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C"). [Figure 5A] FIG. 5A shows Western blotting using anti-tetraspanin membrane protein antibodies (anti-CD63 antibody and anti-CD81 antibody) of samples obtained by immunoprecipitation using anti-tetraspanin membrane protein antibodies (anti-CD63 antibody and anti-CD81 antibody) of serum specimens diluted with PBS, CMC-PBS, EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C") in Example 5. [Figure 5B] Figure 5B shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation with anti-CD147 antibody of serum specimens diluted with PBS, CMC-PBS, EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C") in Example 5. [Figure 6]FIG. 6 shows Western blotting, using biotinylated anti-CD9 antibody, of samples obtained by immunoprecipitation with anti-CD9 antibody from specimens in Example 6 (urine and saliva, two samples each, designated as "#1" and "#2") diluted with PBS, CMC-PBS, EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C"). [Figure 7A] FIG. 7A shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation using anti-CD9 antibody of serum specimens diluted with PBS, various concentrations of HEC-PBS, or CMC-PBS in Example 7. [Figure 7B] FIG. 7B shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation using anti-CD9 antibody of serum specimens diluted with PBS, various concentrations of HPC-PBS, or CMC-PBS in Example 7. [Figure 7C] FIG. 7C shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation using anti-CD9 antibody of serum specimens diluted with PBS, various concentrations of HPMC-PBS, or CMC-PBS in Example 7. [Figure 8] FIG. 8 shows Western blotting, using biotinylated anti-CD9 antibody, of samples obtained by immunoprecipitation with anti-CD9 antibody of serum specimens diluted with PBS or various concentrations of PVP-PBS in Example 8. [Figure 9A] Figure 9A shows Western blotting using biotinylated anti-CD9 antibody of samples obtained by immunoprecipitation of serum specimens diluted with PBS or CMC-PBS with anti-CD9 antibody at temperatures of 35 to 60°C in Example 9. [Figure 9B] FIG. 9B shows Western blotting using anti-CD63 antibody of samples obtained by immunoprecipitation of serum specimens diluted with CMC-PBS with anti-CD63 antibody at each temperature from 35 to 60° C. in Example 9. [Figure 9C] FIG. 9C shows Western blotting with anti-CD81 antibody of samples obtained by immunoprecipitation of serum specimens diluted with CMC-PBS with anti-CD81 antibody at temperatures of 35° C. to 60° C. in Example 9. [Figure 10] FIG. 10 shows the amount of EML4-ALK mRNA detected in samples obtained by immunoprecipitation with anti-CD9 antibody or anti-CD63 antibody from culture supernatants of human lung cancer cells H2228 diluted with PBS, CMC-PBS ("CMC"), EDTA / EGTA-PBS ("ED / EG"), or EDTA / EGTA / CMC-PBS ("ED / EG / C") in Example 11 (expressed as fold change relative to samples diluted with PBS). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

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

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

[0013] The sample containing extracellular vesicles is any sample that contains extracellular vesicles.Preferably, the sample containing extracellular vesicles is a biological liquid sample.The sample containing extracellular vesicles 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 sample containing extracellular vesicles is a culture supernatant. The culture supernatant may be a cell culture supernatant or a tissue culture supernatant. Examples of organisms 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; livestock such as cows, pigs, and goats; and working 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 sample containing extracellular vesicles is a body fluid. The body fluid is derived from an organism as described above. Examples of body fluids include blood samples (e.g., whole blood, serum, and plasma), urine, saliva, lymphatic 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 heparin plasma, citrate plasma, sodium fluoride plasma, plasma containing ACD (acid-citrate-dextrose) or CPD (citrate phosphate dextrose). In general, recovery of extracellular vesicles is difficult from body fluids (e.g., blood, urine, saliva) that contain a larger amount of proteins (e.g., albumin, lysozyme, lactoferrin, histatin, peroxidase, agglutinin, defensin, immunoglobulin) than the culture supernatant. On the other hand, according to the method of the present invention, the amount of extracellular vesicles recovered from a sample containing extracellular vesicles is increased, and even from such body fluids, extracellular vesicles can be recovered with high efficiency and high purity.

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

[0017] In order to achieve the object of the present invention, the polymer may have a viscosity of 1.5 mPa·s or more in a 1-20 wt % aqueous solution at 20-30° C., for example. The viscosity of the polymer under the above conditions may be preferably 5 mPa·s or more, more preferably 10 mPa·s or more, even more preferably 20 mPa·s or more, even more preferably 30 mPa·s or more, and particularly preferably 35 mPa·s or more. The viscosity of the polymer under the above conditions may be preferably 30,000 mPa·s or less, more preferably 20,000 mPa·s or less, even more preferably 10,000 mPa·s or less, even more preferably 5,000 mPa·s or less, and particularly preferably 1,000 mPa·s or less. More specifically, the viscosity of the polymer under the above conditions may be preferably 5 to 30,000 mPa·s, more preferably 10 to 20,000 mPa·s, even more preferably 20 to 10,000 mPa·s, still more preferably 30 to 5,000 mPa·s, and particularly preferably 35 to 1,000 mPa·s.

[0018] In order to achieve the object of the present invention, the polymer may have a viscosity of 1.5 mPa·s or more at 30°C in a phosphate buffered saline (PBS) solution in which the polymer is dissolved in PBS to a concentration of 2% by weight. The viscosity of the polymer under the above conditions may be preferably 5 mPa·s or more, more preferably 10 mPa·s or more, even more preferably 20 mPa·s or more, even more preferably 30 mPa·s or more, and particularly preferably 35 mPa·s or more. The viscosity of the polymer under the above conditions may be preferably 30,000 mPa·s or less, more preferably 20,000 mPa·s or less, even more preferably 10,000 mPa·s or less, even more preferably 5,000 mPa·s or less, and particularly preferably 1,000 mPa·s or less. More specifically, the viscosity of the polymer under the above conditions may be preferably 5 to 30,000 mPa·s, more preferably 10 to 20,000 mPa·s, even more preferably 20 to 10,000 mPa·s, still more preferably 30 to 5,000 mPa·s, and particularly preferably 35 to 1,000 mPa·s.

[0019] The viscosity of a polymer can be measured, for example, by detecting the viscosity friction torque of the liquid generated on the outer periphery of a rotor when a liquid sample is rotated by the rotor (a method using a rotational viscometer), or by allowing a falling weight to fall freely in a measurement tube filled with the sample and measuring the fall time (a method using a falling ball viscometer). In addition, the viscosity of a polymer can be measured, for example, by placing a vibrating body (viscosity sensor) in a liquid sample and vibrating it, and the vibration amplitude of the sensor is suppressed and reduced by the viscous resistance as the liquid viscosity increases, but the vibrating current is increased to overcome this suppressing force and maintain a constant amplitude, and the amount of input current at this time is measured (a method using a vibration viscometer). The viscosity of the polymer can be measured, for example, using a viscosity analyzer (eg, 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] A cellulose derivative is a cellulose derivative in which the hydrogen atom of at least one hydroxyl group of cellulose is replaced with a hydrophilic group. Examples of the hydrophilic group in a cellulose derivative include carboxyalkyl (e.g., carboxy C 1~6 alkyl), hydroxyalkyl (e.g., hydroxy C 1~6 The hydrophilic group in the cellulose derivative is preferably a carboxyalkyl or hydroxyalkyl group. Examples of 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), carboxy t-butyl, carboxypentyl (1-carboxypentyl, 2-carboxypentyl, 3-carboxypentyl, 4-carboxypentyl, 5-carboxypentyl), and carboxyhexyl (1-carboxyhexyl, 2-carboxyhexyl, 3-carboxyhexyl, 4-carboxyhexyl, 5-carboxyhexyl, 6-carboxyhexyl). Examples of 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), and hydroxyhexyl (1-hydroxyhexyl, 2-hydroxyhexyl, 3-hydroxyhexyl, 4-hydroxyhexyl, 5-hydroxyhexyl, 6-hydroxyhexyl). Specific examples of cellulose derivatives include carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), and hydroxypropyl methyl cellulose (HPMC). The cellulose derivatives also include nanocellulose derivatives, which are derivatives of nanocellulose described below.

[0022] The polyvinyl derivative having a hydrophilic group is a polyvinyl derivative in which at least one hydrogen atom is replaced with a hydrophilic group, and a polyvinyl derivative in which one hydrogen atom in a methylene unit is replaced with a hydrophilic group is preferred. 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 (carbon ring or heterocyclic ring)-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 ring-containing hydrophilic group, and more preferably a lactam (e.g., α-lactam, β-lactam, γ-lactam, δ-lactam, ε-lactam). A specific example of the polyvinyl derivative having a hydrophilic group is polyvinylpyrrolidone.

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

[0024] Nanocellulose is a fibrous cellulose having a fiber width on the order of nanometers. The fiber width of 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, even more preferably 10 nm or less, and particularly preferably 5 nm or less.

[0025] The cellulose derivatives, polyvinyl derivatives having a hydrophilic group, and polyether compounds also include their salts, such as 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] In order to achieve the object of the present invention, the polymer may have a weight average molecular weight of, for example, 10 kDa or more. The weight average molecular weight of the polymer may be preferably 12 kDa or more, more preferably 14 kDa or more, even more preferably 16 kDa or more, even more preferably 18 kDa or more, and particularly preferably 20 kDa or more. The weight average molecular weight of the polymer may be preferably 5000 kDa or less, more preferably 3000 kDa or less, even more preferably 2000 kDa or less, even more preferably 1000 kDa or less, and particularly preferably 500 kDa or less. More specifically, the weight average molecular weight of the polymer may be preferably 12 to 5000 kDa, more preferably 14 to 3000 kDa, even more preferably 16 to 2000 kDa, even more preferably 18 to 1000 kDa, and particularly preferably 20 to 500 kDa.

[0027] The concentration of the polymer in the separation step of extracellular vesicles is not particularly limited as long as it is a concentration that allows the extracellular vesicles to be recovered more efficiently than when the polymer is not included and allows the polymer to dissolve in the solution used in the separation step. Such a concentration varies depending on the type of 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 step of separating extracellular vesicles, the extracellular vesicles are separated from the sample containing extracellular vesicles in a liquid phase. That is, the extracellular vesicles are separated from the sample containing extracellular vesicles by a separation method described below without being coprecipitated with a polymer. Therefore, the separation step in the present invention is different from separation by a coprecipitation method using a coprecipitating polymer (e.g., 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, the polymer may be present in the separation step, so the recovery method of the present invention may include combining the sample containing extracellular vesicles with the polymer. For example, the sample containing extracellular vesicles may be combined with the polymer, and then the extracellular vesicles may be separated from the sample containing extracellular vesicles. In addition, for example, when using a separation method using an extracellular vesicle membrane-binding substance described later, the polymer may be added in advance to a solution containing the extracellular vesicle membrane-binding substance, and the sample containing extracellular vesicles may be added to the solution.

[0030] The recovery method of the present invention may further include combining the extracellular vesicle-containing sample with a chelating agent.In this case, in the recovery method of the present invention, it is sufficient to separate the extracellular vesicles from the extracellular vesicle-containing sample in the presence of the polymer and the chelating agent, so for example, the polymer and the chelating agent may be added simultaneously to the extracellular vesicle-containing sample, the chelating agent may be added after the polymer is added to the extracellular vesicle-containing sample, or the polymer may be added after the chelating agent is added to the extracellular vesicle-containing sample.In addition, for example, when using a separation method using an extracellular vesicle membrane-binding substance described later, the polymer and the chelating agent may be added in advance to a solution containing the extracellular vesicle membrane-binding substance, and the extracellular vesicle-containing sample may be added to this.

[0031] The chelating agent is a compound or a salt thereof having a coordination moiety capable of coordinate bonding 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 a group having the above-mentioned coordination atom. The coordination group is preferably a carboxylic acid group or a phosphoric acid 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), glycoletherdiaminetetraacetic acid (EGTA), and salts thereof. Examples of the salt include metal salts (e.g., monovalent metal salts such as sodium salts and potassium salts, and divalent metal salts such as calcium salts and magnesium salts), inorganic salts (e.g., halide salts such as fluorides, chlorides, bromides, and iodides, 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 (eg, 2, 3, 4, 5) chelating agents may be used to separate a sample containing extracellular vesicles.

[0033] Chelating agents are effective in recovering extracellular vesicles at high purity, possibly by suppressing the adsorption of contaminants to the extracellular vesicles (WO 2018 / 070479). Therefore, by separating extracellular vesicles from a sample containing extracellular vesicles in the presence of a polymer and a chelating agent, a synergistic effect is achieved in improving the recovery efficiency of extracellular vesicles. Since all of the above-mentioned chelating agents are effective in recovering extracellular vesicles at high purity, their use in combination with a polymer provides a synergistic effect of the same quality.

[0034] The concentration of the chelating agent in the separation step of extracellular vesicles is not particularly limited as long as it is a concentration that can suppress the adsorption of impurities to extracellular vesicles and can dissolve the chelating agent in the solution used in the separation step. Such a concentration varies depending on the type of chelating agent, but is, for example, 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 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 the sample containing extracellular vesicles in the presence of a polymer (step (1)) can be carried out, for example, by a separation method using an extracellular vesicle membrane-binding substance or ultracentrifugation. Separation using an extracellular vesicle membrane-binding substance may be carried out during an analytical method (e.g., immunoassay described later). When separating extracellular vesicles using an extracellular vesicle membrane-binding substance, the extracellular vesicle membrane-binding substance is mixed with the sample containing extracellular vesicles to bind the extracellular vesicle membrane-binding substance to the extracellular vesicles, and then the extracellular vesicles to which the extracellular vesicle membrane-binding substance is bound are separated from the sample, whereby the extracellular vesicles can be collected. When separating extracellular vesicles by ultracentrifugation, the extracellular vesicles in the sample containing extracellular vesicles are precipitated by ultracentrifugation, and then the supernatant is discarded, whereby the extracellular vesicles can be collected. Furthermore, when the separation of extracellular vesicles is performed during the course of an analysis method, the extracellular vesicles can be separated by removing the solution or washing the solid phase in the analysis method (e.g., immunoassay such as ELISA). Specifically, in an immunoassay, the extracellular vesicles can be separated by binding to an antibody of the extracellular vesicles and removing the solution containing the extracellular vesicle-containing sample and / or washing 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] The extracellular vesicle membrane-binding substance used in the recovery method of the present invention is a substance having affinity for extracellular vesicle markers. Examples of extracellular vesicle markers include tetraspanin membrane proteins (extracellular vesicle membrane-specific four-spanning membrane proteins, e.g., CD9, CD63, CD81), extracellular matrix metalloprotease inducers (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, integrins, ceramide, cholesterol, phosphatidylserine, ALIX, Annexins, Caveolin-I, Flotillin-I, Rab proteins, and EpCAM. The extracellular vesicle marker is preferably a tetraspanin membrane protein or an extracellular matrix metalloprotease inducer. The extracellular vesicle marker is preferably CD9, CD63, CD81, or CD147. Examples of the extracellular vesicle membrane-binding substance include antibodies (e.g., monoclonal antibodies, polyclonal antibodies) and antigen-binding fragments thereof, aptamers, phosphatidylserine-binding proteins, and ceramide-binding proteins. The antigen-binding fragment may be an antibody fragment that maintains binding to the target extracellular vesicle marker, and may include Fab, Fab', F(ab')2, scFv, and the like. The extracellular vesicle membrane-binding substance is preferably an antibody or an antigen-binding fragment thereof, and more preferably a monoclonal antibody or an antigen-binding fragment thereof. The extracellular vesicle membrane-binding substance used in recovering extracellular vesicles may be one or a combination of multiple substances.

[0037] The extracellular vesicle membrane-binding substance may be bound to a solid phase to facilitate the separation of extracellular vesicles. For example, sepharose beads, agarose beads, magnetic beads, or plastic plates can be used as the solid phase. The extracellular vesicle membrane-binding substance can be immobilized on the solid phase 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 may be present at least when the extracellular vesicle membrane-binding substance and the sample containing extracellular vesicles are mixed.For example, when magnetic beads are used, the extracellular vesicle membrane-binding substance bound to the magnetic beads is mixed with the sample containing extracellular vesicles in the presence of a polymer, so that the extracellular vesicle membrane-binding substance is bound to the extracellular vesicles in the sample containing extracellular vesicles, and the magnetic beads are magnetically collected using a permanent magnet or an electromagnet, and then the supernatant containing the components that are not bound to the magnetic beads is discarded, so that the extracellular vesicles bound to the magnetic beads can be separated from the sample containing extracellular vesicles.The method of magnetically collecting magnetic beads can be a method well known to those skilled in the art. In addition, for example, when using sepharose beads or agarose beads, the extracellular vesicle membrane-binding substance bound to these beads is mixed with the extracellular vesicle-containing sample in the presence of a polymer to bind the extracellular vesicle membrane-binding substance to the extracellular vesicles in the extracellular vesicle-containing sample, and the beads are precipitated by centrifugation, and the supernatant containing components that are not bound to the beads is discarded, thereby separating the extracellular vesicles bound to the beads from the extracellular vesicle-containing sample. The method of precipitating the beads by centrifugation can be a method well known to those skilled in the art.

[0039] When separating extracellular vesicles from an extracellular vesicle-containing sample by a separation method using an extracellular vesicle membrane-binding substance, the temperature at which the extracellular vesicle membrane-binding substance and the extracellular vesicle-containing sample are mixed may be any temperature at which the mixture of the extracellular vesicle membrane-binding substance and the extracellular vesicle-containing sample is in liquid form, 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 extracellular vesicle membrane-binding substance and extracellular vesicle is not particularly limited, as long as it is sufficient time for extracellular vesicle membrane-binding substance to bind to extracellular vesicle, and may be, for example, 1 minute or more, 5 minutes or more, 10 minutes or more, or 20 minutes or more.Such time may also be 24 hours or less, 18 hours or less, 8 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less.

[0040] In the recovery method of the present invention, when extracellular vesicles are recovered by ultracentrifugation, the polymer may be present when the extracellular vesicles-containing sample 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 may be, for example, 10,000×g to 200,000×g, and preferably 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., twice or three times).

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

[0042] The analytical method of the present invention comprises: (1) isolating extracellular vesicles from an extracellular vesicle-containing sample in the presence of a polymer; and (2) To analyze isolated extracellular vesicles.

[0043] Step (1) in the analytical 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. Examples of subjects to be analyzed include components contained in the extracellular vesicles (e.g., components contained inside the extracellular vesicles, membrane components of the extracellular vesicles, components present on the membrane surface of the extracellular vesicles), and the extracellular vesicles themselves (particles).

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

[0046] Analysis of the components can be performed by any method known in the art. When the component to be analyzed is a protein, the analysis method may include, for example, immunoassay and mass spectrometry. For example, immunoassay may include direct competitive method, indirect competitive method, and sandwich method. For example, such immunoassay may include chemiluminescence immunoassay (CLIA) (e.g., chemiluminescence enzyme immunoassay (CLEIA)), immunoturbidimetric method (TIA), enzyme immunoassay (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination reaction method, fluorescent immunoassay (FIA), and immunochromatography method, Western blotting, immunostaining, and 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, analytical methods include, for example, hybridization methods using a probe, reverse transcription (RT) reactions using reverse transcriptase, gene amplification methods using primers (e.g., 2, 3, or 4 primers) (e.g., PCR methods such as quantitative PCR and RT-PCR), sequencing, and mass spectrometry. When the components to be analyzed are components other than proteins and nucleic acids, the analytical method may include, for example, immunoassays and mass spectrometry. When multiple components are analyzed, metabolomic analysis may be performed.

[0047] The analysis method of the present invention can be used to detect markers contained in extracellular vesicles. Examples of markers contained in extracellular vesicles include markers that indicate the presence of extracellular vesicles and markers that indicate diseases such as cancer (e.g., diagnostic markers and risk assessment markers for diseases). Markers that indicate the presence of extracellular vesicles include, for example, tetraspanin membrane proteins (extracellular vesicle membrane-specific four-spanning membrane proteins, e.g., CD9, CD63, CD81), extracellular matrix metalloproteinase 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, integrins, ceramide, cholesterol, phosphatidylserine, ALIX, Annexins, Caveolin-I, Flotillin-I, Rab proteins, EpCAM, and nucleic acids (e.g., DNA, RNA) encoding these proteins. Examples of markers that are indicative of disease include proteins (hereinafter referred to as "abnormal proteins"; e.g., mutant proteins, foreign proteins) that are specifically present in extracellular vesicles derived from organisms suffering from diseases such as cancer or extracellular vesicles secreted from abnormal cells such as cancer cells. Examples of mutant proteins include fusion proteins such as EML4-ALK. EML4-ALK has variants such as variants 1, 2, 3a, 3b, 4, 5a, 5b, and 6. Examples of foreign proteins include proteins derived from viruses. Examples of markers that are indicative of disease include the presence or absence of expression of nucleic acids encoding these proteins or changes in the amount thereof, as well as mutations such as SNPs, haplotypes, translocations, the presence or absence of methylation of nucleic acids, and types of variants.

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

[0049] It has been reported that extracellular vesicles may be involved in various diseases such as cancer (WO 2014 / 003053; WO 2014 / 152622; Taylor et al., Gynecologic Oncol, 100 (2008) pp13-21). Therefore, the collection method and analysis method of the present invention are useful, for example, for diagnosis and drug discovery based on extracellular vesicles. For example, detection of EML4-ALK, a cancer marker for lung cancer, etc., by the analysis method of the present invention can be useful as an indicator for determining the administration of ALK tyrosine kinase inhibitors such as crizotinib and alectinib to cancer patients such as lung cancer.

[0050] In another embodiment, the present invention provides a method for analyzing extracellular vesicles, comprising: (1) isolating extracellular vesicles from an extracellular vesicle-containing sample using an extracellular vesicle membrane-binding substance; and (2) Analyzing markers contained in the isolated extracellular vesicles that are indicative of diseases such as cancer.

[0051] In the present embodiment, the extracellular vesicle membrane-binding substance may be a substance having affinity for the above-mentioned extracellular vesicle marker. The extracellular vesicle marker is preferably a tetraspanin membrane protein or an extracellular matrix metalloprotease-inducing substance. The extracellular vesicle marker is preferably CD9, CD63, CD81, or CD147, more preferably CD9 or CD63. The extracellular vesicle membrane-binding substance may be, for example, the above-mentioned antibody (e.g., monoclonal antibody, polyclonal antibody) and its antigen-binding fragment, aptamer, phosphatidylserine-binding protein, or ceramide-binding protein.

[0052] Markers that are indicative of diseases such as cancer include the presence or absence of expression of the above-mentioned abnormal proteins or changes in their amount. Examples of abnormal proteins include fusion proteins such as EML4-ALK. Markers that are indicative of diseases also include the presence or absence of expression of nucleic acids that code for these proteins or changes in their amount, as well as mutations such as SNPs, haplotypes, translocations, the presence or absence of methylation of nucleic acids, and types of variants.

[0053] The present invention also provides a kit comprising the above-mentioned polymer and extracellular vesicle membrane-binding substance.The kit of the present invention may further comprise a chelating agent.The kit of the present invention is useful, for example, for easily carrying out the recovery method and analysis method of the present invention. EXAMPLES

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

[0055] Example 1 We investigated the effect of three commercially available CMC sodium 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. EV recovery was performed using an anti-CD9 antibody. Serum from healthy subjects was centrifuged at 20,000×g for 15 minutes at 4°C, and 200μL of serum was diluted with 200μL of PBS (2.9mM NaH2PO4, 9.0mM Na2HPO4, 137mM NaCl), EDTA / EGTA-PBS (PBS containing EDTA and EGTA so that the final concentration after diluting the serum is 50mM) (ED / EG), or CMC-PBS (PBS in which CMC is dissolved so that the final concentration after diluting the serum is 0.2-2.5% by weight) at a final concentration of 0.2-2.5% by weight, and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) with immobilized anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26mg / mL. After rotating overnight at 4°C, the magnetic beads were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. In the Western blotting samples, samples containing exosomes were treated with SDS, which destroyed the exosomes and released exosome marker proteins (e.g., CD9) into the sample solution. The immunoprecipitation efficiency was analyzed by Western blotting using a biotinylated anti-CD9 antibody (manufactured in-house) (Figure 1). All four types of CMCs showed improved EV recovery efficiency compared to PBS-diluted samples. The physical properties of the CMC used in the examples are summarized in Table 1 below.

[0056] [Table 1]

[0057] Example 2 The effects of CMC (Sigma-Aldrich #C4888) concentration (final concentration of 0.06 wt% to 1.0 wt%) and reaction temperature (4 °C, 37 °C) on EV recovery were examined. Serum from healthy volunteers was centrifuged at 20,000×g for 15 minutes at 4°C, and 200 μL of 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% by weight), and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) with immobilized anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After rotating overnight at 4°C or for 1 hour at 37°C, the magnetic beads were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. The EV recovery efficiency was analyzed by Western blotting using biotinylated anti-CD9 antibody (Figure 2). At reaction temperatures of 4°C and 37°C, the effect of improving EV recovery efficiency was observed compared to the PBS-diluted sample at final CMC concentrations of 0.25% to 1% by weight.

[0058] Example 3 The effect of CMC (Sigma-Aldrich #C4888) on EV recovery was examined by nanoparticle tracking analysis (NanoSight LM10, Quantum Design). 200 μL of healthy human serum was centrifuged at 20,000×g at 4°C for 15 minutes, and the supernatant was diluted with 200 μL of PBS, EDTA / EGTA-PBS (PBS containing EDTA and EGTA so that the final concentration after serum dilution was 50 mM) (ED / EG), or CMC-PBS (PBS in which CMC was dissolved so that the final concentration after serum dilution was 0.5% by weight) (CMC), and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) with immobilized anti-CD9 antibody (produced in-house) were added to a concentration of 0.26 mg / mL. After rotating at 37°C for 30 minutes, the magnetic beads were washed three times with PBS-T, reacted with 40μL BRUB (Britton & Robinson Universal Buffer) (pH 2.6) for 5 minutes, and then neutralized with 20μL of 1M Tris-HCl (pH 8.0) to release the extracellular vesicles from the antibody magnetic bead particles. After quantifying the total protein concentration using Qubit protein assay (Life Technologies), 450μL of PBS was added and the number of particles was analyzed using NanoSight (Figure 3). An increase in the total number of EV recovered 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 serum and plasma containing five types of anticoagulants (heparin, EDTA, citrate, ACD (acid-citrate-dextrose), and CPD (citrate phosphate dextrose)). 200 μL of healthy donor serum and healthy donor plasma containing an anticoagulant were centrifuged at 20,000×g and 4°C for 15 minutes, and the supernatant was diluted with 200 μL of PBS, EDTA / EGTA-PBS (PBS containing EDTA and EGTA so that the final concentration after dilution of serum or plasma is 50 mM) (ED / EG), CMC-PBS (PBS in which CMC is dissolved so that the final concentration after dilution of serum or plasma is 0.5% by weight) (CMC), or EDTA / EGTA / CMC-PBS (PBS containing EDTA, EGTA, and CMC so that the final concentrations after dilution of serum or plasma are 37.5 mM / 37.5 mM / 0.5% by weight) (ED / EG / C), and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) with immobilized anti-CD9 antibody (manufactured in-house) were added to the supernatant to a concentration of 0.26 mg / mL. After rotating 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 using a biotinylated anti-CD9 antibody (Figure 4). Regardless of the type of anticoagulant, the effect of CMC on improving EV recovery efficiency was observed even in plasma samples. In addition, the EV recovery efficiency was further improved by combining CMC with a chelating agent.

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

[0061] Example 6 The effect of CMC (Sigma-Aldrich #C4888) on EV recovery was examined in two types of body fluids (urine and saliva). 200 μL of healthy human urine and saliva (2 samples each, designated as “#1” and “#2”) were centrifuged at 15,000×g at 4°C for 15 minutes and filtered through a 0.22 μm filter. The samples were then diluted with 200 μL of PBS, EDTA / EGTA-PBS (final concentration of urine or saliva after dilution: 50 mM) (ED / EG), CMC-PBS (final concentration of urine or saliva after dilution: 0.5% by weight) (CMC), or EDTA / EGTA / CMC-PBS (final concentration of urine or saliva after dilution: 37.5 mM / 37.5 mM / 0.5% by weight) (ED / EG / C), and 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 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 using a biotinylated anti-CD9 antibody (Figure 6). The effect of CMC in improving EV recovery efficiency 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 (final concentrations of 0.13% by weight to 4.0% by weight) on EV recovery were examined. Serum from healthy volunteers was centrifuged at 20,000 × g for 15 min at 4 ° C., and 200 μL of serum was diluted with 200 μL of PBS, CMC-PBS (final concentration after serum dilution: 0.5 wt%) (CMC), or cellulose derivative dissolved in PBS (final concentration after serum dilution: 0.13 to 4.0 wt%), and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) with immobilized anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After rotating at 37 ° C. for 1 h, the magnetic beads were washed three times with PBS-T and diluted with sample buffer (BIO-RAD) to prepare samples for Western blotting. The EV recovery efficiency was analyzed by Western blotting using biotinylated anti-CD9 antibody (Figure 7A-C). The three cellulose derivatives other than CMC also showed improved EV recovery efficiency compared to the PBS diluted sample (HEC in the range of 0.13 wt% to 2.0 wt%, HPC in the range of 0.25 wt% to 4.0 wt%, and HPMC in the range of 0.25 wt% to 2.0 wt%).

[0063] [Table 2]

[0064] Example 8 The effect of polyvinylpyrrolidone (final concentrations of 1 wt%, 2 wt%, and 4 wt%) shown in Table 3 below on EV recovery was examined. Serum from healthy subjects was centrifuged at 20,000 × g for 15 minutes at 4 ° C., and 200 μL of serum was diluted with 200 μL of PBS or polyvinylpyrrolidone dissolved in PBS (final concentration after serum dilution: 1.0 to 4.0% by weight), and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) with immobilized anti-CD9 antibody (manufactured in-house) were added to a concentration of 0.26 mg / mL. After rotating 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 EV recovery efficiency was analyzed by Western blotting using biotinylated anti-CD9 antibody (Figure 8). The effect of improving EV recovery efficiency was observed when polyvinylpyrrolidone was used in the range of 1.0 to 4.0% by weight, compared to the PBS-diluted sample.

[0065] [Table 3]

[0066] Example 9 The effect of CMC (Sigma-Aldrich #C4888) on EV recovery at each reaction temperature from 35°C to 60°C was investigated. Serum from healthy volunteers was centrifuged at 20,000 × g and 4 ° C for 15 minutes, after which 100 μL of serum was diluted with 100 μL of PBS or CMC-PBS (final concentration after serum dilution: 0.5 wt%), and magnetic beads (Dynabeads M-280 tosylactivated (Life Technologies)) immobilized with anti-CD9 antibody (manufactured in-house), anti-CD63 antibody (8A12: Cosmo Bio Co., Ltd.) or anti-CD81 antibody (12C4: Cosmo Bio Co., Ltd.) were added to a concentration of 0.26 mg / mL. After reacting for 5 minutes at each reaction temperature, the magnetic beads were washed three times with PBS-T and diluted with sample buffer (BIO-RAD Co., Ltd.) to prepare samples for Western blotting. The EV recovery efficiency 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 Co., Ltd.) (Figures 9A to 9C). The addition of CMC was found to improve EV collection efficiency at each reaction temperature from 35°C to 60°C. Furthermore, the addition of CMC was found to be even more effective at improving EV collection efficiency at high temperatures above 40°C. Furthermore, the effect of CMC in improving EV collection efficiency was also observed in short-term reactions.

[0067] Example 10 The viscosity of the cellulose derivative used in Example 7 and the polyvinylpyrrolidone used in Example 8 in a PBS solution was measured. Specifically, each PBS solution was prepared by dissolving each cellulose derivative or polyvinylpyrrolidone in PBS to a concentration of 2% by weight, and the viscosity was calculated as the average of the results measured at 30° C., 60 seconds for measurement, and rotation speeds of 200 rpm, 400 rpm, 600 rpm, and 800 rpm (Table 4).

[0068] [Table 4]

[0069] Example 11 We investigated the effect of immunoprecipitation using CMC (Sigma-Aldrich #C4888) and antibodies against tetraspanin membrane proteins (CD9 and CD63) on EV recovery and detection of marker (EML4-ALK fusion gene) RNA from EVs. 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 at 4 ° C for 5 minutes, filtered through a 0.22 μm filter (Millipore), and then concentrated 100-fold using an Amicon Ultra-15 (Millipore). The concentrate was diluted with an equal amount of PBS, EDTA / EGTA-PBS (final concentration of 50 mM after dilution of the concentrate) (ED / EG), CMC-PBS (final concentration of 0.5% by weight after dilution of the concentrate) (CMC), or EDTA / EGTA / CMC-PBS (final concentration of 37.5 mM / 37.5 mM / 0.5% by weight after dilution of the concentrate) (ED / EG / C), and Dynabeads M-280 tosylactivated (Life Technologies) immobilized with anti-CD9 antibody (manufactured in-house) and anti-CD63 antibody (manufactured in-house) was added to a concentration of 0.26 mg / mL. After rotating overnight at 4°C, the mixture was washed three times with PBS-T and total RNA was purified using the miRNeasy micro kit (manufactured by 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 a fluorescent probe with the sequences shown in Table 5 were used to detect EML4-ALK mRNA. The fluorescent probe used was a double quencher probe having a fluorescent substance HEX at the 5' end, a ZEN quencher inside the probe, and an Iowa Black (registered trademark) quencher (IABkFQ) at the 3' end. By using the primers and fluorescent probe 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 was detected in EVs collected by immunoprecipitation. The use of CMC increased the amount of EML4-ALK mRNA detected, demonstrating an improved EV collection efficiency. Furthermore, the addition of a chelating agent further increased the amount of EML4-ALK mRNA detected, demonstrating an improved EV collection efficiency.

Claims

1. separating extracellular vesicles from an extracellular vesicle-containing sample in a single-phase aqueous solution containing a polymer; the polymer comprises a cellulose derivative, polyvinylpyrrolidone, or a salt thereof; The separation is carried out by a separation method using an extracellular vesicle membrane-bound substance, The extracellular vesicle membrane-binding substance comprises an antibody or a fragment thereof having affinity for an extracellular vesicle marker. Methods for recovering extracellular vesicles.

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

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

4. A method according to any one of claims 1 to 3, wherein the cellulose derivative is carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, or hydroxypropyl methyl cellulose.

5. The method of 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 an extracellular vesicle-containing sample is 0.01 to 10.00% by weight.

7. The method of 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 membrane-binding substance is an antibody against a tetraspanin membrane protein or an antibody against an extracellular matrix metalloproteinase inducer.

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

11. below: (1) separating extracellular vesicles from an extracellular vesicle-containing sample in a single-phase aqueous solution containing a polymer; and (2) analyzing the isolated extracellular vesicles; the polymer comprises a cellulose derivative, polyvinylpyrrolidone, or a salt thereof; The separation is carried out by a separation method using an extracellular vesicle membrane-bound substance, The extracellular vesicle membrane-binding substance comprises an antibody or a fragment thereof having affinity for an extracellular vesicle marker. Methods for analyzing extracellular vesicles.

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

13. The method of claim 11 or 12, wherein proteins or nucleic acids in the separated extracellular vesicles are analyzed.

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