WO2020004436A1 - Method and reagent for destroying exosomes in sample - Google Patents

Abstract

Provided are: a method for destroying exosomes in a sample, characterized by adding at least one surfactant selected from the group consisting of a polyether alkyl amine, a polyoxyethylene-polyoxypropylene alkyl ether, and a polyoxyethylene alkyl phenyl ether having an HLB value of no more than 13; and a reagent for destroying exosomes in a sample, characterized by comprising at least one surfactant selected from the group consisting of a polyether alkyl amine, a polyoxyethylene-polyoxypropylene alkyl ether, and a polyoxyethylene alkyl phenyl ether having an HLB value of no more than 13.

Description

Method for destruction of exosome in sample and reagent for destruction

(4) The present invention relates to a method and a reagent for destruction of exosomes in a sample.

Exosomes are vesicle granules secreted from animal cells and having a lipid bilayer structure of 30 to 200 nm in diameter. It is known that various membrane proteins exist on the exosome surface, similar to general cell surfaces, and microRNA (miRNA) may be contained in the exosome in addition to various proteins such as cytokines. It is known (Non-Patent Document 1). In addition, exosomes have been reported to be secreted from various cells, for example, cells of the immune system and various cancer cells, and function as a mediator of intercellular communication in vivo and are related to physiological phenomena, The association with diseases such as cancer is attracting attention.

In order to analyze the substance contained in the exosome, it is necessary to destroy the lipid bilayer structure of the exosome. Heretofore, as a method of destroying exosomes, a method of isolating exosomes by ultracentrifugation of cell culture supernatant or human serum (110,000 G for 70 minutes) and destroying the isolated exosomes with an antimicrobial peptide (Patent Literature 1), cell culture supernatant was ultracentrifuged (120,000 G, 100 minutes) to isolate exosomes, and the isolated exosomes were non-ionic detergent Triton X-100 (polyoxyethylene octyl) (Non-Patent Document 2) and the like are known.

However, these methods require exosome isolation by ultracentrifugation or the like as a pretreatment for exosome disruption, so that the operation is complicated and the operation requires time. On the other hand, in applying the analysis of substances contained in exosomes to the site of clinical examination, a simple and rapid method for destruction of exosomes is desired.

Further, as a method for separating exosomes, exosomes are contacted with a solid phase carrier to which a ligand recognizing a surface antigen present on the surface of the exosomes is contacted to form a complex between the exosomes and the solid phase carrier. A body forming step and a washing step of washing the complex, wherein at least one of the complex forming step and the washing step is performed in the presence of a nonionic surfactant containing no aromatic group in the molecule. A method for separating exosomes has been reported (Patent Document 2).

International Publication No. WO 2016/143904 WO 2015/068772

目的 An object of the present invention is to provide a method and a reagent for destruction of exosomes in a sample which are simple and rapid.

The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, a sample was made of polyether alkylamine, polyoxyethylene polyoxypropylene alkyl ether (hereinafter referred to as POE-POP alkyl ether), and HLB (Hydrophile). -Exosome can be rapidly destroyed by adding at least one surfactant selected from the group consisting of polyoxyethylene alkyl phenyl ether (hereinafter referred to as POE alkyl phenyl ether) having a Lipophile Balance value of 13 or less. Heading, the present invention has been completed.

That is, the present invention relates to the following [1] to [4].
[1] At least one surfactant selected from the group consisting of polyether alkylamines, POE-POP alkyl ethers, and POE alkyl phenyl ethers having an HLB value of 13 or less is added to the sample. How to destroy exosomes in a sample.
[2] The method according to [1], wherein the sample is serum or plasma.
[3] The sample according to claim 1, further comprising at least one surfactant selected from the group consisting of polyether alkylamines; POE-POP alkyl ethers; and POE alkyl phenyl ethers having an HLB value of 13 or less. Exosomal disruption reagent.
[4] The reagent according to [3], wherein the sample is serum or plasma.

According to the present invention, a method and a reagent for destroying exosomes in a sample in a simple and rapid manner are provided.

It is a gel filtration chromatogram obtained by subjecting serum to gel filtration chromatography. The horizontal axis represents the elution fraction. The left vertical axis represents the luminescence (RLU) obtained by a sandwich method using an anti-CD9 monoclonal antibody (first antibody) and an alkaline phosphatase-labeled anti-CD9 monoclonal antibody (alkaline phosphatase-labeled second antibody), The amount of luminescence in each fraction is indicated by ●. The right vertical axis represents the absorbance at 560 nm obtained by the protein quantification method (BCA method) using bicinchoninic acid (BCA), and the absorbance at 560 nm in each fraction is indicated by □. 4 is a gel filtration chromatogram showing the results of a test for destruction of exosomes in serum using an exosome-disrupting reagent containing a polyetheralkylamine and a control reagent. The horizontal axis represents the elution fraction. The vertical axis represents the luminescence (RLU) obtained by a sandwich method using an anti-CD9 monoclonal antibody (first antibody) and an alkaline phosphatase-labeled anti-CD9 monoclonal antibody (alkaline phosphatase-labeled second antibody). ● represents the amount of luminescence in each fraction when using an exosome-destroying reagent containing Nimeen L-202 (polyetheralkylamine). The symbol 発 光 indicates the amount of luminescence in each fraction when an exosome-disrupting reagent containing Nimeen DT-203 (polyetheralkylamine) was used. □ indicates the amount of luminescence in each fraction when the control reagent was used. 5 is a gel filtration chromatogram showing the results of an exosome disruption test using a exosome disrupting reagent containing POE-POP alkyl ether and a control reagent. The horizontal axis represents the elution fraction. The vertical axis represents the luminescence (RLU) obtained by a sandwich method using an anti-CD9 monoclonal antibody (first antibody) and an alkaline phosphatase-labeled anti-CD9 monoclonal antibody (alkaline phosphatase-labeled second antibody). ● represents the amount of luminescence in each fraction when using an exosome-destroying reagent containing Wandasurf ID-50 (POE-POP alkyl ether). □ indicates the amount of luminescence in each fraction when the control reagent was used. It is a gel filtration chromatogram which shows the result of the exosome destruction test which used the exosome destruction reagent containing the POE alkylphenyl ether whose HLB value is 13 or less, the comparative exosome destruction reagent, and the control reagent. The horizontal axis represents the elution fraction. The vertical axis represents the luminescence (RLU) obtained by a sandwich method using an anti-CD9 monoclonal antibody (first antibody) and an alkaline phosphatase-labeled anti-CD9 monoclonal antibody (alkaline phosphatase-labeled second antibody). ● represents the amount of luminescence in each fraction when using an exosome-destroying reagent containing Nonion NS-204.5 (POE alkylphenyl ether having an HLB value of 13 or less; HLB value of 9.5). The symbol 発 光 indicates the amount of luminescence in each fraction when an exosome-destroying reagent containing nonionic HS-204.5 (POE alkylphenyl ether having an HLB value of 13 or less; HLB value of 9.8) is used. ○ indicates the amount of luminescence in each fraction when an exosome-disrupting reagent containing Triton X-100 (POE alkyl phenyl ether; HLB value 13.5) was used. Δ represents the amount of luminescence in each fraction when an exosome disrupting reagent containing Nonidet P-40 (POE alkylphenyl ether; HLB value 13.1) is used. □ indicates the amount of luminescence in each fraction when the control reagent was used.

1. Exosome Destruction Method The exosome in the sample of the present invention is preferably selected from the group consisting of polyether alkylamine, POE-POP alkyl ether, and POE alkyl phenyl ether having an HLB value of 13 or less. This is a method of breaking using at least one kind of surfactant. Here, `` destroying the exosome '' means that the exosome-specific lipid bilayer structure of vesicle particles is lost and destroyed to the extent that substances contained inside the exosome are eluted outside the exosome. means. In this specification, destruction of an exosome may be expressed as destruction of an exosome membrane, dissolution of an exosome, or dissolution of an exosome membrane.

The method for confirming that the exosome has been destroyed by the method for destroying exosomes of the present invention is not particularly limited as long as it is a method capable of confirming the exosome breakdown, for example, a method using an electron microscope, an exosome that the exosome has on its surface. Examples include a method of measuring a unique antigen by an immunological assay. In the method using an electron microscope, destruction of exosomes can be confirmed by confirming disappearance of the shape of vesicle particles having a lipid bilayer structure specific to exosomes in an electron microscope image. In addition, when the exosomes are examined for destruction of exosomes by measuring an exosome-specific antigen having an exosome on its surface by an immunoassay, the exosomes of the present invention are compared with those before performing the exosome destruction method of the present invention. After the disruption method is performed, the decrease in the measurement signal due to the exosome-specific antigen, that is, the decrease in the measurement signal due to the exosome, can be confirmed to confirm the exosome destruction. The criterion for confirming the exosome destruction can be appropriately set. For example, a decrease of the measurement signal of 50% or more can be set as the criterion. Furthermore, when exosomes are examined for destruction of exosomes by measuring an exosome-specific antigen on the surface thereof by an immunological assay, the mixture obtained by mixing the sample and the destruction reagent of the present invention is gel-gelated. Destruction of exosomes can also be confirmed by subjecting to chromosome chromatography and measuring exosomes in each of the obtained fractions. In this case, the sample itself such as serum or plasma is subjected to gel filtration chromatography to confirm in advance the fraction from which exosomes are eluted, and thereafter, the sample such as serum or plasma and the disrupting reagent of the present invention are mixed. The mixture obtained in (1) is subjected to gel filtration chromatography, the exosomes in the fraction previously confirmed are measured by immunoassay, and the decrease in the measurement signal is confirmed, thereby confirming the exosome destruction. be able to. The criterion for confirming the exosome destruction can be appropriately set. For example, a decrease of the measurement signal of 50% or more can be set as the criterion.

The measurement signal is a signal obtained by an immunoassay for an exosome-specific antigen that the exosome has on its surface, and a exosome having an exosome-specific antigen on its surface, and a label formed by binding a label to an antibody against the exosome-specific antigen. Due to the label in the immune complex containing the conjugated antibody. The measurement signal is not particularly limited as long as it is a signal capable of confirming exosome destruction, and examples thereof include color development (absorbance), fluorescence, and luminescence.
When the measurement signal is color development (absorbance), for example, a peroxidase that is a label is reacted with a combination of its substrate, hydrogen peroxide and an oxidized chromogen, and the absorbance of the reaction solution is measured using a spectrophotometer or a multiwell. A method of measuring with a plate reader or the like, a method of reacting a label β-D-galactosidase with its substrate, and measuring the absorbance of the reaction solution with a spectrophotometer, a multiwell plate reader, or the like can be used. Examples of the oxidative coloring type chromogen include a leuco type chromogen and an oxidative coupling coloring type chromogen.
A leuco-type chromogen is a substance that is converted into a dye alone in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Specifically, tetramethylbenzidine, O-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7- Bis (dimethylamino) -10H-phenothiazine (MCDP), N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine sodium salt (DA-64), 10-N-carboxymethylcarbamoyl-3 , 7-bis (dimethylamino) -10H-phenothiazine sodium salt (DA-67), 4,4'-bis (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] Amine (BCMA) and the like.

An oxidatively coupled chromogen is a substance that produces a dye by oxidative coupling of two compounds in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler with an aniline (Trinder reagent) and a combination of a coupler with a phenol.
Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine.
Examples of anilines include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N-ethyl-N- (2-hydroxy -3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methylaniline, N , N-bis (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl N- (3-sulfopropyl) aniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxyaniline, N- (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl- N- (3-sulfopropyl) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3 -Sulfopropyl) aniline, N-ethyl-N- (3-methylphenyl) -N'succinylethylenediamine (EMSE), N-ethyl-N- (3-methylphenyl) -N'acetylethylenediamine, N-ethyl-N -(2-hydroxy-3-sulfopropyl) -4-fluoro-3,5-dimethoxyaniline (F-DAOS) and the like.
Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.

基質 β-D-galactosidase substrate includes, for example, o-nitrophenyl-β-D-galactopyranoside.

When the measurement signal is fluorescence, for example, a peroxidase that is a label is reacted with a combination of hydrogen peroxide and a fluorescent substrate of peroxidase, and the intensity of the generated fluorescence is measured with a fluorometer, a fluorescence multiwell plate reader, or the like. And a method in which β-D-galactosidase as a label is reacted with a fluorescent substrate of β-D-galactosidase, and the intensity of the generated fluorescence is measured with a fluorometer, a fluorescence multiwell plate reader, or the like. . Examples of the fluorescent substrate for peroxidase include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, coumarin and the like. Examples of the fluorescent substrate of β-D-galactosidase include 4-methylumbelliferyl-β-D-galactopyranoside.

When the measurement signal is luminescence, for example, a method for measuring the intensity of luminescence due to the luminescent substance as a label with a luminescence photometer or luminescence multiwell plate reader, etc., the peroxidase as a label, and the luminescence of hydrogen peroxide and peroxidase A method of measuring the intensity of luminescence generated by reacting with a combination of substrates with a luminescence intensity meter, a luminescence multi-well plate reader, or the like; a method of labeling β-D-galactosidase with a luminescent substrate of β-D-galactosidase; A method of measuring the intensity of the generated luminescence with a luminescence photometer or a luminescence multi-well plate reader, and reacting the alkaline phosphatase that is a label with a luminescent substrate of the alkaline phosphatase, and determining the intensity of the generated luminescence by the luminescence intensity. And a method of measuring with a light-emitting multi-well plate reader or the like. Examples of the luminescent substance include acridinium esters and derivatives thereof, ruthenium complex compounds, lophine and the like. Examples of the luminescent substrate of peroxidase include a luminol compound and a lucigenin compound. Examples of the luminescent substrate of β-D-galactosidase include Galacton-Plus (manufactured by Applied Biosystems) and its analogous compounds. Examples of the luminescent substrate for alkaline phosphatase include 3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), Chloro-5- {4-methoxyspiro [1,2-dioxetane-3,2 ′ (5′-chloro) tricyclo [3.3.1.1 ^3.7 ] decane] -4-yl} phenylphosphate. Sodium salt (CDP-Star ^™ ), 3- {4-methoxyspiro [1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.1 ^3.7 ] decane]- 4'-yl} phenyl phosphate disodium salt ^(CSPD TM), 9 - [(phenyloxy) (phosphoryloxy) methylidene] -10-methyl-acridan-disodium, - [(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methyl-acridan-disodium (Lumigen ^TM APS-5), and the like.

Examples of a method for confirming exosome destruction by measuring an exosome specific antigen on the surface of the exosome by an immunoassay include, for example, a method including the following steps.
<Method for confirming exosome destruction (1)>
(1A) a step of adding at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkyl phenyl ether having an HLB value of 13 or less to a sample;
(2A) the sample obtained in the step (1A) is treated with a first antibody or an antibody fragment that binds to a first exosome specific antigen that the exosome has on its surface, and a second exosome that the exosome has on its surface An immune complex 1 comprising the second antibody or the antibody fragment that binds to a unique antigen and an aqueous medium, and reacting with the first antibody or the antibody fragment, exosomes, and the second antibody or the antibody fragment. Generating a;
(3A) measuring the immune complex generated in the step (2A);
(4A) Except that in the above step (1A), no surfactant of any of polyether alkylamine, POE-POP alkyl ether, and POE alkylphenyl ether having an HLB value of 13 or less was added to the sample. Measuring the immune complex generated by the same step as the above steps (1A) to (3A); and
(5A) The measured signal obtained in the step (3A) is compared with the measured signal obtained in the step (4A), and the measured signal obtained in the step (3A) is compared with the measured signal obtained in the step (4A). A step of judging that the exosome has been destroyed when the measured signal has decreased compared to the measurement signal obtained in the step.

In the above step (1A), at least one surfactant selected from the group consisting of polyetheralkylamine, POE-POP alkylether, and POEalkylphenylether having an HLB value of 13 or less is added to the sample Exosome destruction reagent of the present invention containing at least one surfactant selected from the group consisting of polyether alkylamine, POE-POP alkyl ether, and POE alkyl phenyl ether having an HLB value of 13 or less, It can be performed by addition. In this case, the step (4A) is a control exosome-destroying reagent that does not contain any of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkyl phenyl ether having an HLB value of 13 or less. Can be added to the sample. The criterion for exosome destruction in the step (5A) can be set as appropriate. For example, the measurement signal obtained in the step (3A) is compared with the measurement signal obtained in the step (4A). , A criterion for determining that the exosome has been destroyed when the exosome is reduced by 50% or more can be set.
In the step (2A), the first antibody or the antibody fragment that binds to the first exosome-specific antigen that the exosome has on its surface may or may not be immobilized on the insoluble carrier, It is preferably immobilized. The insoluble carrier is not particularly limited as long as the first antibody or the antibody fragment that binds to the first exosome-specific antigen that the exosome has on its surface is immobilized and the exosome can be destroyed by the method of the present invention. For example, various types of plates such as synthetic resin plates such as microtiter plates, glass or synthetic resin granules (beads), glass or synthetic resin spheres (balls), latex, magnetic particles, nitrocellulose membrane, etc. Examples include membranes and test tubes made of synthetic resin. Examples of the synthetic resin plate include a polyethylene plate, a polypropylene plate, and a polystyrene plate.

The immobilization of the first antibody or the antibody fragment that binds to the first exosome-specific antigen that the exosome has on its surface to the insoluble carrier is not particularly limited as long as the immobilization enables the method for destruction of the exosome of the present invention. But immobilization by physical adsorption, chemical bonding and the like. Examples of the physical adsorption include an electrostatic bond, a hydrogen bond, and a hydrophobic bond. Examples of the chemical bond include a covalent bond and a coordinate bond.
The first antibody or the antibody fragment that binds to the first exosome-specific antigen that the exosome has on its surface can be directly immobilized on the insoluble carrier by physical adsorption and / or chemical bonding, or indirectly immobilized on the insoluble carrier. You may. As an indirect immobilization method, for example, the first exosome binds to the first exosome-specific antigen on the surface thereof through specific binding of biotin and avidins (avidin, streptavidin, neutravidin, etc.). Examples include a method of immobilizing the antibody or the antibody fragment on an insoluble carrier. In addition, the first antibody or the antibody fragment that binds to the first exosome specific antigen that the exosome has on its surface may be immobilized on an insoluble carrier by covalent bonding via a linker.
In the step (2A), the second antibody or the antibody fragment that binds to the second exosome specific antigen that the exosome has on its surface may or may not bind to the label. Is preferred. Examples of the label include the above-mentioned labels and the like. When the second antibody or the antibody fragment that binds to the second exosome-specific antigen that the exosome has on its surface is bound to the label, the measurement of the generated immune complex in the above step (3A) is performed by measuring the generated immune complex. It can be performed by measuring the label in the complex.
When no label is bound to the second antibody or the antibody fragment, a third antibody bound to the second antibody or the antibody fragment or a labeled third antibody bound to a label to the antibody fragment or the antibody Using the fragment, the destruction of the exosome in the sample can be similarly confirmed. That is, the labeled third antibody or the antibody fragment is reacted with the second antibody or the antibody fragment in the immune complex 1, and the first antibody or the antibody fragment, the exosome, and the second antibody or the antibody fragment are reacted. And the labeled third antibody or the antibody fragment to form an immune complex 2, and measuring the label in the immune complex 2 by the above-described method, thereby obtaining the immune complex generated in the step (2A). One quantity can be measured. Examples of the third antibody include an antibody that binds to the Fc region of the second antibody or an antibody fragment thereof. Examples of the label include the aforementioned labels.

The combination of the first exosome-specific antigen and the second exosome-specific antigen is not particularly limited as long as the combination enables the method of destructing exosomes of the present invention, and examples thereof include combinations shown in Table 1 below. No.

The combination of the first antibody that binds to the first exosome-specific antigen and the second antibody that binds to the second exosome-specific antigen is not particularly limited as long as the combination enables the method of destroying exosomes of the present invention. Instead, for example, the combinations shown in Table 2 below can be mentioned.

In the present invention, as the antibody fragment of the first antibody that binds to the first exosome-specific antigen, any antibody fragment that binds to the first exosome-specific antigen and enables the exosome-destroying method of the present invention can be used. There is no limitation, for example, Fab obtained by treating the antibody with papain, F (ab ') ₂ obtained by treating the antibody with pepsin, Fab' obtained by treating the antibody with pepsin and reducing, and the like. Examples include an antibody fragment from which the Fc portion has been removed, an antibody fragment from which the Fc portion has been removed by genetic engineering techniques, and the like.
In the present invention, as the antibody fragment of the second antibody that binds to the second exosome-specific antigen, any antibody fragment that binds to the second exosome-specific antigen and enables the exosome destruction method of the present invention can be used. There is no limitation, for example, Fab obtained by treating the antibody with papain, F (ab ') ₂ obtained by treating the antibody with pepsin, Fab' obtained by treating the antibody with pepsin and reducing, and the like. Examples include an antibody fragment from which the Fc portion has been removed, an antibody fragment from which the Fc portion has been removed by genetic engineering techniques, and the like.

In the present invention, the first antibody that binds to the first exosome specific antigen is not particularly limited as long as it is an antibody that binds to the first exosome specific antigen and enables the exosome destruction method of the present invention. Both polyclonal antibodies and monoclonal antibodies can be used.
In the present invention, the second antibody that binds to the second exosome specific antigen is not particularly limited as long as it is an antibody that binds to the second exosome specific antigen and enables the exosome destruction method of the present invention. Both polyclonal antibodies and monoclonal antibodies can be used.

市 販 A commercially available antibody can be used for both the first antibody that binds to the first exosome specific antigen and the second antibody that binds to the second exosome specific antigen. Examples of commercially available antibodies include anti-CD9 monoclonal antibody (clone A100-4; manufactured by Institute of Medical Biology), Purified Mouse Anti-Human CD9 (manufactured by Becton Dickinson Japan), Purified Mouse Anti-Human CD63 (manufactured by Becton Japan) Dickinson), Purified Mouse Anti-Human CD81 (manufactured by Becton Dickinson Japan, clone: JS-81), anti-CD147 antibody [MEM-M6 / 1] (manufactured by Novous biologics) and the like.

<Method for confirming exosome destruction (2)>
(1B) a step of adding to the sample at least one surfactant selected from the group consisting of polyetheralkylamines, POE-POP alkylethers, and POEalkylphenylethers having an HLB value of 13 or less;
(2B) subjecting the sample obtained in the step (1B) to gel filtration chromatography to separate a fraction containing exosomes from other fractions;
(3B) the exosome-containing fraction obtained in the step (2B) is treated with a first antibody or an antibody fragment thereof, which binds to a first exosome-specific antigen that the exosome has on its surface, and the exosome on its surface. Reacting the second antibody or the antibody fragment that binds to the second exosome-specific antigen with an aqueous medium, the first antibody or the antibody fragment, the exosome, and the second antibody or the antibody fragment; Generating an immune complex 1;
(4B) a step of measuring the immune complex 1 generated in the step (3B);
(5B) In the step (1B), at least one surfactant selected from the group consisting of polyetheralkylamine, POE-POP alkylether, and POEalkylphenylether having an HLB value of 13 or less is not added. Measuring the immune complex generated by the same steps as the above steps (1B) to (4B), except for; and
(6B) The measurement signal obtained in the step (4B) is compared with the measurement signal obtained in the step (5B), and the measurement signal obtained in the step (4B) is compared with the measurement signal obtained in the step (5B). A step of determining that the exosome has been destroyed when the measured signal has decreased compared to the measurement signal obtained in the step.

In the above step (1B), the addition of at least one or more surfactants selected from the group consisting of polyether alkylamines, POE-POP alkyl ethers, and POE alkyl phenyl ethers having an HLB value of 13 or less to the sample comprises: Exosome destruction reagent of the present invention containing at least one surfactant selected from the group consisting of polyether alkylamine, POE-POP alkyl ether, and POE alkyl phenyl ether having an HLB value of 13 or less, It can be performed by addition. In this case, the step (5B) does not contain at least one or more surfactants selected from the group consisting of polyether alkylamines, POE-POP alkyl ethers, and POE alkyl phenyl ethers having an HLB value of 13 or less. It can be performed by adding a control exosome disrupting reagent to the sample. In addition, the criterion for exosome destruction in step (6B) can be appropriately set. For example, the measurement signal obtained in step (4B) is compared with the measurement signal obtained in step (5B). , A criterion for determining that the exosome has been destroyed when the exosome is reduced by 50% or more can be set.
In the above step (3B), the first antibody or the antibody fragment that binds to the first exosome-specific antigen that the exosome has on its surface may or may not be immobilized on the insoluble carrier, It is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carriers and the like.

Examples of the immobilization of the first antibody or the antibody fragment that binds to the first exosome-specific antigen that the exosome has on its surface to an insoluble carrier include the above-described immobilization.
In the step (3B), the second antibody or the antibody fragment that binds to the second exosome-specific antigen that the exosome has on its surface may or may not bind to the label. Is preferred. Examples of the label include the above-mentioned labels and the like. When the second antibody or the antibody fragment that binds to the second exosome-specific antigen that the exosome has on its surface is bound to the label, the measurement of the generated immune complex 1 in the step (4B) is performed. It can be performed by measuring the label in the immune complex 1.
When no label is bound to the second antibody or the antibody fragment, a third antibody bound to the second antibody or the antibody fragment or a labeled third antibody bound to a label to the antibody fragment or the antibody Using the fragment, the destruction of the exosome in the sample can be similarly confirmed. That is, the labeled third antibody or the antibody fragment is reacted with the second antibody or the antibody fragment in the immune complex 1, and the first antibody or the antibody fragment, the exosome, and the second antibody or the antibody fragment are reacted. And the labeled third antibody or the antibody fragment to form an immune complex 2, and measuring the label in the immune complex 2 by the above-described method, whereby the immune complex generated in the step (3B) is obtained. One quantity can be measured. Examples of the third antibody include an antibody that binds to the Fc region of the second antibody or an antibody fragment thereof. Examples of the label include the aforementioned labels.

Examples of the combination of the first exosome specific antigen and the second exosome specific antigen include the above-described combinations and the like.
Examples of the combination of the first antibody that binds to the first exosome-specific antigen and the second antibody that binds to the second exosome-specific antigen include, for example, the combinations described above.
As both the first antibody that binds to the first exosome-specific antigen and the second antibody that binds to the second exosome-specific antigen, commercially available antibodies can be used. Examples of commercially available antibodies include the aforementioned antibodies and the like.

エ The exosome in the present invention is a vesicle granule secreted from animal cells and having a lipid bilayer structure of 30 to 200 nm in diameter.

The sample in the present invention means a sample on which exosome isolation operation such as ultracentrifugation has not been performed, and as an example, a biological sample containing exosomes (for example, whole blood, serum, plasma, urine, saliva, Body fluid samples such as milk, seminal plasma, cerebrospinal fluid, and feces), and cell culture media, and the like. Biological samples are preferred, and serum and plasma are particularly preferred.

The polyetheralkylamine in the exosome breaking method of the present invention is not particularly limited as long as it is a polyetheralkylamine that enables the exosome breaking method of the present invention, and a polyetheralkylamine having an HLB value of 13 or less is preferable. preferable. The polyetheralkylamine in the present invention has a structure in which at least one of the hydrogen atoms bonded to the nitrogen atom of the alkylamine has been substituted with polyoxyethylene. For example, polyoxyalkylamine (hereinafter referred to as POE) Alkylamine), polyoxyethylene alkyl propylene diamine (hereinafter referred to as POE alkyl propylene diamine) and the like.
Examples of the alkyl in the polyetheralkylamine include an alkyl having 8 to 24 carbon atoms, and an alkyl having 10 to 20 carbon atoms is preferable. Examples of the alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonyl, decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), Oleyl, nonadecyl, icosyl, heneicosyl, docosyl (behenyl), tricosyl, tetracosyl and the like can be mentioned. Examples of the alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecyl, and icosyl. No.

Among the commercially available polyetheralkylamines, commercially available POE alkylamines include, for example, Nymeen L-201 [oxyethylene dodecylamine; HLB value 3.8], Nymeen L-202 [POE dodecylamine; HLB value 6] .4], Nimeen L-207 [POE dodecylamine; HLB value 12.5], Nimeen S-204 [POE stearylamine; HLB value 8.0], Nimeen S-220 [POE stearylamine; HLB value 15.4] ], Nymin T2-210 [POE alkyl (tallow) amine; HLB value 12.5], Nymin F-202 [POE alkyl (coconut) amine; HLB value 6.1] (all manufactured by NOF CORPORATION), Brownon L -205 [POE dodecylamine; HLB value 10.4], Brownon L-210 [POE Dodecylamine; HLB value 13.6] (above, manufactured by Aoki Yushi Kogyo Co., Ltd.). Commercially available POE alkyl propylene diamines include, for example, Nimeen DT-203 [POE alkyl propylene diamine; HLB value 6.0]. , Nimeen DT-208 [POE alkyl propylene diamine; HLB value 10.7] (above, manufactured by NOF CORPORATION), BROWNON DT-03 [POE alkyl (tallow) propylene diamine; HLB value 5.9], BROWNON DT-15 [POE alkyl (tallow) propylene diamine; HLB value 13.4] (all manufactured by Aoki Yushi Kogyo KK) and the like.
As a commercially available product of a polyetheralkylamine having an HLB value of 13 or less, for example, POE alkylamines include Nymene L-201 [oxyethylene dodecylamine; HLB value 3.8] and Nymene L-202 [POE dodecylamine; HLB] 6.4], Nimeen L-207 [POE dodecylamine; HLB value 12.5], Nimeen S-204 [POE stearylamine; HLB value 8.0], Nimeen T2-210 [POE alkyl (tallow) amine; HLB value 12.5], Nymein F-202 [POE alkyl (coconut) amine; HLB value 6.1] (all manufactured by NOF CORPORATION), Brownon L-205 [POE dodecylamine; HLB value 10.4] ( Aoki Yushi Kogyo Co., Ltd.). Commercially available POE alkyl propylene diamines having an HLB value of 13 or less include, for example, Nymein DT-203 [POE alkyl propylene diamine; HLB value 6.0], Nymein DT-208 [POE alkyl propylene diamine; HLB value 10.7] (Above, manufactured by NOF CORPORATION) and Brownon DT-03 [POE alkyl (tallow) propylene diamine; HLB value: 5.9] (above, manufactured by Aoki Yushi Kogyo Co., Ltd.).

The POE-POP alkyl ether in the method for destroying exosomes of the present invention is not particularly limited as long as it is a POE-POP alkyl ether that enables the method for destroying exosomes of the present invention, but POE-POP having an HLB value of 13 or less. Alkyl ethers are particularly preferred.
Examples of the alkyl in the POE-POP alkyl ether include an alkyl having 8 to 24 carbon atoms, and an alkyl having 10 to 20 carbon atoms is preferable. Examples of the alkyl having 8 to 24 carbon atoms include the aforementioned alkyl having 8 to 24 carbon atoms. Examples of the alkyl having 10 to 20 carbon atoms include the aforementioned alkyl having 10 to 20 carbon atoms.

Commercially available POE-POP alkyl ethers include, for example, Nonion HT-505 [POE-POP alkyl ether; HLB value 5], Nonion HT-510 [POE-POP alkyl ether; HLB value 10], Nonion HT-512 [ POE-POP alkyl ether; HLB value 12], Nonion HT-515 [POE-POP alkyl ether; HLB value 15] (all manufactured by NOF CORPORATION), Wandasurf ID-50 [POE-POP isodecyl ether; HLB value 10.5], Wandasurf ID-70 [POE-POP isodecyl ether; HLB value 12.1], Wandasurf ID-90 [POE-POP isodecyl ether; HLB value 13.2] (above, Aoki Yushi Kogyo Co., Ltd.) Co., Ltd.), Neugen TDS-30 [POE-POP tridecyl ether HLB value 8], Neugen TDS-50 [POE-POP tridecyl ether; HLB value 10.5], Neugen TDS-70 [POE-POP tridecyl ether; HLB value 12.1] (Daiichi Kogyo Seiyaku Co., Ltd.) Manufactured).
Commercially available POE-POP alkyl ethers having an HLB value of 13 or less include, for example, Nonion HT-505 [POE-POP alkyl ether; HLB value 5], Nonion HT-510 [POE-POP alkyl ether; HLB value 10], Nonion HT-512 [POE-POP alkyl ether; HLB value 12] (above, manufactured by NOF CORPORATION), Wandasurf ID-50 [POE-POP isodecyl ether; HLB value 10.5], Wandasurf ID -70 [POE-POP isodecyl ether; HLB value 12.1] (above, manufactured by Aoki Yushi Kogyo Co., Ltd.), Neugen TDS-30 [POE-POP tridecyl ether; HLB value 8], Neugen TDS-50 [POE- POP tridecyl ether; HLB value 10.5], Neugen TDS-70 [POE- OP tridecyl ether; HLB value 12.1 (or higher, Dai-ichi Kogyo Seiyaku Co., Ltd.).

The POE alkyl phenyl ether having an HLB value of 13 or less in the method for destroying exosomes of the present invention is not particularly limited as long as it is a POE alkyl phenyl ether having an HLB value of 13 or less that enables the method for destroying exosomes of the present invention.
Examples of the alkyl in the POE alkyl phenyl ether having an HLB value of 13 or less include an alkyl having 8 to 9 carbon atoms. Examples of the alkyl having 8 to 9 carbon atoms include octyl and nonyl.

Commercially available POE alkyl phenyl ethers having an HLB value of 13 or less include, for example, Nonion HS-204.5 [POE octyl phenyl ether; HLB value 9.8], Nonionic HS-206 [POE octyl phenyl ether; HLB value 11 .2], nonionic NS-202 [POE nonylphenyl ether; HLB value 5.7], nonionic NS-204.5 [POE nonylphenyl ether; HLB value 9.5], nonionic NS-208.5 [POE nonylphenyl] Ether; HLB value 12.6] (all manufactured by NOF Corporation), Brownon NK-8055 [POE octyl phenyl ether; HLB value 10.8], Brownon NK-808 [POE octyl phenyl ether; HLB value 12.5] , Brownon N-502 [POE Nonylphenyl A HLB value 5.7], Brownon N-506 [POE nonyl phenyl ether; HLB value 10.9], Brownon DT-9 [POE dodecyl phenyl ether; HLB value 12.0] (all manufactured by Aoki Yushi Kogyo Co., Ltd.) ) And the like.

As a method for adding at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkyl phenyl ether having an HLB value of 13 or less to the sample in the method of destroying exosomes of the present invention, Is not particularly limited as long as it is an addition method that enables the method of destroying exosomes of the present invention. For example, a method of directly adding the surfactant to a sample; a method of dissolving the surfactant in an aqueous medium Adding the aqueous solution of the surfactant to a sample; adding the sample to an aqueous solution of the surfactant prepared by dissolving the surfactant in an aqueous medium; A method in which an aqueous solution of the surfactant prepared by dissolving in an aqueous medium is added to a composition obtained by freeze-drying, and the like. An aqueous solution was prepared by dissolving a method of adding to the sample is preferred.
The aqueous medium is not particularly limited as long as it is an aqueous medium that enables the exosome destruction method of the present invention, and examples thereof include deionized water, distilled water, and a buffer, and a buffer is preferable. The pH of the aqueous medium is, for example, 4 to 10. When a buffer is used as the aqueous medium, it is preferable to use a buffer suitable for the set pH. The buffer used in the preparation of the buffer is not particularly limited as long as it has a buffering capacity.Examples include a lactate buffer, a citrate buffer, an acetate buffer, a succinate buffer, a phthalate buffer, Phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, carbonate buffer And a good buffer.

Examples of good buffers include 2-morpholinoethanesulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, and tris (hydroxymethyl) aminomethane (Tris) Buffer, N- (2-acetamido) iminodiacetic acid (ADA) buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES) buffer, 2- [N- (2-acetamido) Amino] ethanesulfonic acid (ACES) buffer, 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) buffer, 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) buffer Agent, 3-morpholinopropanesulfonic acid (MOPS) buffer, 2- {N- [tris (hydroxymethyl B) methyl] aminodiethanesulfonic acid (TES) buffer, N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (HEPES) buffer, 3- [N, N-bis (2- Hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO) buffer, 2-hydroxy-3-{[N-tris (hydroxymethyl) methyl] amino} propanesulfonic acid (TAPSO) buffer, piperazine-N, N'-bis (2-hydroxypropane-3-sulfonic acid) (POPSO) buffer, N- (2-hydroxyethyl) -N '-(2-hydroxy-3-sulfopropyl) piperazine (HEPPSO) buffer, N- (2-hydroxyethyl) -N ′-(3-sulfopropyl) piperazine (EPPS) buffer, [N-tris (hydroxymethyl B) Methylglycine] (Trisine) buffer, [N, N-bis (2-hydroxyethyl) glycine] (Bicine) buffer, 3- [N-tris (hydroxymethyl) methyl] aminopropanesulfonic acid (TAPS) Buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxypropanesulfonic acid (CAPSO) buffer, 3- (N-cyclohexylamino) propane Sulfonic acid (CAPS) buffer and the like.
The aqueous medium may contain salts, metal ions, sugars, preservatives, proteins and the like. Examples of the salts include lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ammonium chloride, lithium bromide, sodium bromide, potassium bromide, calcium bromide, magnesium bromide, and ammonium bromide. . Examples of the metal ion include a magnesium ion, a manganese ion, a zinc ion and the like. Examples of the saccharide include mannitol and sorbitol. Examples of preservatives include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), Bioace, Proclin 300, Proxel GXL and the like. Examples of the protein include bovine serum albumin and the like.

In the method for destroying exosomes according to the present invention, the reaction between the sample and at least one surfactant selected from the group consisting of polyether alkylamine, POE-POP alkyl ether, and POE alkyl phenyl ether having an HLB value of 13 or less. The reaction temperature is not particularly limited as long as it allows the method for disrupting exosomes of the present invention, and is usually 0 to 50 ° C, preferably 4 to 45 ° C, and particularly preferably 15 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it allows the exosome destruction method of the present invention, and is usually 10 seconds to 24 hours, preferably 30 seconds to 3 hours, and preferably 1 minute to 2 hours. Is particularly preferred.

In the method for destroying exosomes of the present invention, at least one surfactant selected from the group consisting of polyether alkylamine, POE-POP alkyl ether, and POE alkyl phenyl ether having an HLB value of 13 or less was added to a sample. The subsequent concentration is not particularly limited as long as the concentration allows the method of destroying exosomes of the present invention, and is usually 0.001 to 10% (w / v), and 0.01 to 5% (w / v). ) Is preferred, and 0.04 to 1% (w / v) is particularly preferred.

2. Exosome-destroying reagent The exosome-destroying reagent in the sample of the present invention is a reagent used for the method of destructing exosomes in the sample of the present invention, and includes an exosome-disrupting agent in a sample, a exosome-destroying composition in a sample, and a sample. It can also be expressed as a reagent for lysing exosomes in a sample, an agent for lysing exosomes in a sample, and a composition for lysing exosomes in a sample. Examples of the sample containing exosomes to be destroyed using the disrupting reagent of the present invention include the above-described samples.
The polyether alkylamine, POE-POP alkyl ether, and POE alkylphenyl ether having an HLB value of 13 or less in the exosome-destroying reagent of the present invention include, for example, the aforementioned polyether alkylamine, POE-POP alkyl ether, And POE alkyl phenyl ethers having an HLB value of 13 or less, respectively.

エ The exosome-destroying reagent of the present invention may be in a liquid state or a lyophilized state. In the case of a liquid breaking reagent, at least one surfactant selected from the group consisting of polyether alkylamines, POE-POP alkyl ethers, and POE alkyl phenyl ethers having an HLB value of 13 or less is prepared in advance by using the aforementioned aqueous solution. It is dissolved in the medium. In the case of a lyophilized breaking reagent, at least one surfactant selected from the group consisting of polyether alkylamines, POE-POP alkyl ethers, and POE alkyl phenyl ethers having an HLB value of 13 or less is the aforementioned aqueous solution. It may be used by being dissolved in a medium or by directly contacting the sample. The breaking reagent of the present invention may contain the above-mentioned salts, metal ions, saccharides, preservatives, proteins and the like.

The content of at least one surfactant selected from the group consisting of polyether alkylamines, POE-POP alkyl ethers, and POE alkyl phenyl ethers having an HLB value of 13 or less in the exosome-destroying reagent of the present invention is as follows. There is no particular limitation as long as the content allows the destruction method, and the concentration after mixing with the sample is usually 0.001 to 10% (w / v), and 0.01 to 5%. (W / v) is preferred, and a content of 0.04 to 1% (w / v) is particularly preferred.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
<Material>
Superdex200HR 10/30 (GE Healthcare), disodium hydrogen phosphate (phosphate buffer; manufactured by Kanto Kagaku), sodium dihydrogen phosphate (phosphate buffer; manufactured by Kanto Kagaku), sodium chloride (sum Sodium azide (manufactured by Wako Pure Chemical Industries), BCA protein assay kit (manufactured by Thermo Fisher Scientific), MES (manufactured by Dojindo Laboratories), bovine serum albumin (BSA; Oriental) Yeast Industry Co., Ltd.), streptavidin-bound magnetic particles (Dynabeads MyOne Streptavidin C1; manufactured by Thermo Fisher Scientific), Biotin Labeling Kit-NH ₂ (manufactured by Dojindo Laboratories), anti-CD9 monoclonal antibody (clone A100-4) ; Medicine Manufactured goods Institute Inc.), MES (Dojin Chemical Laboratories), manufactured by Alkaline Phosphatase Labeling Kit-NH ₂ (Dojin Chemical Laboratories), MOPS (manufactured by Dojindo Laboratories), magnesium chloride hexahydrate (Manufactured by Wako Pure Chemical Industries, Ltd.), Tween 20 (polyoxyethylene sorbitan monolaurate; manufactured by Dojindo Laboratories, manufactured by Wako Pure Chemical Industries, Ltd.), 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10 -Methylacridan disodium salt (Lumigen ^™ APS-5; manufactured by Oriental Yeast Co., Ltd.), Nimeen L-202 [POE laurylamine (polyetheralkylamine); manufactured by NOF CORPORATION], Nimeen DT-203 [POE alkyl Propylenediamine (polyether alkylamine); NOF Corporation ], Wandasurf ID-50 [POE-POP isodecyl ether (POE-POP alkyl ether); manufactured by Aoki Yushi Kogyo Co., Ltd.], Nonion NS-204.5 [POE nonylphenyl ether (POE alkylphenyl having an HLB value of 13 or less) Nonyl HS-204.5 [POE octyl phenyl ether (POE alkyl phenyl ether having an HLB value of 13 or less); NOF]; Triton X-100 [POE octyl phenyl ether (HLB) Value 13.5); manufactured by Sigma-Aldrich], Nonidet P-40 [POE nonylphenyl ether (HLB value 13.1); manufactured by Sigma-Aldrich].

[Reference Example 1]
<Identification of exosome-containing fraction obtained when serum is subjected to gel filtration chromatography>
(1) Preparation of Serum Whole blood collected from healthy individuals belonging to Kyowa Medex Co., Ltd. was centrifuged at 2,000 rpm and 25 ° C. for 20 minutes to prepare serum.
(2) Separation of Serum Components by Gel Filtration Chromatography The serum (500 μL) prepared in the above (1) was applied to Superdex200HR 10/30, which is a column for gel filtration chromatography, and then sodium azide 0 was used as an eluate. 0.2 g / L of PBS [phosphate-buffered saline (10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2)] was flowed at a flow rate of 0.5 mL / min. As the elution fraction, fraction No. 1-80 (0.5 mL each) were obtained.
(3) Measurement of each fraction obtained in the above (2) using a BCA protein assay kit The fractions 1 to 80 were reacted using a BCA protein assay kit, which is a kit for measuring the amount of protein, according to the instruction manual for the kit, and the absorbance (560 nm) was measured. The result is shown in FIG.

(4) Preparation of Reagent for Measurement Used in Sandwich Method Using Antigen-Antibody Reaction A streptavidin-conjugated magnetic particle solution, a biotin-conjugated anti-CD9 monoclonal antibody solution, and an ALP (alkaline phosphatase) -labeled anti-CD9 monoclonal antibody solution were prepared.
-Streptavidin-binding magnetic particle solution A streptavidin-binding magnetic particle solution having the following composition was prepared using commercially available streptavidin-binding magnetic particles (Dynabeads MyOne Streptavidin C1).
MES (pH 6.5) 0.05 mol / L
BSA 0.1% (w / v)
Sodium chloride 0.1mol / L
Streptavidin-bound magnetic particles 0.225mg / mL
-Using a biotin-conjugated anti-CD9 monoclonal antibody solution Biotin Labeling Kit-NH ₂ (manufactured by Dojindo Laboratories) and following the instructions for use of the kit, an anti-CD9 monoclonal antibody (clone A100-4; Medical Biology Laboratories) Was labeled with biotin to prepare a biotin-conjugated anti-CD9 monoclonal antibody. Using the obtained biotin-conjugated anti-CD9 monoclonal antibody, a biotin-conjugated anti-CD9 monoclonal antibody solution having the following composition was prepared.
MES (pH 6.5) 0.05 mol / L
Biotin-conjugated anti-CD9 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1mol / L
· ALP using a labeled anti-CD9 monoclonal antibody solution Alkaline Phosphatase Labeling Kit-NH ₂ (manufactured by Dojin Chemical Laboratory Co., Ltd.), according to the instruction manual of the kit, the anti-CD9 monoclonal antibodies (clone A100-4; Medical & Biological Laboratories Was labeled with ALP to prepare an ALP-labeled anti-CD9 monoclonal antibody. Using the obtained ALP-labeled anti-CD9 monoclonal antibody, an ALP-labeled anti-CD9 monoclonal antibody solution having the following composition was prepared.
MES (pH 6.5) 0.05 mol / L
ALP-labeled anti-CD9 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1mol / L

(5) Measurement of each fraction obtained in the above (2) by a sandwich method using an antigen-antibody reaction. For 1 to 80, each fraction (10 μL) was added to the streptavidin-bound magnetic particle solution (30 μL) prepared in the above (4), the biotin-bound anti-CD9 monoclonal antibody solution (30 μL) prepared in the above (4), and The ALP-labeled anti-CD9 monoclonal antibody solution (30 μL) prepared in the above (4) was added, stirred, and reacted at 37 ° C. for 10 minutes. Next, the magnetic particles were collected by magnetic force, the reaction solution other than the magnetic particles was removed, and the collected magnetic particles were washed five times with the following washing liquid.
・ Washing liquid MOPS (pH 7.3) 0.005mol / L
Sodium chloride 0.3mol / L
Magnesium chloride hexahydrate 0.2 mmol / L
Tween 20 0.075% (w / v)
Thereafter, the collected and washed magnetic particles were subjected to luminescence mainly composed of 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium salt (Lumigen ^™ APS-5). 100 μL of the substrate solution was added and stirred, and the generated luminescence (RLU) was measured. The result is shown in FIG.

(6) Identification of Fraction Containing Exosome As is clear from FIG. 18 and fractions before and after 18 showed a large measurement signal by the sandwich method using an anti-CD9 monoclonal antibody. Therefore, the fraction No. It was found that exosome having CD9 on its surface was contained in 18 and the fractions before and after it.
In serum, monomolecule CD9 may be present in addition to exosomes having CD9 on its surface. However, the sandwich method (5) uses a sandwich method using an anti-CD9 monoclonal antibody as a primary antibody and an ALP-labeled anti-CD9 monoclonal antibody as a labeled secondary antibody, that is, a primary antibody and a secondary antibody. In this method, a single molecule of CD9 cannot be sandwiched because both antibodies use an antibody that recognizes the same epitope. Therefore, the fact that a signal is obtained by the sandwich method in FIG. 1 means that only the exosome having CD9 on its surface is detected, and the single molecule CD9 itself is not detected.

In addition, as is apparent from FIG. In Nos. 20 to 40, since a large measurement signal was obtained by measurement using the BCA protein assay kit, fraction Nos. It was found that 20 to 40 contained a large amount of protein. These fractions were subjected to SDS-PAGE. As a result, these fractions contained large amounts of proteins corresponding to the molecular weights of albumin and gamma globulin (principal molecular weights of about 66 kDa and 150 kDa, respectively) in serum. Was found to be contained.
Here, in gel filtration chromatography, a molecule having a larger molecular weight elutes faster. However, as is evident from FIG. 1, fraction No. 1 showing a signal derived from CD9 (molecular weight: about 24 kDa). The fraction around 18 elutes faster than albumin (molecular weight about 66 kDa) and gamma globulin (molecular weight about 150 kDa). Therefore, the fraction No. showing a signal attributable to CD9. It was found that the fraction around 18 contained exosomes having CD9 on its surface instead of single molecule CD9.

<Preparation of reagent for exosome disruption>
[Example 1]
Each of the five surfactants shown in Table 3 below was added to PBS so as to have a concentration of 10% (w / v) to prepare exosome disrupting reagents A1 to A5.
[Comparative Example 1]
Two surfactants shown in Table 3 below, namely, Triton X-100 and Nonidet P-40 surfactants were added to PBS so as to be 10% (w / v), and the surfactants were used for exosome disruption. Reagents B1 and B2 were prepared, respectively.
[Comparative Example 2]
PBS was used as a control reagent B0.

[Example 2]
<Destruction of exosomes using exosome destruction reagent containing polyetheralkylamine>
(1) Preparation of Serum and Treatment of Serum with Surfactant Whole blood collected from a healthy person belonging to Kyowa Medex Co., Ltd., which is different from the healthy person in Reference Example 1, was subjected to 2,000 rpm at 25 ° C. for 20 minutes. After centrifugation, serum was prepared.
To the prepared serum (900 μL), the exosome-disrupting reagent A1 (100 μL) prepared in Example 1 was added, and the mixture was allowed to stand for 10 minutes. Similarly, the exosome-disrupting reagent A2 (100 μL) prepared in Example 1 was added to the prepared serum (900 μL), and the mixture was allowed to stand for 10 minutes.
(2) Separation Operation of Serum Components by Gel Filtration Chromatography Serum components prepared and surfactant-treated in (1) above using Superdex200HR 10/30 (manufactured by GE Healthcare) which is a column for gel filtration chromatography. Was performed. First, 500 μL of the serum treated with the surfactant was applied to the column, and then PBS [phosphate-buffered saline (0.15 mol / L chloride) containing 0.2 g / L of sodium azide was used as an eluent. Sodium-containing 10 mmol / L phosphate buffer, pH 7.2)] at a flow rate of 0.5 mL / min. 1-80 (0.5 mL each) were obtained.

(3) Preparation of measurement reagent used for sandwich method using antigen-antibody reaction Streptavidin-conjugated magnetic particle solution, biotin-conjugated anti-CD9 monoclonal antibody solution, and ALP labeling were performed in the same manner as in (4) of Reference Example 1 above. An anti-CD9 monoclonal antibody solution was prepared.
(4) Measurement of each fraction obtained in the above (2) by a sandwich method using an antigen-antibody reaction. Among the fraction Nos. 1 to 80, For 15 to 25, 30 μL of the streptavidin-conjugated magnetic particle solution prepared in (3) above, 30 μL of the biotin-conjugated anti-CD9 monoclonal antibody solution prepared in (3) above, and 10 μL of each fraction prepared in (3) above 30 μL of the prepared ALP-labeled anti-CD9 monoclonal antibody solution was added, stirred, and reacted at 37 ° C. for 10 minutes. Next, the magnetic particles were collected by magnetic force, the reaction solution other than the magnetic particles was removed, and the collected magnetic particles were washed five times with the washing solution used in (5) of Reference Example 1 described above. Thereafter, the collected and washed magnetic particles were subjected to luminescence mainly composed of 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium salt (Lumigen ^™ APS-5). 100 μL of the substrate solution was added and stirred, and the generated luminescence (RLU) was measured. The result is shown in FIG.

[Comparative Example 3]
(1) Preparation of Serum and Treatment of Serum with Control Reagent B0 Serum (900 μL) prepared in (1) of Example 2 above was added to control reagent B0 (100 μL) prepared in Comparative Example 2 above. Was added and allowed to stand for 10 minutes.
(2) Measurement by sandwich method using gel filtration chromatography-antigen-antibody reaction Except for using the serum obtained in (1) above as a sample for gel filtration chromatography, (2) to (4) of Example 2 described above. ), Separation of serum components by gel filtration chromatography, and fraction No. eluted by gel filtration chromatography. 15 to 25 were measured by a sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Example 3]
The fraction No. obtained in (2) of Comparative Example 3 above. Assuming that the luminescence amount with respect to No. 18 was 100, the fraction No. obtained from the serum treated with the exosome disrupting reagent A1 in (4) of Example 2 described above. The light emission amount relative to 18 was calculated. Similarly, the fraction No. obtained from the serum treated with the exosome disrupting reagent A2 obtained in (4) of Example 2 above. The light emission amount relative to 18 was calculated. Table 4 shows the results.

As is clear from FIG. 2 and Table 4, when the serum was treated for 10 minutes with the exosome disrupting reagent containing Nimeen L-202 or Nimeen DT-203, the exosome-containing fraction (fraction No. 18) was removed. The luminescence amount is remarkably reduced as compared with the case where the serum was treated with the control reagent B0. Exosome is destroyed in a short time by adding Niamin L-202 or Niamin DT-203 to the serum. It has been found. Therefore, it was revealed that the addition of polyetheralkylamine to serum can easily and quickly destroy exosomes in serum without isolating exosomes in serum.

[Example 4]
<Destruction of exosomes using exosome destruction reagent containing POE-POP alkyl ether>
(1) Preparation of Serum and Treatment of Serum with Surfactant Whole blood collected from a healthy person belonging to Kyowa Medex Co., Ltd., which is different from the healthy person in Reference Example 1 and Example 2, was subjected to 2,000 rpm, 25 Centrifugation was performed at 20 ° C. for 20 minutes to prepare serum.
To the prepared serum (900 μL), the exosome-disrupting reagent A3 (100 μL) prepared in Example 1 was added, and the mixture was allowed to stand for 10 minutes.
(2) Measurement by sandwich method using gel filtration chromatography-antigen-antibody reaction Except for using the serum obtained in (1) above as a sample for gel filtration chromatography, (2) to (4) of Example 2 described above. ), The exosomes are separated from the other components by gel filtration chromatography, and the fraction No. 1 obtained by the gel filtration chromatography is separated. Exosomes in 15 to 25 were measured by a sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Comparative Example 4]
(1) Preparation of Serum and Treatment of Serum with Control Reagent B0 Serum (900 μL) prepared in (1) of Example 4 above was added to control reagent B0 (100 μL) prepared in Comparative Example 2 above. Was added and allowed to stand for 10 minutes.
(2) Measurement by sandwich method using gel filtration chromatography-antigen-antibody reaction Except for using the serum obtained in (1) above as a sample for gel filtration chromatography, (2) to (4) of Example 2 described above. ), The exosomes are separated from the other components by gel filtration chromatography, and the fraction No. 1 obtained by the gel filtration chromatography is separated. Exosomes in 15 to 25 were measured by a sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Example 5]
The fraction No. obtained in (2) of Comparative Example 4 above. Assuming that the luminescence amount with respect to No. 18 was 100, the fraction No. obtained from the serum treated with the exosome disrupting reagent A3 in (2) of Example 4 described above. The light emission amount relative to 18 was calculated. Table 5 shows the results.

As is clear from FIG. 3 and Table 5, when the serum was treated with the exosome disrupting reagent containing Wandasurf ID-50 for 10 minutes, the luminescence amount for the exosome-containing fraction (fraction No. 18) was: Compared with the case where the serum was treated with the control reagent B0, the exosome was significantly reduced, and it was found that the addition of Wandasurf ID-50 to the serum destroyed the exosome in a short time. Therefore, it was revealed that the addition of POE-POP alkyl ether to serum can easily and quickly destroy exosomes in serum without isolating exosomes in serum.

[Example 6]
<Destruction of exosomes using exosome destruction reagent containing POE alkyl phenyl ether having HLB value of 13 or less>
(1) Preparation of Serum and Treatment of Serum with Surfactant Whole blood collected from a healthy person belonging to Kyowa Medex Co., Ltd., which is different from the healthy person in Reference Example 1, Example 2 and Example 4, was collected. The mixture was centrifuged at 2,000 rpm at 25 ° C. for 20 minutes to prepare a serum.
To the prepared serum (900 μL), the exosome-disrupting reagent A4 (100 μL) prepared in Example 1 was added, and the mixture was allowed to stand for 10 minutes. Similarly, to the prepared serum (900 μL), the exosome-disrupting reagent A5 (100 μL) prepared in Example 1 described above was added and left to stand for 10 minutes.
(2) Measurement by sandwich method using gel filtration chromatography-antigen-antibody reaction Except for using the serum obtained in (1) above as a sample for gel filtration chromatography, (2) to (4) of Example 2 described above. ), The exosomes are separated from the other components by gel filtration chromatography, and the fraction No. 1 obtained by the gel filtration chromatography is separated. Exosomes in 15 to 25 were measured by a sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Comparative Example 5]
<Destruction of exosomes using exosome destruction reagent containing POE alkyl phenyl ether having HLB value of more than 13>
(1) Preparation of Serum and Treatment of Serum with Surfactant Serum (900 μL) prepared in (1) of Example 6 was added to exosome disrupting reagent B1 (100 μL) prepared in Comparative Example 1 above. ) Was added and allowed to stand for 10 minutes. Similarly, the exosome-disrupting reagent B2 (100 μL) prepared in Comparative Example 1 was added to the serum (900 μL) prepared in (1) of Example 6 described above, and allowed to stand for 10 minutes.
(2) Measurement by sandwich method using gel filtration chromatography-antigen-antibody reaction Except for using the serum obtained in (1) above as a sample for gel filtration chromatography, (2) to (4) of Example 2 described above. ), The exosomes are separated from the other components by gel filtration chromatography, and the fraction No. 1 obtained by the gel filtration chromatography is separated. Exosomes in 15 to 25 were measured by a sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Comparative Example 6]
(1) Preparation of Serum and Treatment of Serum with Control Reagent B0 Serum (900 μL) prepared in (1) of Example 6 was added to control reagent B0 (100 μL) prepared in Comparative Example 2 above. Was added and allowed to stand for 10 minutes.
(2) Measurement by sandwich method using gel filtration chromatography-antigen-antibody reaction Except for using the serum obtained in (1) above as a sample for gel filtration chromatography, (2) to (4) of Example 2 described above. ), The exosomes are separated from the other components by gel filtration chromatography, and the fraction No. 1 obtained by the gel filtration chromatography is separated. Exosomes in 15 to 25 were measured by a sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Example 7]
The fraction No. obtained in (2) of Comparative Example 6 above. Assuming that the amount of luminescence with respect to No. 18 was 100, the fraction No. obtained from the serum treated with the exosome disrupting reagent A4 in (2) of Example 6 described above. The light emission amount relative to 18 was calculated. Similarly, the fraction No. obtained from the serum treated with the exosome disrupting reagent A5 obtained in (2) of Example 6 above. The light emission amount relative to 18 was calculated. Table 6 shows the results.
In addition, the fraction No. obtained in (2) of Comparative Example 6 above. Assuming that the luminescence amount with respect to No. 18 was 100, the fraction No. obtained from the serum treated with the exosome-destroying reagent B1 obtained in (2) of Comparative Example 5 above was used. The light emission amount relative to 18 was calculated. Similarly, the fraction No. obtained from the serum treated with the exosome disrupting reagent B2 obtained in (2) of Comparative Example 5 above. The light emission amount relative to 18 was calculated. Table 6 shows the results.

As is clear from FIG. 4 and Table 6, when the serum was treated for 10 minutes with the exosome disrupting reagent containing Nonion NS-204.5 or Nonion HS-204.5, the fraction containing exosomes (fraction No. .18) was significantly reduced as compared to the case where the serum was treated with the control reagent B0, and the addition of Nonion NS-204.5 or Nonion HS-204.5 to the serum resulted in Exosomes were found to be destroyed in a short time. Therefore, it was revealed that by adding POE alkylphenyl ether having an HLB value of 13 or less to serum, exosomes in serum can be easily and quickly destroyed without isolating exosomes in serum.
As is clear from FIG. 4 and Table 6, when the serum was treated for 10 minutes with the exosome disrupting reagent containing Nonion NS-204.5 or Nonion HS-204.5, the fraction containing the exosome (fraction The luminescence amount for the image No. 18) was higher than that in the case where the serum was treated with an exosome-disrupting reagent containing Triton X-100 or Nonidet P-40, which is a POE alkylphenyl ether having an HLB value of more than 13. Notably low. Therefore, compared to the addition of Triton X-100 or Nonidet P-40, which are POE alkyl phenyl ethers having an HLB value of more than 13, to Nonion NS-204.5 or Nonion HS-204.5 to serum. It was found that the addition significantly destroyed the exosome. Therefore, it was revealed that by adding POE alkylphenyl ether having an HLB value of 13 or less to serum, exosomes in serum can be easily and quickly destroyed without isolating exosomes in serum.

Example 8
<Sample difference: serum and plasma>
(1) Preparation of Serum and Treatment of Serum Whole blood collected from a healthy person 1 belonging to Kyowa Medex Co., which is different from a healthy person in Reference Example 1, Example 2, Example 4 and Example 6, Serum 1 was prepared by centrifugation at 2,000 rpm for 20 minutes at 25 ° C.
To the prepared serum 1 (900 μL), the exosome-disrupting reagent A4 (100 μL) prepared in Example 1 was added, and the mixture was allowed to stand for 10 minutes to obtain serum 1 _A4 .
Further, to the prepared serum 1 (900 μL), the control reagent B0 (100 μL) prepared in Comparative Example 2 was added and allowed to stand for 10 minutes to obtain serum 1 _B0 .
(2) Preparation of a measuring reagent used in a sandwich method using an antigen-antibody reaction A streptavidin-conjugated magnetic particle solution, a biotin-conjugated anti-CD9 monoclonal antibody solution, and an ALP label were prepared in the same manner as in (4) of Reference Example 1 above. An anti-CD9 monoclonal antibody solution was prepared.
(3) Measurement by sandwich method using antigen-antibody reaction To each serum (10 μL) of serum 1 _A4 and serum 1 _B0 prepared in (1) above, a solution of streptavidin-bound magnetic particles (30 μL) prepared in (2) above Then, the biotin-conjugated anti-CD9 monoclonal antibody solution (30 μL) prepared in (2) and the ALP-labeled anti-CD9 monoclonal antibody solution (30 μL) prepared in (2) are added, stirred, and reacted at 37 ° C. for 10 minutes. I let it. Next, the magnetic particles were collected by magnetic force, the reaction solution other than the magnetic particles was removed, and the collected magnetic particles were washed five times with the washing solution used in (5) of Reference Example 1 described above. Thereafter, the collected and washed magnetic particles were subjected to luminescence mainly composed of 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium salt (Lumigen ^™ APS-5). the substrate solution 100μL was added and stirred, the light emission quantity _{1 A4} to serum _{1 A4,} and were measured an emission amount _{1 B0} to serum _{1 B0.}

(4) Measurement of blank luminescence in sandwich method and calculation of S / N ratio Blank luminescence was measured by the same measurement as in (3) above, except that physiological saline was used as a sample.
Next, the S / N ratio (signal / noise ratio) was calculated from the blank light emission amount, the light emission amount 1 _A4 and the light emission amount 1 _B0 obtained in the above (3) by the following equation.

S / N ratio 1 _A4 = light emission amount 1 _A4 / blank light emission amount S / N ratio 1 _B0 = light emission amount 1 _B0 / blank light emission amount

(5) Calculation of S / N ratio residual ratio With the S / N ratio 1 _B0 obtained in the above (4) being 100, the relative value of the S / N ratio 1 _A4 obtained in the above (4) is calculated. The residual ratio of S / N was set to 1. Table 7 shows the results.
Here, the closer the S / N ratio residual rate is to 100, the more the exosome is not destroyed. That is, the closer the S / N ratio residual ratio is to 0, the more the exosome is destroyed.
(6) Calculation of residual ratio of S / N ratio in plasma 1 and serum 2 and plasma 2 Except for using EDTA plasma 1 obtained from healthy person 1 in (1) above as a sample, The S / N ratio residual ratio 1 ′ was calculated in the same manner as in 5). Table 7 shows the results.
In addition, except that serum 2 or EDTA plasma 2 obtained from a healthy person 2 different from the healthy person 1 in the above (1) is used as a sample, S / S is obtained in the same manner as in the above (1) to (5). The N ratio residual ratio 2 and the S / N ratio residual ratio 2 ′ were calculated respectively. Table 7 shows the results.

As is clear from Table 7, it was found that the addition of nonionic NS-204.5 destroyed the exosomes in both the serum and plasma samples obtained from the healthy subject 1 in a short time. did. In addition, it was found that the addition of Nonion NS-204.5 destroyed the exosomes in the serum and plasma samples obtained from the healthy subject 2 in a short time. Therefore, it has been clarified that the exosome in the sample can be easily and quickly destroyed by the exosome-destroying reagent of the present invention in any of serum and plasma samples regardless of the difference between healthy individuals.

[Example 9]
<Preparation of reagent for exosome disruption>
Nonionic NS-204.5 was added to PBS so as to have the concentration shown in Table 8 below, to prepare exosome disrupting reagents A6 to A8, respectively.

[Example 10]
<Destruction of exosomes in serum by exosome disrupting reagents A6 to A8>
(1) Preparation of Serum and Treatment of Serum All samples collected from healthy persons belonging to Kyowa Medex Co., Ltd., which are different from the healthy persons in Reference Example 1, Example 2, Example 4, Example 6, and Example 8 Blood was centrifuged at 2,000 rpm at 25 ° C. for 20 minutes to prepare serum.
To each of the prepared serum (900 μL), each of the exosome disrupting reagents A6 to A8 (100 μL) prepared in Example 9 described above was added and allowed to stand for 10 minutes to obtain serum _A6 , serum _A7 and serum _A8 , respectively. Was.
Further, the control reagent B0 (100 μL) prepared in Comparative Example 2 was added to the prepared serum (900 μL), and the _mixture was allowed to stand for 10 minutes to obtain serum _B0 .
(2) Preparation of a measuring reagent used in a sandwich method using an antigen-antibody reaction A streptavidin-conjugated magnetic particle solution, a biotin-conjugated anti-CD9 monoclonal antibody solution, and an ALP label were prepared in the same manner as in (4) of Reference Example 1 above. An anti-CD9 monoclonal antibody solution was prepared.
(3) Measurement by Sandwich Method Using Antigen-Antibody Reaction To 10 μL of serum _A6 , serum _A7 , serum _A8 and serum _B0 prepared in (1) above, the streptavidin-bound magnetic particle solution (30 μL) prepared in (2) above, The biotin-conjugated anti-CD9 monoclonal antibody solution (30 μL) prepared in (2) and the ALP-labeled anti-CD9 monoclonal antibody solution (30 μL) prepared in (2) are added, and the mixture is stirred and reacted at 37 ° C. for 10 minutes. Was. Next, the magnetic particles were collected by magnetic force, the reaction solution other than the magnetic particles was removed, and the collected magnetic particles were washed five times with the washing solution used in (5) of Reference Example 1 described above. Thereafter, the collected and washed magnetic particles were subjected to luminescence mainly composed of 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium salt (Lumigen ^™ APS-5). The substrate solution (100 μL) was added and stirred, and the luminescence _A6 , luminescence _A7 , luminescence _A8 and luminescence _B0 were measured respectively.

(4) Measurement of blank luminescence in sandwich method and calculation of S / N ratio Blank luminescence was measured by the same measurement as in (3) above, except that physiological saline was used as a sample.
Next, the S / N ratio was calculated from the blank light emission amount and each of the light emission amounts _A6 , _A7 , _A8 and _B0 obtained in the above (3) by the following equation.

S / N ratio _A6 = light emission amount _A6 / blank light emission amount S / N ratio _A7 = light emission amount _A7 / blank light emission amount S / N ratio _A8 = light emission amount _A8 / blank light emission amount S / N ratio _B0 = light emission amount _B0 / blank Light output

(5) Calculation of S / N ratio residual ratio When the S / N ratio _B0 obtained in (4) above is set to 100, the S / N ratio _A6 and S / N obtained in (4) above are obtained. The relative values of the ratio _A7 and the S / N ratio _A8 were calculated, and were defined as the S / N ratio residual ratio _A6 , the S / N ratio residual ratio _A7, and the S / N ratio residual ratio _A8 , respectively. Table 9 shows the results. Here, the closer the S / N ratio residual rate is to 100, the more the exosome is not destroyed. That is, the closer the S / N ratio residual ratio is to 0, the more the exosome is destroyed.

As is clear from Table 9, in a concentration range of 0.04 to 1% of nonionic NS-204.5 after mixing with serum, the S / N ratio remaining rate was less than 50%, and exosomes in serum Turned out to be destroyed. That is, it was found that a wide range of concentrations of nonion NS-204.5 was effective for destroying exosomes in serum. Therefore, it was revealed that exosomes in serum can be easily and quickly destroyed by using the reagent for disrupting exosomes of the present invention.

According to the present invention, a method and a reagent for destruction of exosomes in a sample which are simple and quick are provided.

Claims (4)

1. The sample is characterized by adding at least one surfactant selected from the group consisting of polyether alkylamine, polyoxyethylene polyoxypropylene alkyl ether, and polyoxyethylene alkyl phenyl ether having an HLB value of 13 or less. To destroy exosomes in a sample.
2. The method according to claim 1, wherein the sample is serum or plasma.
3. A sample comprising at least one surfactant selected from the group consisting of polyether alkylamines, polyoxyethylene polyoxypropylene alkyl ethers, and polyoxyethylene alkyl phenyl ethers having an HLB value of 13 or less. Exosome destruction reagents inside.
4. The reagent according to claim 3, wherein the sample is serum or plasma.

Images