Method for collecting extracellular vesicles and blood collection tube

Abstract

The present invention provides a technique useful in the recovery of extracellular vesicles. More specifically, the present invention provides items (I) and (II) below. (I) An extracellular vesicle recovery method that includes the following: (1) mixing whole blood with a nonionic surfactant and a chelating agent and generating a mixture containing extracellular vesicles, the nonionic surfactant, and the chelating agent; and (2) separating the extracellular vesicles from the mixture. (II) A blood collection tube containing a nonionic surfactant and a chelating agent.

Description

Technical Field

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

Background Art

[0002] Extracellular vesicles (EVs) are minute vesicles with a membrane structure that are secreted from various types of cells and are present in body fluids such as blood. As extracellular vesicles secreted outside the cell, for example, exosomes, ectosomes, and apoptotic blebs are known. Since extracellular vesicles contain various substances that function in cell-to-cell communication and the like, they are being analyzed for purposes such as diagnosis and drug discovery. Therefore, there is a demand for the development of a method useful for recovering extracellular vesicles that can be applied to such analyses.

[0003] For example, Patent Document 1 describes that non-specific adsorption in an immune reaction between extracellular vesicles and anti-CD antibodies in a buffer (more specifically, an immune reaction between an extracellular vesicle-containing buffer in which the liquid medium has been exchanged from serum to a buffer by using magnetic particles and a washing buffer, and an anti-CD antibody) can be suppressed by a nonionic surfactant (Test Example 4, Figure 2).

[0004] Patent Document 2 describes that although the recovery amount of extracellular vesicles can be improved in the presence of EDTA (a chelating agent) at a low concentration of less than about 3.0 mg / mL (about 8 mM), not only can the recovery amount of extracellular vesicles not be improved in the presence of a higher concentration of EDTA, but rather, the recovery amount is significantly decreased (Example 1, Figures 1A to 1C).

[0005] By the way, in the clinical blood test field, plasma and serum that can be prepared from whole blood are widely used as blood samples. Also, since it is widely known that hemolysis caused by the destruction of red blood cells affects the results of blood tests, hemolysis is recognized as something to be avoided. Furthermore, in the treatment of whole blood for preparing blood samples, blood collection tubes are widely used in the clinical field from the viewpoints of simplicity and speed of treatment, etc.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a method useful for the recovery of extracellular vesicles.

Means for Solving the Problems

[0008] As a result of intensive studies, the present inventors have found that by mixing a nonionic surfactant and a chelating agent with whole blood, the recovery amount of extracellular vesicles from whole blood can be significantly improved.

[0009] Generally, surfactants are recognized in the clinical field as something to be avoided in the treatment of whole blood for blood tests because they cause hemolysis due to the destruction of red blood cell membranes. However, among surfactants, by selecting and using nonionic surfactants (especially specific nonionic surfactants), it becomes easier to avoid hemolysis. Therefore, by using such nonionic surfactants in combination with chelating agents, not only can the recovery amount of extracellular vesicles from whole blood be significantly improved, but also reduction or avoidance of hemolysis can be expected. Therefore, by applying nonionic surfactants and chelating agents to whole blood, it is possible to prepare a highly versatile blood sample that can be used not only for the analysis of extracellular vesicles but also for normal blood tests.

[0010] As described above, Patent Document 1 describes that nonspecific adsorption in the immune reaction between extracellular vesicles and anti-CD antibodies in a buffer can be suppressed by nonionic surfactants. However, Patent Document 1 does not describe mixing whole blood with nonionic surfactants (acting nonionic surfactants on whole blood) and that the recovery amount of extracellular vesicles can be improved by such mixing. Also, in light of the fact that the improvement in the recovery amount of extracellular vesicles from whole blood by mixing nonionic surfactants and chelating agents with whole blood and the suppression of nonspecific adsorption in the immune reaction are considered to be independent and separate effects (Example 5), Patent Document 1 does not teach or suggest the present invention.

[0011] The inventors of the present invention have also found that by using both nonionic surfactants and chelating agents, it is possible to produce a blood collection tube that can significantly improve the recovery amount of extracellular vesicles from whole blood, and a blood collection tube having high versatility that can be used not only for the analysis of extracellular vesicles but also for normal blood tests, and thus have completed the present invention.

[0012] That is, the present invention is as follows. 〔1〕A method for recovering extracellular vesicles, comprising the following: (1) Mixing whole blood with a nonionic surfactant and a chelating agent to produce a mixture containing extracellular vesicles, a nonionic surfactant, and a chelating agent; and (2) Separating extracellular vesicles from the mixture. 〔2〕The method according to 〔1〕, wherein the nonionic surfactant is a nonionic surfactant having a polyoxyethylene alcohol structure. 〔3〕The method according to 〔2〕, wherein the nonionic surfactant having a polyoxyethylene alcohol structure is an alcohol ethoxylate. 〔4〕The method according to 〔2〕, wherein the nonionic surfactant having a polyoxyethylene alcohol structure is a polyoxyethylene-polyoxyalkylene block copolymer. 〔5〕The method according to any one of 〔1〕 to 〔4〕, wherein the nonionic surfactant is a nonionic surfactant having an HLB of 18 or more. 〔6〕The method according to any one of 〔1〕 to 〔5〕, wherein the concentration of the nonionic surfactant in the mixture is 0.01 to 10% by weight / volume. 〔7〕The method according to any one of 〔1〕 to 〔6〕, wherein the chelating agent is hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetra(methylenephosphonic acid) (EDTMP), glycol ether diamine tetraacetic acid (EGTA), or a salt thereof. 〔8〕The method according to any one of 〔1〕 to 〔7〕, wherein the concentration of the chelating agent in the mixture is 10 mM to 1000 mM. 〔9〕The method according to any one of 〔1〕 to 〔8〕, wherein the mixing is performed after putting whole blood into a blood collection tube containing a nonionic surfactant and a chelating agent. 〔10〕The method according to any one of 〔1〕 to 〔9〕, wherein the separation is performed by any one of the following (A) to (C): (A) Collecting the supernatant from the mixture; (B) Using a substance that binds to the surface marker of extracellular vesicles for the mixture; or (C) Collecting the supernatant from the mixture and using a substance that binds to the surface marker of extracellular vesicles for the supernatant. The method according to any one of [1] to

[10] , wherein the extracellular vesicle is an exosome.

[12] A method for analyzing extracellular vesicles, comprising: (1) Mixing whole blood with a nonionic surfactant and a chelating agent to produce a mixture containing extracellular vesicles, a nonionic surfactant, and a chelating agent; (2) Separating extracellular vesicles from the mixture; and (3) Analyzing the extracellular vesicles.

[13] A blood collection tube containing a nonionic surfactant and a chelating agent.

[14] The blood collection tube according to

[13] , wherein the amount of the nonionic surfactant in the blood collection tube is 0.1 to 2000 mg.

[15] The blood collection tube according to

[13] or

[14] , wherein the amount of the chelating agent in the blood collection tube is 3.8 to 3800 mg. [Effect of the Invention]

[0013] The method of the present invention can significantly improve the recovery amount of extracellular vesicles from whole blood, and can also be expected to reduce or avoid hemolysis. The blood collection tube of the present invention can significantly improve the recovery amount of extracellular vesicles from whole blood, and has high versatility in that it can be used not only for the analysis of extracellular vesicles but also for ordinary blood tests. [Mode for Carrying Out the Invention]

[0014] The present invention provides a method for recovering extracellular vesicles, comprising: (1) Mixing whole blood with a nonionic surfactant and a chelating agent to produce a mixture containing extracellular vesicles, a nonionic surfactant, and a chelating agent; and (2) Separating extracellular vesicles from the mixture.

[0015] Whole blood can be collected from animals. Examples of animals include mammals (e.g., primates such as humans and monkeys; rodents such as mice, rats, and rabbits; ungulates such as cows, pigs, goats, horses, and sheep; carnivores such as dogs and cats), and birds (e.g., chickens). Preferably, the animal is a mammal such as a human.

[0016] Whole blood contains extracellular vesicles. Therefore, by mixing whole blood with a non-ionic surfactant and a chelating agent, a mixture containing extracellular vesicles, non-ionic surfactant, and chelating agent is produced.

[0017] Extracellular vesicles are tiny vesicles with a membrane structure 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 - 1000 nm, preferably 50 - 300 nm, more preferably 80 - 200 nm. The size of extracellular vesicles can be measured by NanoSight (manufactured by Malvern Instruments).

[0018] Extracellular vesicles can also be defined by extracellular vesicle markers. Such extracellular vesicle markers include, for example, intracellular markers and surface markers of extracellular vesicles. Examples of intracellular markers of extracellular vesicles include carcinoembryonic antigen (CEA), CA125, CA15-3, actin family, TSG101, ALIX, Charged multivesicular body protein (CHMP) family, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), poly(ADP-ribose)polymerase (PARP) family, AFP, CA19-9, Flotillin-I, Flotillin-II, Rab proteins, programmed cell death (PDCD)6, phospholipid scramblase (PLSCR) family, cytochrome C oxidase (COX) family, lamin family, proliferating cell nuclear antigen (PCNA), tubulin family, TATA-binding protein (TBP), voltage-dependent anion channel protein (VDAC), amyloid beta, tau protein. Examples of surface markers of extracellular vesicles include tetraspanin membrane proteins (extracellular vesicle membrane-specific four-pass transmembrane proteins, e.g., CD9, CD63, CD81), extracellular matrix metalloproteinase inducer (e.g., CD147), heat shock protein (HSP)70, HSP90, major histocompatibility complex (MHC)I, lysosome-associated membrane protein (LAMP)1, intercellular adhesion molecule (ICAM)-1, integrin, ceramide, cholesterol, phosphatidylserine, Annexins, Caveolin-I, EpCAM. The extracellular vesicle marker is preferably an extracellular vesicle surface marker, more preferably a tetraspanin membrane protein, and even more preferably CD9.

[0019] In the present invention, by using a nonionic surfactant among surfactants, it becomes possible to reduce or avoid hemolysis. As the nonionic surfactant, any nonionic surfactant can be used, but preferably, a nonionic surfactant having a polyoxyethylene alcohol structure is used.

[0020] The nonionic surfactant having a polyoxyethylene alcohol structure is preferably a linear or branched compound (a compound not containing a cyclic structure) containing a structure represented by -O-(-CH 2 -CH 2 -O-) x -H (polyoxyethylene alcohol structure). x is an integer of 1 or more, preferably an integer of 10 or more, more preferably an integer of 20 or more, even more preferably an integer of 30 or more, and particularly preferably an integer of 40 or more. x is also an integer of 400 or less, preferably an integer of 350 or less, more preferably an integer of 300 or less, even more preferably an integer of 280 or less, and particularly preferably an integer of 270 or less. More specifically, x may be an integer of 1 to 400, preferably an integer of 10 to 350, more preferably an integer of 20 to 300, even more preferably an integer of 30 to 280 or less, and particularly preferably an integer of 40 to 270 or less. In the present specification, the value of x shall represent an average value.

[0021] The nonionic surfactant having a polyoxyethylene alcohol structure may consist only of single bonds or may contain an unsaturated bond (double / triple bond), but a compound consisting only of single bonds is preferred. Such a compound may contain, as a structure other than the polyoxyethylene alcohol structure, for example, an alkyl structure (linear or branched), and / or a polyoxyalkylene structure (linear or branched). The alkyl structure may be, for example, C 6~31 an alkyl structure, and a linear alkyl structure is preferred. The alkylene in the polyoxyalkylene structure may be, for example, C 6~31 an alkylene. C 1~6 an alkylene may also be used. C1~6 Examples of the alkylene include, for example, C 1 alkylene (methylene), C 2 alkylene (ethylene group, ethylidene group), C 3 alkylene (propylidene, propylene, trimethylene, isopropylidene), C 4 alkylene (e.g., tetramethylene), C 5 alkylene (e.g., pentamethylene), C 6 alkylene (e.g., hexamethylene) can be mentioned. Such compounds may consist of carbon atoms, hydrogen atoms, and oxygen atoms.

[0022] More specifically, examples of the nonionic surfactant having a polyoxyethylene alcohol structure include alcohol ethoxylate and polyoxyethylene-polyoxyalkylene block copolymer (e.g., polyoxyethylene-polyoxypropylene block copolymer).

[0023] Alcohol ethoxylate is also called poly(oxyethylene) alkyl ether, and is a compound in which a hydrophilic polyoxyethylene (POE) chain and a hydrophobic alkyl group are bonded by an ether bond. Alcohol ethoxylate can be represented by the following formula (1).

[0024] [Chemical formula] (In the formula, x1 is an integer of 1 or more; y1 is an integer of 0 or more; z1 is an integer of 0 or more; y1 ≥ z1). In this specification, the values of x1, y1, and z1 represent average values.

[0025] x1 is an integer of 1 or more, preferably an integer of 10 or more, more preferably an integer of 20 or more, even more preferably an integer of 30 or more, and particularly preferably an integer of 40 or more. x1 may also be an integer of 300 or less, preferably an integer of 250 or less, more preferably an integer of 200 or less, even more preferably an integer of 150 or less, and particularly preferably an integer of 100 or less, 80 or less, 60 or less, or 50 or less. More specifically, x1 may be an integer from 1 to 300, preferably an integer from 10 to 250, more preferably an integer from 20 to 200, even more preferably an integer from 30 to 150 or less, and particularly preferably an integer from 40 to 100, 40 to 80, 40 to 60, or 40 to 50.

[0026] y1 is an integer of 0 or more, preferably an integer of 1 or more, and more preferably an integer of 5 or more. y1 may also be an integer of 30 or less, preferably an integer of 20 or less, and even more preferably an integer of 13 or less. More specifically, y1 may be an integer from 0 to 30, preferably an integer from 1 to 20, and even more preferably an integer from 5 to 13.

[0027] z1 is an integer of 0 or more, preferably an integer of 1 or more. z1 may also be an integer of 15 or less, preferably an integer of 10 or less, or 7. More specifically, z1 may be an integer from 0 to 15, preferably an integer from 1 to 10, and even more preferably an integer from 1 to 7.

[0028] Preferably, y1 and z1 may satisfy the relationship y1 ≥ z1. y1 and z1 may also satisfy the relationship that y1 + z1 is an integer from 5 to 30. Preferably, y1 + z1 may satisfy the relationship that it is an integer from 10 to 14.

[0029] The alcohol ethoxylate may be either a branched-chain alcohol ethoxylate or a linear alcohol ethoxylate. Preferably, the alcohol ethoxylate may be a branched-chain alcohol ethoxylate.

[0030] The branched-chain alcohol ethoxylate corresponds to the compound represented by the above formula (1) in which y1 is an integer of 1 or more and z1 is an integer of 1 or more. Examples of such a branched-chain alcohol ethoxylate include polyoxyethylene (40) sec-tridecyl ether. Specific examples of such a branched-chain alcohol ethoxylate include compounds of the TERGITOL (registered trademark) series such as Tergitol 15-S-40.

[0031] The straight-chain alcohol ethoxylate corresponds to the compound represented by the above formula (1) in which z1 is 0. Specific examples of the straight-chain alcohol ethoxylate include compounds of the BRIJ (registered trademark) series such as BrijS100. The straight-chain alcohol ethoxylate can also be represented by the following formula (1’).

[0032]

Chemical formula

[0033] Preferred examples of x1 and y1 in formula (1’) are the same as the preferred examples of x1 and y1 in formula (1), respectively.

[0034] Preferably, the compound represented by formula (1) is as follows. x1 is an integer from 40 to 100; y1 is an integer of 0 or more; z1 is an integer of 0 or more; y1 ≥ z1; y1 + z1 is an integer from 5 to 30. (The preferred ranges of x1, y1, and z1 are the same as described above.)

[0035] More specifically, examples of the alcohol ethoxylate corresponding to the compound represented by the formula (1) include Tergitol 15-S-40 and BrijS100. Preferably, the alcohol ethoxylate is Tergitol 15-S-40.

[0036] The "polyoxyethylene-polyoxyalkylene block copolymer" is a block copolymer containing a polyoxyethylene block and a polyoxyalkylene block. The alkylene in the polyoxyalkylene block may be, for example, C 1~6 alkylene, for example, C 1 alkylene (methylene), C 2 alkylene (ethylene, ethylidene), C 3 alkylene (propylidene, propylene, trimethylene, isopropylidene), C 4 alkylene (e.g., tetramethylene), C 5 alkylene (e.g., pentamethylene), C 6 alkylene (e.g., hexamethylene). Examples of the polyoxyethylene-polyoxyalkylene block copolymer include block copolymers having a structure of HO-[polyoxyethylene block]-[polyoxyalkylene block]-[polyoxyethylene block]-OH. The polyoxyethylene-polyoxyalkylene block copolymer may preferably be a polyoxyethylene-polyoxypropylene block copolymer.

[0037] The "polyoxyethylene-polyoxypropylene block copolymer" is a compound represented by the following formula (2).

[0038]

Chemical formula

[0039] x2 and z2 are each an integer of 1 or more. The sum of x2 and z2 is an integer of 2 or more, preferably an integer of 20 or more, more preferably an integer of 80 or more, and particularly preferably an integer of 150 or more. The sum of x2 and z2 may also be an integer of 400 or less, preferably an integer of 350 or less, more preferably an integer of 300 or less, and particularly preferably an integer of 270 or less. More specifically, the sum of x2 and z2 may be an integer from 2 to 400, preferably an integer from 20 to 350, more preferably an integer from 80 to 300, and particularly preferably an integer from 150 to 270.

[0040] y2 is an integer of 1 or more, preferably an integer of 5 or more, more preferably an integer of 10 or more, even more preferably an integer of 15 or more, and particularly preferably an integer of 20 or more. y2 may also be an integer of 200 or less, preferably an integer of 150 or less, more preferably an integer of 100 or less, even more preferably an integer of 80 or less, and particularly preferably an integer of 70 or less. More specifically, x1 may be an integer from 1 to 200, preferably an integer from 5 to 150, more preferably an integer from 10 to 100, even more preferably an integer from 15 to 80 or less, and particularly preferably an integer from 20 to 70.

[0041] Examples of the polyoxyethylene-polyoxypropylene block copolymer include poloxamer 188, poloxamer 388, poloxamer 407, and the like. Specific examples of the polyoxyethylene-polyoxypropylene block copolymer include compounds of the PLURONIC (registered trademark) series such as Pluronic F68, Pluronic F108, and Pluronic F127.

[0042] Preferably, the compound represented by formula (2) is as follows. x2 is an integer of 1 or more; y2 is an integer from 15 to 80; z2 is an integer of 1 or more; x2 + z2 is an integer from 80 to 280. (The preferred ranges of x2, y2, and z2 are the same as those described above.)

[0043] More specifically, examples of the polyoxyethylene-polyoxypropylene block copolymer corresponding to the compound represented by formula (2) include poloxamer 188 (e.g., Pluronic F68), poloxamer 108 (e.g., Pluronic F38), poloxamer 217 (e.g., Pluronic F77), poloxamer 237 (e.g., Pluronic F87), poloxamer 238 (e.g., Pluronic F88), poloxamer 288 (e.g., Pluronic F98), poloxamer 388 (e.g., Pluronic F108), and poloxamer 407 (e.g., Pluronic F127). Preferably, the polyoxyethylene-polyoxypropylene block copolymer includes poloxamer 188 (e.g., Pluronic F68), poloxamer 388 (e.g., Pluronic F108), and poloxamer 407 (e.g., Pluronic F127).

[0044] In a specific embodiment, the nonionic surfactant may be a nonionic surfactant having an HLB of 18 or more. If the HLB value is 18 or more, extracellular vesicles can be recovered while more easily avoiding the lysis of blood cell components. In particular, red blood cells contain EV-related proteins such as tetraspanin proteins including CD9 and other proteins that may become contaminants. Therefore, when recovering extracellular vesicles, it is desirable to avoid the lysis of red blood cells, which are blood cell components. The HLB value can be determined by the Griffin method.

[0045] For reference, the relationship between the nonionic surfactant (alcohol ethoxylate / polyoxyethylene-polyoxypropylene block copolymer) and the compound represented by the above formula (1) or (2) is as follows.

[0046]

Table A

[0047]

Table B

[0048]

Table C

[0049] In the present invention, one type of nonionic surfactant may be used, or a plurality of types (e.g., two types, three types, four types) of nonionic surfactants may be used in combination.

[0050] When the nonionic surfactant acts on whole blood, the concentration of the nonionic surfactant is not particularly limited as long as it is a concentration that can improve the recovery amount of extracellular vesicles from whole blood by using the nonionic surfactant in combination with a chelating agent. In the present invention, in light of the fact that a mixed solution containing extracellular vesicles, a nonionic surfactant, and a chelating agent is produced by mixing whole blood with a nonionic surfactant and a chelating agent, the concentration of the nonionic surfactant when the nonionic surfactant acts on whole blood is the same as the concentration of the nonionic surfactant in such a mixed solution. Therefore, in the present invention, the concentration of the chelating agent when the nonionic surfactant acts on whole blood can also be defined as the concentration of the nonionic surfactant in such a mixed solution. Such a concentration varies depending on factors such as the type and concentration of the chelating agent used in combination with the nonionic surfactant, but is, for example, 0.01 to 10% by weight / volume% (W / V%). Preferably, the concentration of the nonionic surfactant may be 0.02 W / V% or more, 0.04 W / V% or more, 0.06 W / V% or more, 0.08 W / V% or more, 0.1 W / V% or more, 0.2 W / V% or more, or 0.5 W / V% or more. Such a concentration may also be 8 W / V% or less, 6 W / V% or less, 5 W / V% or less, 4 W / V% or less, 3 W / V% or less, 2 W / V% or less, or 1 W / V% or less. More specifically, the concentration of the nonionic surfactant may be 0.02 to 8 W / V%, 0.04 to 6 W / V%, 0.06 to 5 W / V%, 0.08 to 4 W / V%, 0.1 to 3 W / V%, 0.2 to 2 W / V%, or 0.5 to 1 W / V%.

[0051] A 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 coordinating atom as the coordinating moiety include an oxygen atom, a phosphorus atom, a nitrogen atom, a sulfur atom, and a chlorine atom. The coordinating atom is preferably an oxygen atom or a phosphorus atom, and more preferably an oxygen atom. Examples of the coordinating group as the coordinating moiety include a group having the above-mentioned coordinating atom. The coordinating group is preferably a carboxylic acid group or a phosphoric acid group, and more preferably a carboxylic acid group.

[0052] Examples of chelating agents include 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 salts 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 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).

[0053] In certain embodiments, the chelating agent may be a chelating agent commonly used as a component contained in blood collection tubes for clinical tests. Examples of such chelating agents include EDTA, EGTA, NTA, HEDTA, EDTMP, HIDA, citric acid, and salts thereof. In the present invention, the use of such chelating agents is also desirable from the perspective of clinical applications.

[0054] In the present invention, one type of chelating agent may be used alone, or multiple types (e.g., two types, three types, four types) of chelating agents may be used in combination.

[0055] When the chelating agent acts on whole blood, the concentration of the chelating agent is not particularly limited as long as it is a concentration that can improve the recovery amount of extracellular vesicles from whole blood by using the chelating agent in combination with a nonionic surfactant. In the present invention, in view of the fact that a mixed solution containing extracellular vesicles, a nonionic surfactant, and a chelating agent is generated by mixing whole blood with a nonionic surfactant and a chelating agent, the concentration of the chelating agent when the chelating agent acts on whole blood is the same as the concentration of the chelating agent in such a mixed solution. Therefore, in the present invention, the concentration of the chelating agent when the chelating agent acts on whole blood can also be defined as the concentration of the chelating agent in such a mixed solution. Such a concentration varies depending on factors such as the type and concentration of the nonionic surfactant used in combination with the chelating agent, but is, for example, 10 mM to 1000 mM. Preferably, the concentration of the chelating agent may be 20 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 80 mM or more, or 100 mM or more. Such a concentration may also be 800 mM or less, 600 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, or 300 mM or less. More specifically, the concentration of the chelating agent may be 20 to 800 mM, 30 to 600 mM, 40 to 500 mM, 50 to 450 mM, 60 to 400 mM, 80 to 350 mM, or 100 to 300 mM.

[0056] The mixing in step (1) can be carried out in any manner. For example, the mixing can be carried out by tumbling mixing and / or stirring. The order of mixing whole blood, as well as nonionic surfactant and chelating agent, is not particularly limited. For example, the mixing may be carried out by first mixing whole blood with one of the nonionic surfactant or chelating agent and then mixing with the other. The mixing may also be carried out by mixing whole blood with a mixture of nonionic surfactant and chelating agent. The mixing time is not particularly limited as long as it is sufficient for the mixing of whole blood, as well as nonionic surfactant and chelating agent, and may be, for example, 1 second to 30 minutes, preferably 30 seconds to 5 minutes. The temperature during mixing is, for example, 4 to 37°C, and preferably 15 to 30°C. After mixing, the mixed solution may be allowed to stand. The standing time may be, for example, 1 second to 60 minutes, preferably 5 seconds to 20 minutes.

[0057] In a specific embodiment, the mixing can be carried out after putting whole blood into a blood collection tube containing a nonionic surfactant and a chelating agent (preferably a vacuum blood collection tube sealed by a sealing means). In this case, tumbling mixing is preferred as the mixing. Details of the blood collection tube are as described below.

[0058] The separation in step (2) can be carried out in any manner. For example, the separation may be carried out by any of the following (A) to (C): (A) Collection of the supernatant from the mixed solution; (B) Use of a substance that binds to the surface marker of extracellular vesicles for the mixed solution; or (C) Collection of the supernatant from the mixed solution and use of a substance that binds to the surface marker of extracellular vesicles for the supernatant.

[0059] (A) The separation of the supernatant from the mixed solution will be described. In the present invention, it has been demonstrated that the amount of extracellular vesicles present in the supernatant separated from whole blood (mixed solution) to which a chelating agent and a surfactant are added is significantly increased compared to the amount of extracellular vesicles present in the supernatant separated from whole blood to which neither the chelating agent nor the surfactant is added, or only one of them is added (Examples). Therefore, according to this separation method, a high-concentration extracellular vesicle (supernatant rich in extracellular vesicles) from which a large amount of contaminants in whole blood have been excluded can be easily and quickly recovered.

[0060] The separation of the supernatant from the mixed solution can be carried out by any method. Such methods include, for example, centrifugation of the mixed solution and standing of the mixed solution. From the viewpoint of quickly obtaining a clear supernatant, it is preferable to separate the supernatant after centrifuging the mixed solution. Centrifugation can be carried out under normal centrifugation conditions. Such conditions vary depending on factors such as the rotation radius of the rotor of the centrifuge, but a rotation speed of 100 to 100,000 × g (preferably 200 to 5,000 × g) for 10 seconds to 1 hour (preferably 1 to 10 minutes) at 40°C or lower (preferably 30°C or lower) can be adopted. The supernatant thus obtained can contain a chelating agent and a surfactant in addition to extracellular vesicles. The concentrations of the chelating agent and the surfactant in the supernatant thus obtained can be the same as those in the mixed solution.

[0061] (B) The use of a substance that binds to the surface marker of extracellular vesicles for the mixed solution will be described. In the present invention, it has been demonstrated that the amount of extracellular vesicles measured using the surface marker of extracellular vesicles in a sample obtained from whole blood (mixed solution) to which a chelating agent and a surfactant are added is significantly increased compared to the amount of extracellular vesicles measured using the surface marker of extracellular vesicles in a sample obtained from whole blood to which neither the chelating agent nor the surfactant is added, or only one of them is added (Examples). Therefore, according to this separation method, a large amount of high-purity extracellular vesicles can be easily and quickly recovered.

[0062] Examples of substances that bind to the surface markers of extracellular vesicles include, for example, antibodies against the above-described extracellular vesicle surface markers, aptamers, phosphatidylserine-binding proteins, and ceramide-binding proteins. In the present invention, as the substance that binds to the surface marker of the extracellular vesicle, a single substance or a plurality of substances (e.g., 2, 3, or 4 kinds) can be used.

[0063] Preferably, the substance that binds to the surface marker of the extracellular vesicle may be an antibody against the extracellular vesicle surface marker from the viewpoints of ensuring specificity for the extracellular vesicle surface marker and ease of preparation. Examples of the antibody include full-length antibodies (e.g., monoclonal antibodies, polyclonal antibodies) and antigen-binding fragments thereof. The antigen-binding fragment may be any antibody fragment that maintains binding to the target extracellular vesicle surface marker, such as Fab, Fab’, F(ab’) 2 , scFv, etc. In the present invention, a single antibody or a plurality of antibodies (e.g., 2, 3, or 4 kinds) can be used.

[0064] A substance (preferably, an antibody) that binds to a surface marker of extracellular vesicles may be immobilized on a solid phase for facilitating the separation of extracellular vesicles. Examples of the solid phase include beads and particles (e.g., Sepharose beads, agarose beads, magnetic beads (magnetic particles)), and supports (e.g., plates such as plastic plates, membranes). The immobilization of the substance on the solid phase can be performed by any method. By mixing the substance that binds to the extracellular vesicle surface marker immobilized on the solid phase with a mixed solution containing extracellular vesicles to form a complex of the substance that binds to the extracellular vesicle surface marker and the extracellular vesicles, and separating this complex from the mixed solution, the extracellular vesicles can be separated from the mixed solution. For example, when using beads or particles as the solid phase, after the formation of the complex, centrifugation is performed to remove the supernatant, thereby separating the extracellular vesicles from the mixed solution. When using magnetic beads as the solid phase, after the formation of the complex, magnetic collection is performed to remove the supernatant, and the extracellular vesicles may be separated from the mixed solution. When using a support as the solid phase, after the formation of the complex, the mixed solution is removed, thereby separating the extracellular vesicles from the mixed solution.

[0065] (C) The fractionation of the supernatant from the mixed solution and the use of the substance that binds to the surface marker of extracellular vesicles for the supernatant can be performed by combining the above (A) and (B).

[0066] The present invention also provides a method for analyzing extracellular vesicles, comprising: (1) Mixing whole blood with a nonionic surfactant and a chelating agent to produce a mixed solution containing extracellular vesicles, the nonionic surfactant, and the chelating agent; (2) Separating extracellular vesicles from the mixed solution; and (3) Analyzing the extracellular vesicles.

[0067] Steps (1) and (2) in the analysis method of the present invention can be performed in the same manner as steps (1) and (2) in the recovery method of the present invention.

[0068] In step (3), the analysis of extracellular vesicles can be performed after or simultaneously with the isolation of extracellular vesicles. The analysis targets in the analysis of extracellular vesicles 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).

[0069] The analysis of components contained in extracellular vesicles can be performed qualitatively or quantitatively. Such analysis is also an 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. According to the present invention, since the recovery amount of extracellular vesicles increases, the components in extracellular vesicles can be analyzed with high precision.

[0070] The analysis of components can be performed by any method known in the art. When the component to be analyzed is a protein, examples of the analysis method include immunoassay and mass spectrometry. Examples of immunoassay include direct competitive method, indirect competitive 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 competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination reaction method, fluorescence immunoassay (FIA), and 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 the analysis method include hybridization method using a probe, gene amplification method using primers (e.g., 2, 3, or 4 primers), and mass spectrometry. When the component to be analyzed is a component other than protein and nucleic acid, examples of the analysis method include immunoassay and mass spectrometry. When multiple components are analyzed, metabolome analysis may be performed.

[0071] The analysis of extracellular vesicles themselves (particles) can also be performed qualitatively or quantitatively. For example, the analysis of extracellular vesicles can be performed using equipment such as particle analyzers, electron microscopes, and flow cytometers. In this case, the number of particles of extracellular vesicles, the size and shape of the particles, and their distribution can be analyzed.

[0072] It has been reported that extracellular vesicles can be involved in various diseases such as cancer (International Publication No. 2014 / 003053; International Publication No. 2014 / 152622; Taylor et al., Gynecologic Oncol, 100 (2008) pp13-21). Therefore, the present invention is useful, for example, for diagnosis and drug discovery based on extracellular vesicles.

[0073] The present invention also provides a blood collection tube containing a nonionic surfactant and a chelating agent.

[0074] As the blood collection tube, any form can be used, but a tubular form is preferably used. The cross-section of the tubular blood collection tube is preferably annular (e.g., circular, substantially circular). Preferably, the blood collection tube is a blood collection tube for clinical examination.

[0075] In addition, as the blood collection tube, an unsealed blood collection tube and a vacuum blood collection tube sealed with a sealing means such as a rubber stopper or a film sheet can be used. From the viewpoints of easy collection of a certain amount of blood from a syringe and retention of the nonionic surfactant and chelating agent contained in the blood collection tube, a vacuum blood collection tube sealed with the above sealing means is preferable. The degree of decompression in the vacuum blood collection tube can be appropriately set according to factors such as the blood collection volume.

[0076] As the material of the blood collection tube, any material can be used, and a colorless and transparent material can be preferably used. Examples of such materials include glass and plastic (e.g., polyethylene terephthalate).

[0077] The size of the blood collection tube used in the present invention can be defined by the volume of whole blood that it can accommodate. For example, as the blood collection tube, those with a size suitable for accommodating 1 to 10 mL of whole blood are widely used in clinical tests. Therefore, in the present invention, such sized blood collection tubes can be preferably used. Of course, in the present invention, depending on the purpose, blood collection tubes with a size of less than 1 mL (e.g., 0.5 mL or more and less than 1.0 mL), or blood collection tubes with a size exceeding 10 mL (e.g., more than 10 mL and 15 mL or less) can also be appropriately used.

[0078] Details of the nonionic surfactant and chelating agent contained in the blood collection tube are the same as those described above in the method of the present invention. As long as the nonionic surfactant and chelating agent are contained in the blood collection tube, their forms are not particularly limited. For example, the nonionic surfactant and chelating agent may be contained in the blood collection tube in the form of a liquid or a solid (e.g., powder), respectively. The nonionic surfactant and chelating agent may also be provided in a manner of being accumulated as a liquid or a solid at the bottom in the blood collection tube, or may be provided in a manner of being coated or impregnated inside the blood collection tube (and the sealing means). The nonionic surfactant and chelating agent contained in the blood collection tube may be each one kind or a plurality of kinds (e.g., 2 kinds, 3 kinds, 4 kinds).

[0079] When whole blood is added to and mixed with the blood collection tube, the nonionic surfactant and the chelating agent are preferably contained in the blood collection tube in an amount capable of achieving the above concentrations of the nonionic surfactant and the chelating agent in the mixed solution as described above. The amount of such a nonionic surfactant varies depending on factors such as the size of the blood collection tube and the volume of whole blood added. For example, it may be 0.1 to 2000 mg (0.1 mg to 200 mg per 1 mL of whole blood), preferably 0.2 to 1500 mg (0.2 mg to 150 mg per 1 mL of whole blood), more preferably 0.5 to 1000 mg (0.5 mg to 100 mg per 1 mL of whole blood), even more preferably 1 to 700 mg (1 mg to 70 mg per 1 mL of whole blood), and particularly preferably 2 mg to 500 mg (2 mg to 50 mg per 1 mL of whole blood). The amount of such a chelating agent also varies depending on factors such as the size of the blood collection tube and the volume of whole blood added. For example, it may be 3.8 to 3800 mg (3.8 to 380 mg per 1 mL of whole blood), preferably 7.6 to 1900 mg (7.6 to 190 mg per 1 mL of whole blood), more preferably 15.2 to 1500 mg (15.2 to 150 mg per 1 mL of whole blood), even more preferably 30.4 to 1200 mg (30.4 to 120 mg per 1 mL of whole blood), and particularly preferably 60.8 to 1000 mg (60.8 to 100 mg per 1 mL of whole blood).

[0080] In a specific embodiment, the chelating agent may be the above-mentioned chelating agent commonly used as a component contained in a blood collection tube for clinical tests. The use of such a chelating agent in the blood collection tube is also desirable from the perspective of clinical application.

[0081] In another specific embodiment, the blood collection tube may further contain one or more commonly used components other than the chelating agent contained in the blood collection tube for clinical tests. Therefore, the blood collection tube may further contain one or more commonly used components selected from the group consisting of heparin or its salts, and fluorides (e.g., sodium fluoride). Examples of the salts include those described above.

[0082] The blood collection tube of the present invention containing a nonionic surfactant and a chelating agent can significantly improve the recovery amount of extracellular vesicles from whole blood, and thus is useful for the preparation of blood samples for the analysis of extracellular vesicles. The blood collection tube of the present invention also contains a chelating agent, which is a blood coagulation prevention component contained in a blood collection tube for ordinary blood tests, and a nonionic surfactant that enables reduction or avoidance of hemolysis. Therefore, it is useful for the preparation of highly versatile blood samples that can be used not only for the analysis of extracellular vesicles but also for ordinary blood tests. The blood collection tube of the present invention can also be suitably used in the method of the present invention, and thus is useful for the simple and rapid implementation of the method of the present invention.

Examples

[0083] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples. Note that all percentages described in the examples are W / V%.

[0084] (Materials and Methods) (1) Preparation of antibody-immobilized particles (solid-phase antibody) To 0.01 g / mL of magnetic particles in 10 mM MES buffer (pH 5.0), 0.2 mg / mL of a mouse anti-CD9 monoclonal antibody (manufactured by Fujirebio Inc.), which recognizes CD9, a surface marker of extracellular vesicles (EV), was added, and the mixture was incubated at 25°C for 1 hour with gentle stirring. After the reaction, the magnetic particles were magnetically collected, and the particles were washed with a washing solution (50 mM Tris buffer, 150 mM NaCl 2 , 2.0% BSA, pH 7.2) to obtain anti-CD9 antibody-immobilized particles. Each antibody-immobilized particle was suspended in a particle diluent (50 mM Tris buffer, 1 mM EDTA·2Na, 0.1% NaN 3 , 2.0% BSA, pH 7.2) to obtain each antibody-immobilized particle solution.

[0085] (2) Preparation of alkaline phosphatase-labeled antibody Desalted alkaline phosphatase (ALP) was mixed with N-(4-maleimidobutyryloxy)-succinimide (GMBS) (final concentration 0.3 mg / mL), and allowed to stand at 30 °C for 1 hour to maleimidize ALP. Next, in a coupling reaction solution (100 mM phosphate buffer, 1 mM EDTA·2Na, pH 6.3), the Fab’-modified mouse anti-CD9 monoclonal antibody and maleimidized ALP were mixed at a molar ratio of 1:1 and reacted at 25 °C for 1 hour. The reaction solution was purified using Superdex 200 column chromatography (General Electric) to obtain an ALP-labeled anti-CD9 antibody. Each ALP-labeled antibody was suspended in a label diluent (50 mM MES buffer, 150 mM NaCl 2 , 0.3 mM ZnCl 2 , 1 mM MgCl 2 , 0.1% NaN 3 , 2.0% BSA, pH 6.8) to obtain each labeled antibody solution.

[0086] (3) Method for measuring EV surface marker (CD9) The method for measuring the EV surface marker (CD9) in the following examples is as follows. 20 μL of the sample for measurement and 50 μL of the anti-CD9 antibody immobilized particle solution were dispensed into a reaction vessel and stirred. Then, the resulting mixture was incubated at 37 °C for 8 minutes (primary reaction), and B (bound) / F (free) separation and washing were performed. 50 μL of the ALP-labeled antibody was dispensed into the reaction vessel, stirred, incubated at 37 °C for 8 minutes, and B / F separation and washing were performed. Then, 200 μL of a Lumi-Pulse (registered trademark) substrate solution (Fujirebio) containing 3-(2’-spiroadamantane)-4-methoxy-4-(3’’-phosphoryloxy)phenyl-1,2-dioxetane·2 sodium salt (AMPPD), a chemiluminescent substrate, was dispensed into the reaction vessel, stirred, incubated at 37 °C for 4 minutes, and then the luminescence amount was measured with a luminometer. A count value was obtained as the measurement value. The measurement of the luminescence amount was performed using a fully automated chemiluminescent enzyme immunoassay system (Lumi-Pulse L2400 (Fujirebio)). The antibody immobilized on magnetic particles and the ALP-labeled antibody used antibodies that recognize the same epitope in CD9. Since multiple CD9s are present on a single extracellular vesicle, it is possible to measure extracellular vesicles presenting multiple CD9s as surface markers rather than free CD9 by a sandwich immunoassay using an antibody that recognizes the same epitope as the immobilized antibody as the ALP-labeled antibody.

[0087] Example 1: Recovery of Extracellular Vesicles (EVs) from Whole Blood Added with Chelating Agents Chelating agents were added to human whole blood, and after centrifugation, CD9 in the supernatant was measured. As chelating agents, disodium ethylenediaminetetraacetate (EDTA·2Na), glycol ether diamine tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), hydroxyethylethylenediamine triacetic acid (HEDTA), ethylenediamine tetra(methylenephosphonic acid) (EDTMP), and hydroxyethyliminodiacetic acid (HIDA) were used. More specifically, 900 μL of whole blood after blood collection was added to a 5 mL polypropylene test tube (manufactured by Nunc) containing 100 μL of D-PBS(-) added with each chelating agent. The chelating agent was added so that the final concentration after mixing with the whole blood was 15 mM. After adding the whole blood, the test tube was inverted and mixed, left standing at room temperature for 30 minutes, centrifuged (HITACHI CF5RE, 3000 rpm (2130 xg), 5 minutes, room temperature), the supernatant was recovered, and CD9, which is a surface marker protein of extracellular vesicles (EVs), was measured by the method described in (Materials and Methods) to obtain a count value. As a control (not added), the same measurement was performed using D-PBS(-) without adding a chelating agent. As a result, for all the chelating agents examined, the supernatant prepared from whole blood added with the chelating agent showed a significantly higher count value compared to the supernatant prepared from whole blood without adding the chelating agent (Table 1). This indicates that the amount of EVs present in the supernatant prepared from whole blood added with the chelating agent is significantly increased compared to the amount of EVs present in the supernatant prepared from whole blood without adding the chelating agent. Therefore, it was shown that the addition of a chelating agent to whole blood can significantly increase the recovery of EVs.

[0088]

Table 1

[0089] Example 2: Confirmation of the presence or absence of hemolysis in whole blood added with a surfactant Surfactants were added to human whole blood, and the presence or absence of hemolysis was confirmed. As nonionic surfactants, Tergitol 15-S-30, Brij 35, Pluronic F68, TWEEN20, and PVPK30 were used. As cationic surfactants, octyltrimethylammonium bromide (C8TAB) and hexadecyltrimethylammonium chloride (C16TAC) were used. As anionic surfactants, sodium N-decanoyl sarcosinate (N-DSS (NDS)) and sodium N-lauroyl sarcosinate hydrate (NLS) were used. As zwitterionic surfactants, CHAPS and N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C12APS) were used. More specifically, 900 μL of whole blood after blood collection was added to a 5 mL polypropylene test tube (manufactured by nunc) containing 100 μL of D-PBS(-) added with a surfactant. The surfactant was added so that the final concentration after mixing with the whole blood would be 0.5 W / V%. After adding the whole blood, the test tube was inverted and mixed, left standing at room temperature for 30 minutes, centrifuged (3000 rpm, 5 minutes), the supernatant was collected, and the presence or absence of hemolysis was visually confirmed. As a result, it was confirmed that nonionic surfactants such as Tween, Brij, and Tergitol did not cause hemolysis (Table 2).

[0090]

Table 2

[0091] Example 3: Recovery of EVs from whole blood added with a chelating agent and a surfactant Chelating agents and surfactants were added to human whole blood, and CD9 in the supernatant after centrifugation was measured. As the chelating agents, EDTA·2Na and EGTA were used. As the surfactant, Tergitol 15-S-40 was used. First, 100 μL of D-PBS(-) with a chelating agent added and 100 μL of D-PBS(-) with a surfactant added were placed in a 5 mL polypropylene test tube (manufactured by Nunc) to prepare a blood collection tube model containing the chelating agent and the surfactant. In the preparation of this blood collection tube model, the chelating agent was set so that the final concentration after mixing with whole blood would be 60 mM. The surfactant was set so that the final concentration after mixing with whole blood would be 0.5%. Next, 800 μL of whole blood after blood collection was added to this test tube (blood collection tube model). After adding the whole blood, the test tube was inverted and mixed, allowed to stand at room temperature for 30 minutes, centrifuged (3000 rpm, 5 minutes), the supernatant was collected, and CD9, which is a surface marker protein of extracellular vesicles (EVs), was measured by the method described in (Materials and Methods). Also, as a comparison, a blood collection tube containing neither a chelating agent nor a surfactant and a blood collection tube containing a chelating agent but no surfactant were prepared, and CD9 was measured in the same manner. As a result, regardless of the type of chelating agent, the supernatant prepared from whole blood to which a chelating agent and a surfactant were added showed significantly higher count values compared to the supernatant prepared from whole blood to which a chelating agent was added but no surfactant was added (Table 3). This indicates that the amount of EVs present in the supernatant prepared from whole blood to which a chelating agent and a surfactant were added is significantly increased compared to the amount of EVs present in the supernatant prepared from whole blood to which a chelating agent was added but no surfactant was added. Therefore, it was shown that the recovery amount of EVs can be significantly increased by adding a chelating agent and a surfactant to whole blood.

[0092]

Table 3

[0093] Example 4: Examination of the Concentrations of Chelating Agents and Surfactants Various concentrations of chelating agents and surfactants were added to human whole blood, and CD9 in the supernatant after centrifugation was measured. First, 100 μL of D-PBS(-) with various concentrations of EGTA added and 100 μL of D-PBS(-) with various concentrations of Tergitol 15-S-40 added were placed in a 5 mL polypropylene test tube (manufactured by Nunc) to prepare a blood collection tube model containing a chelating agent and a surfactant. In the preparation of this blood collection tube model, the final concentration of EGTA after mixing with whole blood was set to 0, 10, 50, 100, 200 mM. The final concentration of Tergitol 15-S-40 after mixing with whole blood was set to 0, 0.1, 0.25, 1.0, 2.0%. Next, 800 μL of whole blood after blood collection was added to this test tube (blood collection tube model). After adding the whole blood, the test tube was inverted and mixed, left standing at room temperature for 30 minutes, centrifuged (3000 rpm, 5 minutes), the supernatant was collected, and CD9, which is a surface marker protein of extracellular vesicles (EVs), was measured by the method described in (Materials and Methods). As a result, the chelating agent increased the reactivity in a concentration-dependent manner (Table 4). The surfactant alone did not increase the reactivity, but in combination with the chelating agent, it increased the reactivity (Table 5). Also, with the combination of the chelating agent, the higher the concentration of the surfactant, the higher the reactivity (Tables 4 and 5). This indicates that the amount of EVs present in the supernatant prepared from whole blood added with a chelating agent and a surfactant increases depending on the concentrations of the chelating agent and the surfactant. Therefore, it was shown that the recovery amount of EVs can be increased by adding a high concentration of chelating agent and a high concentration of surfactant to whole blood.

[0094]

Table 4

[0095]

Table 5

[0096] Example 5: Examination of the effects of chelating agents and surfactants It was confirmed whether the effects of chelating agents and surfactants were on whole blood or on the immune response. First, 125 μL of D-PBS(-) containing a chelating agent (EGTA) and 125 μL of D-PBS(-) containing a surfactant (tergitol 15-S-40) were placed in a 5 mL polypropylene test tube (manufactured by Nunc) to prepare a blood collection tube model containing a chelating agent and a surfactant. In the preparation of this blood collection tube model, the chelating agent and the surfactant were set so that the final concentration after mixing with whole blood would be the concentration shown in Table 6. Next, 1000 μL of whole blood after blood collection was added to this test tube (blood collection tube model). After adding the whole blood, the test tube was inverted and mixed, allowed to stand at room temperature for 30 minutes, centrifuged (3000 rpm, 5 minutes), the supernatant was collected, and CD9, which is a surface marker protein of extracellular vesicles (EV), was measured by the method described in (Materials and Methods). Also, when measuring CD9, the measurement was also carried out with 100 mM EGTA added to the CD9 antibody-immobilized particle solution. When 100 mM EGTA is added to the CD9 antibody-immobilized particle solution (particle diluent), the EGTA concentration during the primary reaction is 71.4 mM. When EGTA is added so that the final concentration after mixing with whole blood in the blood collection tube is 200 mM, the EGTA concentration during the primary reaction is 57.1 mM. If EGTA acts during the immune response, the count value should be high even when EGTA is added to the CD9 antibody-immobilized particle solution. First, the conditions using the supernatant prepared from whole blood supplemented with EGTA at a final concentration of 200 mM [200 mM EGTA and 0% tergitol 15-s-40, and 200 mM EGTA and 1% tergitol 15-S-40] both showed significantly higher count values compared to the condition using the supernatant prepared from whole blood without adding EGTA of the same concentration [0 mM EGTA and 0% tergitol 15-s-40] (see “% vs. non-added” in Table 6). On the other hand, when EGTA was added to the CD9 antibody-immobilized particle solution, it did not show a significantly higher count value compared to the case where EGTA was not added to the CD9 antibody-immobilized particle solution (see “% vs. without EGTA” in Table 6). This indicates that the actions of the chelating agent and the surfactant are on whole blood rather than on the immune reaction in the CD9 measurement system. Therefore, it was demonstrated that the addition of a chelating agent and a surfactant to whole blood can improve the recovery amount of extracellular vesicles.

[0097]

Table 6

[0098] Example 6: Examination of the type of surfactant It was confirmed whether various nonionic surfactants can act on whole blood. Tergitol 15-S-40, Pluronic F68, Pluronic F108, and Pluronic F127 were used as the surfactants. First, 125 μL of D-PBS(-) supplemented with EGTA and 125 μL of D-PBS(-) supplemented with various surfactants were placed in a 5 mL polypropylene test tube (manufactured by Nunc) to prepare a blood collection tube model containing a chelating agent and a surfactant. In the preparation of this blood collection tube model, the chelating agent and the surfactant were set so that the final concentration after mixing with whole blood would be the concentration shown in Table 7. Next, 1000 μL of whole blood after blood collection was added to this test tube (blood collection tube model). After adding the whole blood, the test tube was inverted and mixed, left standing at room temperature for 30 minutes, centrifuged (3000 rpm, 5 minutes), and the supernatant was collected. CD9, a surface marker protein of extracellular vesicles (EVs), was measured by the method described in (Materials and Methods). As a result, regardless of the type of surfactant, the supernatant prepared from whole blood to which a chelating agent and a surfactant were added showed significantly higher count values than the supernatant prepared from whole blood to which a chelating agent was added and no surfactant was added (Table 7). This indicates that regardless of the type of surfactant, the amount of EVs present in the supernatant prepared from whole blood to which a chelating agent and a surfactant were added was significantly increased compared to the amount of EVs present in the supernatant prepared from whole blood to which a chelating agent was added and no surfactant was added. Therefore, it was shown that the recovery amount of EVs can be significantly increased by adding a chelating agent and various surfactants to whole blood.

[0099]

Table 7

Industrial Applicability

[0100] For example, the present invention is useful for clinical tests of extracellular vesicles.

Claims

1. A method for recovering extracellular vesicles from whole blood, comprising: (1) mixing whole blood with a nonionic surfactant and a chelating agent to produce a mixture containing extracellular vesicles, the nonionic surfactant, and the chelating agent; and (2) separating the extracellular vesicles from the mixture.

2. The method according to claim 1, wherein the nonionic surfactant is a nonionic surfactant having a polyoxyethylene alcohol structure.

3. The method according to claim 2, wherein the nonionic surfactant having a polyoxyethylene alcohol structure is an alcohol ethoxylate.

4. The method according to claim 2, wherein the nonionic surfactant having a polyoxyethylene alcohol structure is a polyoxyethylene-polyoxyalkylene block copolymer.

5. The method according to any one of claims 1 to 4, wherein the nonionic surfactant is a nonionic surfactant having an HLB of 18 or more.

6. The method according to any one of claims 1 to 5, wherein the concentration of the nonionic surfactant in the mixture is 0.01 to 10% by weight / volume.

7. The method according to any one of claims 1 to 6, wherein the chelating agent is hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetra(methylenephosphonic acid) (EDTMP), glycol ether diamine tetraacetic acid (EGTA), or a salt thereof.

8. The method according to any one of claims 1 to 7, wherein the concentration of the chelating agent in the mixture is 10 mM to 1000 mM.

9. The method according to any one of claims 1 to 8, wherein the mixing is performed after putting whole blood into a blood collection tube containing the nonionic surfactant and the chelating agent.

10. The method according to any one of claims 1 to 9, wherein the separation is performed by any one of the following (A) to (C): (A) collecting the supernatant from the mixture; (B) using a substance that binds to the surface marker of the extracellular vesicles for the mixture; or (C) collecting the supernatant from the mixture and using a substance that binds to the surface marker of the extracellular vesicles for the supernatant.

11. The method according to any one of claims 1 to 10, wherein the extracellular vesicles are exosomes.

12. A method for analyzing extracellular vesicles, comprising: (1) Recovering extracellular vesicles from whole blood by the method according to any one of claims 1 to 11; (2) Analyzing the extracellular vesicles recovered from whole blood.

13. A reagent for recovering extracellular vesicles from whole blood, comprising a blood collection tube containing a nonionic surfactant and a chelating agent.

14. The recovery reagent according to claim 13, wherein the amount of the nonionic surfactant in the blood collection tube is 0.1 to 2000 mg.

15. The recovery reagent according to claim 13 or 14, wherein the amount of the chelating agent in the blood collection tube is 3.8 to 3800 mg.

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