JPWO2020004436A1 - Method of destroying exosomes in a sample and reagents for destruction - Google Patents

Abstract

The sample is characterized by adding 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. Method of destroying exosomes in a sample; 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. A reagent for destroying exosomes in a sample, which comprises.

Description

The present invention relates to a method for destroying an exosome in a sample and a reagent for destroying the exosome.

Exosomes are vesicle granules secreted by animal cells and having a lipid bilayer structure with a diameter of 30 to 200 nm. It is known that various membrane proteins are present on the surface of exosomes as well as on the surface of general cells, and the inside of exosomes may contain microRNA (miRNA) in addition to various proteins such as cytokines. It is known (Non-Patent Document 1). In addition, it has been reported that exosomes are secreted from various cells, such as cells of the immune system and various cancer cells, and they function as mediators of cell-cell communication in the living body and are associated with physiological phenomena. Attention is being paid to its association with diseases such as cancer.

In order to analyze the substances contained inside the exosome, it is necessary to destroy the lipid bilayer structure of the exosome. So far, as a method for destroying exosomes, a method in which cell culture supernatant, human serum, etc. are ultracentrifuged (110,000 G for 70 minutes) to isolate exosomes, and the isolated exosomes are destroyed by an antibacterial peptide (Patent). Document 1), the cell culture supernatant is ultracentrifuged (120,000 G for 100 minutes) to isolate exosomes, and the isolated exosomes are triton X-100 (polyoxyethylene octyl) which is a nonionic surfactant. A method of destroying by phenyl ether) (Non-Patent Document 2) and the like are known.

However, these methods are complicated to operate and require time because they require isolation of exosomes by ultracentrifugation or the like as a pretreatment for exosome destruction. On the other hand, in applying the analysis of substances contained inside exosomes to the field of clinical examination, a simple and rapid method for destroying exosomes is desired.

Further, as a method for separating exosomes, a complex in which an exosome is brought into contact with a solid phase carrier to which a ligand that recognizes a surface antigen existing on the surface of the exosome is bound to form a complex of the exosome and the solid phase carrier. A body forming step and a washing step of washing the complex are included, and at least one of the complex forming step and the washing step is carried out 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. 2016/143904 International Publication No. 2015/068772

Int. J. Biol. Sci. 2013, 9; 10: 1021-31 Sci. Rep. 6, 18346; doi: 10.1038 / srep18346 (2016)

An object of the present invention is to provide a simple and rapid method for destroying exosomes in a sample and a reagent for destroying the exosome.

As a result of diligent studies to solve the above problems, the present inventors have prepared samples such as polyether alkylamine, polyoxyethylene polyoxypropylene alkyl ether (hereinafter referred to as POE-POP alkyl ether), and HLB (Hydrophile). -By adding at least one surfactant selected from the group consisting of polyoxyethylene alkyl phenyl ethers (hereinafter referred to as POE alkyl phenyl ethers) having a Lipophile Balance) value of 13 or less, exosomes can be rapidly destroyed. Find out and complete the present invention.

That is, the present invention relates to the following [1] to [4].
[1] The sample is characterized by adding 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. How to destroy exosomes in a sample.
[2] The method according to [1], wherein the sample is serum or plasma.
[3] A sample containing at least one surfactant selected from the group consisting of a polyether alkylamine; a POE-POP alkyl ether; and a POE alkylphenyl ether having an HLB value of 13 or less. Exome-destroying reagent.
[4] The reagent according to [3], wherein the sample is serum or plasma.

INDUSTRIAL APPLICABILITY The present invention provides a simple and rapid method for destroying exosomes in a sample and a reagent for destroying the exosome.

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 amount (RLU) obtained by the 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 light emitted 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 represented by □. 3 is a gel filtration chromatogram showing the results of an exosome destruction test in serum using a reagent for destroying exosomes containing a polyether alkylamine and a reagent for control. The horizontal axis represents the elution fraction. The vertical axis represents the luminescence amount (RLU) obtained by the 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 an exosome-destroying reagent containing nymine L-202 (polyether alkylamine) is used. ▲ represents the amount of luminescence in each fraction when an exosome-destroying reagent containing nymine DT-203 (polyether alkylamine) is used. □ represents the amount of luminescence in each fraction when the control reagent is used. 3 is a gel filtration chromatogram showing the results of an exosome destruction test using a reagent for destroying exosomes containing POE-POP alkyl ether and a reagent for control. The horizontal axis represents the elution fraction. The vertical axis represents the luminescence amount (RLU) obtained by the 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 an exosome-destroying reagent containing Wandasurf ID-50 (POE-POP alkyl ether) is used. □ represents the amount of luminescence in each fraction when the control reagent is used. 3 is a gel filtration chromatogram showing the results of an exosome destruction test using an exosome destruction reagent containing a POE alkyl phenyl ether having an HLB value of 13 or less, a comparative exosome destruction reagent, and a control reagent. The horizontal axis represents the elution fraction. The vertical axis represents the luminescence amount (RLU) obtained by the 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 an exosome-destroying reagent containing nonion NS-204.5 (POE alkyl phenyl ether having an HLB value of 13 or less; HLB value of 9.5) is used. ▲ represents the amount of luminescence in each fraction when an exosome-destroying reagent containing nonionic HS-204.5 (POE alkyl phenyl ether having an HLB value of 13 or less; HLB value of 9.8) is used. ◯ represents the amount of luminescence in each fraction when an exosome-destroying reagent containing Triton X-100 (POE alkyl phenyl ether; HLB value 13.5) is used. Δ represents the amount of luminescence in each fraction when an exosome-destroying reagent containing nonidet P-40 (POE alkyl phenyl ether; HLB value 13.1) was used. □ represents the amount of luminescence in each fraction when the control reagent is used.

1. 1. Method of destroying exosomes In the method of destroying exosomes in the sample of the present invention, the exosomes in the sample are selected from the group consisting of polyether alkylamines, POE-POP alkyl ethers, and POE alkylphenyl ethers having an HLB value of 13 or less. It is a method of destroying with at least one kind of surfactant. Here, "destroying an exosome" means that the shape of vesicle particles having a lipid bilayer structure peculiar to an exosome is lost, and the substance contained inside the exosome is destroyed to the extent that it is eluted to the outside of the exosome. means. As used herein, exosome disruption may also be referred to as exosome membrane disruption, exosome lysis, and exosome membrane lysis.

The method for confirming that the exosome has been destroyed by the method for destroying the exosome of the present invention is not particularly limited as long as the method can confirm the destruction of the exosome. Examples thereof include a method of measuring a specific antigen by an immunoassay method. In the method using an electron microscope, the destruction of exosomes can be confirmed by confirming the disappearance of the shape of the vesicle particles having a lipid bilayer structure peculiar to exosomes on an electron microscope image. In addition, when the destruction of exosomes is confirmed by measuring the exosome-specific antigens on the surface of the exosomes by an immunological measurement method, the exosomes of the present invention are compared with those before the method for destroying exosomes of the present invention. After performing the destruction method, the destruction of exosomes can be confirmed by confirming the decrease in the measurement signal caused by the exosome-specific antigen, that is, the decrease in the measurement signal caused by the exosome. The criterion for confirming the destruction of the exosome can be appropriately set, and for example, a reduction of 50% or more of the measurement signal can be set as the criterion. Furthermore, when the destruction of exosomes is confirmed by measuring the exosome-specific antigens on the surface of the exosomes by an immunological measurement method, the mixture obtained by mixing the sample and the destroying reagent of the present invention is gelled. Destruction of exosomes can also be confirmed by subjecting to filtration chromatography and measuring the 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 the fraction from which the exosome is eluted in advance, and then the sample such as serum or plasma is mixed with the destroying reagent of the present invention. The mixture obtained by is subjected to gel filtration chromatography, the exosomes in the fraction confirmed in advance are measured by an immunological measurement method, and the destruction of the exosomes is confirmed by confirming the decrease in the measurement signal. be able to. The criterion for confirming the destruction of the exosome can be appropriately set, and for example, a reduction of 50% or more of the measurement signal can be set as the criterion.

The measurement signal is a signal obtained by an immunological measurement method of an exosome-specific antigen on the surface of an exosome, and is a label formed by binding an exosome having an exosome-specific antigen on the surface and an antibody against the exosome-specific antigen. It is due to labeling in the immune complex containing the exosome antibody. The measurement signal is not particularly limited as long as it is a signal capable of confirming the destruction of exosomes, and examples thereof include color development (absorbance), fluorescence, and luminescence.
When the measurement signal is color development (absorbance), for example, the label peroxidase is reacted with a combination of hydrogen peroxide as a substrate and an oxidation color-developing chromogen, and the absorbance of the reaction solution is measured by a spectrophotometer or multi-well. Examples thereof include a method of measuring with a plate reader or the like, a method of reacting β-D-galactosidase which is a label with its substrate, and measuring the absorbance of the reaction solution with a spectrophotometer, a multi-well plate reader or the like. Examples of the oxidative color-developing chromogen include a leuco-type chromogen, an oxidative-coupling chromogen, and the like.
The leuco-type chromogen is a substance that is independently converted into a dye in the presence of an active peroxide 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 can be mentioned.

The oxidative coupling color-developing chromogen is a substance in which two compounds oxidatively couple to form a dye in the presence of an active peroxide substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and anilines (Trinder's reagent), a combination of a coupler and phenols, and the like.
Examples of the coupler include 4-aminoantipyrine (4-AA), 3-methyl-2-benzothiazolinone hydrazine and the like.
Examples of anilins include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), and 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.

Examples of the substrate for β-D-galactosidase include o-nitrophenyl-β-D-galactopyranoside.

When the measurement signal is fluorescence, for example, the label peroxidase 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 fluorescent multi-well plate reader, or the like. Examples thereof include a method of reacting β-D-galactosidase, which is a label, with a fluorescent substrate of β-D-galactosidase, and measuring the intensity of the generated fluorescence with a fluorometer, a fluorescent multi-well plate reader, or the like. .. Examples of the fluorescent substrate of 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 of measuring the intensity of luminescence caused by a luminescent substance as a label with a luminescence photometer, a luminescent multi-well plate reader, or the like, peroxidase as a label, and luminescence of hydrogen peroxide and peroxidase. A method of reacting with a combination of substrates and measuring the intensity of the generated luminescence with a luminescence intensity meter, a luminescence multi-well plate reader, etc., a label β-D-galactosidase and a luminescent substrate of β-D-galactosidase. A method of measuring the intensity of luminescence generated by reacting with a luminescence photometer, a luminescent multi-well plate reader, etc. Examples thereof include a method of measuring with a meter, a light emitting multi-well plate reader, and the like. Examples of the luminescent substance include acridinium ester and its derivative, ruthenium complex compound, loffin and the like. Examples of the luminescent substrate of peroxidase include luminol compounds and lucigenin compounds. Examples of the luminescent substrate of β-D-galactosidase include Galacton-Plus (manufactured by Applied Biosystems) and similar compounds thereof. Examples of the luminescent substrate for alkaline phosphatase include 3- (2'-spirodamantan) -4-methoxy-4- (3'-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), 2-. chloro-5- {4-methoxy-spiro [1,2-dioxetane-3,2 '(5'-chloro) tricyclo [3.3.1.1 ^3.7] decan] -4-yl} phenyl phosphate and secondary sodium salt ^{(CDP-Star TM), 3-} {4- methoxy-spiro [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-methylacridan disodium, 9-[(4-chlorophenylthio) (phosphoryl) Oxy) methylidene] -10-methylacridan disodium (Lumigen ^TM APS-5) and the like.

Examples of the method for confirming the destruction of an exosome by measuring the exosome-specific antigen on the surface of the exosome by an immunological measurement method include a method including the following steps.
<Method of 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 alkylphenyl ether having an HLB value of 13 or less to a sample;
(2A) The sample obtained in the step (1A) is used as a first antibody or an antibody fragment thereof 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 composed of a second antibody or the antibody fragment that binds to a specific antigen, reacting in an aqueous medium, the first antibody or the antibody fragment, an exosome, and the second antibody or the antibody fragment. Process to generate;
(3A) A step of measuring the immune complex produced in the step (2A);
(4A) Except that in the step (1A), no surfactant of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkylphenyl ether having an HLB value of 13 or less is added to the sample. , The step of measuring the immune complex produced by the same steps as the steps (1A) to (3A);
(5A) The measurement signal obtained in the step (3A) is compared with the measurement signal obtained in the step (4A), and the measurement signal obtained in the step (3A) is the measurement signal obtained in the step (4A). A step of determining that an exosome has been destroyed when the signal is reduced as compared with the measurement signal obtained in.

In the above step (1A), addition of at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkylphenyl ether having an HLB value of 13 or less to a sample is performed. To a sample of the exosome-destroying reagent of the present invention containing at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkylphenyl ether having an HLB value of 13 or less. It can be done by addition. In this case, the step (4A) is a control exosome disrupting reagent that does not contain any surfactant of the polyether alkylamine, the POE-POP alkyl ether, and the POE alkylphenyl ether having an HLB value of 13 or less. Can be added to the sample. Further, the criteria for determining exosome destruction in step (5A) can be appropriately set. For example, the measurement signal obtained in step (3A) is compared with the measurement signal obtained in step (4A). , It is possible to set a criterion for judging that an exosome is destroyed when the amount is reduced by 50% or more.
In the above 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 particularly limited as long as it is an insoluble carrier that immobilizes a first antibody or an antibody fragment thereof that binds to a first exosome-specific antigen on the surface of the exosome and enables the method for destroying the exosome of the present invention. Various types such as synthetic resin plates such as microtiter plates, glass or synthetic resin granules (beads), glass or synthetic resin spheres (balls), latexes, magnetic particles, nitrocellulose films, etc. Examples include a membrane and a test tube made of synthetic resin. Examples of the synthetic resin plate include a polyethylene plate, a polypropylene plate, a polystyrene plate and the like.

Immobilization of the first antibody or the antibody fragment thereof that binds to the first exosome-specific antigen on the surface of the exosome to an insoluble carrier is particularly limited as long as it enables the method for destroying the exosome of the present invention. However, physical adsorption, immobilization by chemical bonds, etc. can be mentioned. Examples of the physical adsorption include electrostatic bonds, hydrogen bonds, hydrophobic bonds and the like. Examples of the chemical bond include a covalent bond and a coordination bond.
The first antibody or antibody fragment thereof that binds to the first exosome-specific antigen that the exosome has on its surface is immobilized on the insoluble carrier directly or indirectly on the insoluble carrier by physical adsorption and / or chemical bonding. You may. As an indirect immobilization method, for example, a first method of binding to a first exosome-specific antigen on the surface of an exosome via a specific binding between biotin and avidins (avidin, streptavidin, neutravidin, etc.). Examples thereof include a method of immobilizing an antibody or the antibody fragment on an insoluble carrier. Further, the first antibody or the antibody fragment thereof that binds to the first exosome-specific antigen that the exosome has on its surface may be immobilized on the insoluble carrier by covalent bonding via a linker.
Further, in the above 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, but it does. Is preferable. Examples of the sign include the above-mentioned signs and the like. When the second antibody or 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 the measurement of the generated immunity. This can be done by measuring the labeling in the complex.
If the label is not bound to the second antibody or the antibody fragment, the third antibody that binds to the second antibody or the antibody fragment or the labeled third antibody or the antibody to which the label is bound to the antibody fragment. Fragments can also be used to confirm disruption of exosomes in a sample. 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, the first antibody or the antibody fragment, the exosome, and the second antibody or the antibody fragment. The immune complex produced in step (2A) was formed by forming an immune complex 2 composed of the labeled third antibody or the antibody fragment thereof and measuring the label in the immune complex 2 by the above-mentioned method. The amount of 1 can be measured. Examples of the third antibody include an antibody that binds to the Fc region of the second antibody, an antibody fragment thereof, and the like. Examples of the sign include the above-mentioned signs and the like.

The combination of the first exosome-specific antigen and the second exosome-specific antigen is not particularly limited as long as it enables the method for destroying exosomes of the present invention. For example, the combinations shown in Table 1 below are used. Can be mentioned.

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 particularly limited as long as it is a combination that enables the method for destroying exosomes of the present invention. However, for example, the combinations shown in Table 2 below can be mentioned.

The antibody fragment of the first antibody that binds to the first exosome-specific antigen in the present invention is particularly any antibody fragment that binds to the first exosome-specific antigen and enables the method for destroying the exosome of the present invention. There are no restrictions, 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-reduction, and the like. Examples thereof include an antibody fragment from which the Fc portion has been removed, an antibody fragment from which the Fc portion has been removed by a genetic engineering technique, and the like.
The antibody fragment of the second antibody that binds to the second exosome-specific antigen in the present invention is particularly any antibody fragment that binds to the second exosome-specific antigen and enables the method for destroying the exosome of the present invention. There are no restrictions, 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-reduction, and the like. Examples thereof include an antibody fragment from which the Fc portion has been removed, an antibody fragment from which the Fc portion has been removed by a genetic engineering technique, and the like.

The first antibody that binds to the first exosome-specific antigen in the present invention is not particularly limited as long as it is an antibody that binds to the first exosome-specific antigen and enables the method for destroying exosomes of the present invention. Both polyclonal antibodies and monoclonal antibodies can be used.
The second antibody that binds to the second exosome-specific antigen in the present invention is not particularly limited as long as it binds to the second exosome-specific antigen and enables the method for destroying the exosome of the present invention. Both polyclonal antibodies and monoclonal antibodies can be used.

Commercially available antibodies can be used 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. Examples of commercially available antibodies include anti-CD9 monoclonal antibody (clone A100-4; manufactured by Medical & Biological Laboratories), Purified Mouse Anti-Human CD9 (manufactured by Becton Dickinson, Japan), Purified Mouse Anti-Human CD63 (Becton, Japan). (Made by Dickinson), Purified Mouse Anti-Human CD81 (manufactured by Becton Dickinson, Japan, clone: JS-81), anti-CD147 antibody [MEM-M6 / 1] (manufactured by Novous biopharmacy) and the like.

<Method of confirming exosome destruction (2)>
(1B) 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 alkylphenyl ether having an HLB value of 13 or less to a sample;
(2B) A step of subjecting the sample obtained in the above step (1B) to gel filtration chromatography to separate the fraction containing exosomes from the other fractions;
(3B) The fraction containing the exosome obtained in the step (2B) is subjected to the first antibody or the antibody fragment thereof that binds to the first exosome-specific antigen that the exosome has on its surface, and the exosome is on its surface. The second antibody or the antibody fragment that binds to the second exosome-specific antigen to be possessed is reacted in an aqueous medium, and the first antibody or the antibody fragment, the exosome, and the second antibody or the antibody fragment are separated from each other. Step of producing the immune complex 1;
(4B) A step of measuring the immune complex 1 produced in the step (3B);
(5B) In the step (1B), at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkylphenyl ether having an HLB value of 13 or less is not added. Except for the step of measuring the immune complex produced by the same steps as the steps (1B) to (4B);
(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 the measurement signal obtained in the step (5B). A step of determining that an exosome has been destroyed when it is reduced compared to the measurement signal obtained in.

In the above step (1B), addition of at least one surfactant selected from the group consisting of polyether alkylamines, POE-POP alkyl ethers, and POE alkylphenyl ethers having an HLB value of 13 or less to a sample is performed. To a sample of the exosome-destroying reagent of the present invention containing at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkylphenyl ether having an HLB value of 13 or less. It can be done by addition. In this case, the step (5B) does not contain at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkylphenyl ether having an HLB value of 13 or less. This can be done by adding a control exosome disrupting reagent to the sample. Further, the criteria for determining 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). , It is possible to set a criterion for judging that an exosome is destroyed when the amount is reduced by 50% or more.
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 above-mentioned insoluble carrier and the like.

Examples of the immobilization of the first antibody or the antibody fragment thereof that binds to the first exosome-specific antigen on the surface of the exosome to the insoluble carrier include the above-mentioned immobilization.
Further, in the above 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, but it does bind. Is preferable. Examples of the sign include the above-mentioned signs and the like. When the second antibody or 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 produced immune complex 1 in the above step (4B) was produced. This can be done by measuring the label in the immune complex 1.
If the label is not bound to the second antibody or the antibody fragment, the third antibody that binds to the second antibody or the antibody fragment or the labeled third antibody or the antibody to which the label is bound to the antibody fragment. Fragments can also be used to confirm disruption of exosomes in a sample. 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, the first antibody or the antibody fragment, the exosome, and the second antibody or the antibody fragment. The immune complex produced in step (3B) was formed by forming an immune complex 2 composed of the labeled third antibody or the antibody fragment thereof and measuring the label in the immune complex 2 by the above-mentioned method. The amount of 1 can be measured. Examples of the third antibody include an antibody that binds to the Fc region of the second antibody, an antibody fragment thereof, and the like. Examples of the sign include the above-mentioned signs and the like.

Examples of the combination of the first exosome-specific antigen and the second exosome-specific antigen include the above-mentioned 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 the above-mentioned combinations.
Commercially available antibodies can be used 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. Examples of commercially available antibodies include the above-mentioned antibodies.

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

The sample in the present invention means a sample in which an 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, etc.) Body fluid samples such as milk, exosomes, cerebrospinal fluid, and feces), cell culture fluid, and the like are mentioned, and biological samples are preferable, and serum and plasma are particularly preferable.

The polyether alkylamine in the method for destroying exosomes of the present invention is not particularly limited as long as it is a polyether alkylamine that enables the method for destroying exosomes of the present invention, but a polyether alkylamine having an HLB value of 13 or less is used. preferable. The polyether alkylamine in the present invention has a structure in which at least one of the hydrogen atoms bonded to the nitrogen atom of the alkylamine is substituted with polyoxyethylene, and for example, polyoxyethylene alkylamine (hereinafter, POE). (Hereinafter referred to as alkylamine), polyoxyethylene alkylpropylene diamine (hereinafter referred to as POE alkylpropylene diamine) and the like.
Examples of the alkyl in the polyether alkylamine include alkyl having 8 to 24 carbon atoms, and alkyl having 10 to 20 carbon atoms is preferable. Examples of alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonadecylic, decyl, isodecyl, undecyl, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), and the like. Examples thereof include oleyl, nonadecylic acid, icosyl, heneicosyl, docosyl (behenyl), tricosylic acid, and tetracosyl. Examples of alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecylic, and icosyl. Can be mentioned.

Among the commercially available polyether alkylamine products, the commercially available POE alkylamines include, for example, Nymine L-201 [oxyethylene dodecylamine; HLB value 3.8] and Nymeen L-202 [POE dodecylamine; HLB value 6]. .4], Nymine L-207 [POE dodecylamine; HLB value 12.5], Nymeen S-204 [POE stearylamine; HLB value 8.0], Nymeen S-220 [POE stearylamine; HLB value 15.4] ], Nymeen T2-210 [POE alkyl (beef fat) amine; HLB value 12.5], Nymeen F-202 [POE alkyl (palm) amine; HLB value 6.1] (above, manufactured by Nichiyu Co., Ltd.), Braunon L -205 [POE dodecylamine; HLB value 10.4], Blaunon L-210 [POE dodecylamine; HLB value 13.6] (above, manufactured by Aoki Yushi Kogyo Co., Ltd.), etc., are commercially available products of POE alkylpropylene diamine. Examples thereof include Nymine DT-203 [POE alkylpropylene diamine; HLB value 6.0], Nymeen DT-208 [POE alkylpropylene diamine; HLB value 10.7] (all manufactured by Nichiyu Co., Ltd.), Blaunon DT-03. [POE alkyl (beef fat) propylenediamine; HLB value 5.9], Blaunon DT-15 [POE alkyl (beef fat) propylenediamine; HLB value 13.4] (all manufactured by Aoki Oil & Fat Industry Co., Ltd.) and the like can be mentioned.
Commercially available products of polyether alkylamines with an HLB value of 13 or less, for example, as POE alkylamines, include nymine L-201 [oxyethylene dodecylamine; HLB value 3.8] and nymine L-202 [POE dodecylamine; HLB. Value 6.4], Nymine L-207 [POE dodecylamine; HLB value 12.5], Nymeen S-204 [POE stearylamine; HLB value 8.0], Nymeen T2-210 [POE alkyl (beef fat) amine; HLB value 12.5], Nymine F-202 [POE alkyl (palm) amine; HLB value 6.1] (all manufactured by Nichiyu Co., Ltd.), Blaunon L-205 [POE dodecylamine; HLB value 10.4] ( Aoki Oil & Fat Industry Co., Ltd.) and the like. Examples of commercially available POE alkyl propylene diamines having an HLB value of 13 or less include Nymine DT-203 [POE alkyl propylene diamine; HLB value 6.0] and Nymeen DT-208 [POE alkyl propylene diamine; HLB value 10.7]. ] (The above is manufactured by Nichiyu Co., Ltd.), Braunon DT-03 [POE alkyl (beef fat) propylene diamine; HLB value 5.9] (the above is manufactured by Aoki Oil & Fat Industry Co., Ltd.) and the like.

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 the POE-POP having an HLB value of 13 or less is not particularly limited. Alkyl ethers are particularly preferred.
Examples of the alkyl in the POE-POP alkyl ether include alkyl having 8 to 24 carbon atoms, and alkyl having 10 to 20 carbon atoms is preferable. Examples of the alkyl having 8 to 24 carbon atoms include the above-mentioned alkyl having 8 to 24 carbon atoms. Examples of the alkyl having 10 to 20 carbon atoms include the above-mentioned alkyl having 10 to 20 carbon atoms.

Examples of commercially available POE-POP alkyl ethers include Nonion HT-505 [POE-POP alkyl ether; HLB value 5], Nonion HT-510 [POE-POP alkyl ether; HLB value 10], and Nonion HT-512 [. POE-POP alkyl ether; HLB value 12], Nonion HT-515 [POE-POP alkyl ether; HLB value 15] (all manufactured by Nichiyu Co., Ltd.), 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 Oil & Fat Industries) , 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; Ether; HLB value 12.1] (above, manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) and the like.
Examples of commercially available POE-POP alkyl ethers having an HLB value of 13 or less include Nonion HT-505 [POE-POP alkyl ether; HLB value 5] and Nonion HT-510 [POE-POP alkyl ether; HLB value. 10], Nonion HT-512 [POE-POP alkyl ether; HLB value 12] (all manufactured by Nichiyu Co., Ltd.), 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-POP tridecyl ether; HLB value 12.1] (all manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) and the like can be mentioned.

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 alkyl having 8 to 9 carbon atoms. Examples of the alkyl having 8 to 9 carbon atoms include octyl and nonyl.

Examples of commercially available POE alkylphenyl ethers having an HLB value of 13 or less include Nonion HS-204.5 [POE octylphenyl ether; HLB value 9.8] and Nonion HS-206 [POE octylphenyl ether; HLB value 11]. .2], Nonion NS-202 [POE nonylphenyl ether; HLB value 5.7], Nonion NS-204.5 [POE nonylphenyl ether; HLB value 9.5], Nonion NS-208.5 [POE nonylphenyl] Ether; HLB value 12.6] (above, manufactured by Nichiyu Co., Ltd.), Blaunon NK-8055 [POE octylphenyl ether; HLB value 10.8], Blaunon NK-808 [POE octylphenyl ether; HLB value 12.5] , Blaunon N-502 [POE nonylphenyl ether; HLB value 5.7], Blaunon N-506 [POE nonylphenyl ether; HLB value 10.9], Blaunon DT-9 [POE dodecylphenyl ether; HLB value 12.0] ] (The above is manufactured by Aoki Oil & Fat Industry Co., Ltd.) and the like.

As a method of adding at least one surfactant selected from the group consisting of polyether alkylamine, POE-POP alkyl ether, and POE alkylphenyl ether having an HLB value of 13 or less in the method for destroying exosomes of the present invention to a sample. Is not particularly limited as long as it is an addition method capable of destroying the exosome of the present invention, for example, a method of directly adding the surfactant to a sample; prepared by dissolving the surfactant in an aqueous medium. Method of adding the aqueous solution of the surfactant to the sample; method of adding the sample to the aqueous solution of the surfactant prepared by dissolving the surfactant in an aqueous medium; Examples thereof include a method of adding an aqueous solution of the surfactant prepared by dissolving it in an aqueous medium to a composition obtained by freeze-drying, and an aqueous solution prepared by dissolving the surfactant in an aqueous medium is used as a sample. The method of addition is preferable.
The aqueous medium is not particularly limited as long as it is an aqueous medium that enables the method for destroying exosomes of the present invention, and examples thereof include deionized water, distilled water, and a buffer solution, and a buffer solution is preferable. The pH of the aqueous medium is, for example, 4 to 10. When a buffer solution is used as the aqueous medium, it is preferable to use a buffer solution suitable for the pH to be set. The buffer used for preparing the buffer is not particularly limited as long as it has a buffering ability. For example, a lactic acid buffer, a citrate buffer, an acetate buffer, a succinic acid buffer, a phthalate buffer, etc. Phosphoric acid buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, imidazole buffer, malic acid buffer, oxalate buffer, glycine buffer, borate buffer, carbon dioxide buffer , Good buffer, etc.

Good buffers include, for example, 2-morpholinoetan sulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, tris (hydroxymethyl) aminomethane (Tris). Buffer, N- (2-acetamide) iminodiacetic acid (ADA) buffer, piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES) buffer, 2- [N- (2-acetamide) 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) methyl] amino} ethanesulfonic acid (TES) buffer, N- (2-hydroxyethyl) -N'- (2-sulfoethyl) piperazin (HEPES) buffer, 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO) buffer, 2-hydroxy-3-{[N -Tris (Hydroxymethyl) Methyl] Amino} Propane Sulfonic Acid (TAPSO) Buffer, Piperazin-N, N'-Bis (2-Hydroxy Propane-3-Sulfonic Acid) (POPSO) Buffer, N- (2-Hydroxy) Ethyl) -N'-(2-hydroxy-3-sulfopropyl) piperazin (HEPPSO) buffer, N- (2-hydroxyethyl) -N'-(3-sulfopropyl) piperazin (EPPS) buffer, [N -Tris (hydroxymethyl) methylglycine] (Trisine) buffer, [N, N-bis (2-hydroxyethyl) glycine] (Bicine) buffer, 3- [N-Tris (hydroxymethyl) methyl] aminopropanesulfon Acid (TAPS) buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxypropanesulfonic acid (CAPSO) buffer, 3- (N-N-) Cyclohexylamino) propanesulfonic acid (CAPS) buffers and the like can be mentioned.
The aqueous medium may contain salts, metal ions, sugars, preservatives, proteins and the like. Examples of salts include lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ammonium chloride, lithium bromide, sodium bromide, potassium bromide, calcium bromide, magnesium bromide, ammonium bromide and the like. .. Examples of the metal ion include magnesium ion, manganese ion, zinc ion and the like. Examples of saccharides 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 an exosome of the present invention, a reaction between a sample and at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkylphenyl ether having an HLB value of 13 or less. The reaction temperature of the above is not particularly limited as long as it enables the method for destroying the exosome of the present invention, and is usually 0 to 50 ° C., preferably 4 to 45 ° C., particularly preferably 15 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables the method for destroying exosomes of the present invention, and is usually 10 seconds to 24 hours, preferably 30 seconds to 3 hours, preferably 1 minute to 2 hours. Is particularly preferable.

At least one surfactant selected from the group consisting of polyether alkylamines, POE-POP alkyl ethers, and POE alkylphenyl ethers having an HLB value of 13 or less in the method for destroying exosomes of the present invention was added to a sample. The latter concentration is not particularly limited as long as it enables the method for destroying the exosome of the present invention, and is usually 0.001 to 10% (w / v) and 0.01 to 5% (w / v). ) Is preferable, and 0.04 to 1% (w / v) is particularly preferable.

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

The reagent for destroying exosomes of the present invention may be in a liquid state or in a lyophilized state. In the case of a liquid destructive reagent, at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkylphenyl ether having an HLB value of 13 or less is previously described as an aqueous surfactant. It is dissolved in the medium. In the case of a lyophilized destroying reagent, at least one surfactant selected from the group consisting of a polyether alkylamine, a POE-POP alkyl ether, and a POE alkylphenyl ether having an HLB value of 13 or less is the above-mentioned aqueous surfactant. It may be dissolved in a medium and used, or it may be used by direct contact with a sample. The destructive reagent of the present invention may contain the above-mentioned salts, metal ions, sugars, 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 alkylphenyl ethers having an HLB value of 13 or less in the exosome disrupting reagent of the present invention is the present invention. There is no particular limitation as long as the content enables the method of disrupting the above, and the concentration after being mixed with the sample is usually 0.001 to 10% (w / v), which is 0.01 to 5%. The content of (w / v) is preferable, and the content of 0.04 to 1% (w / v) is particularly preferable.

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 (manufactured by GE Healthcare), disodium hydrogen phosphate (manufactured by Kanto Chemical Co., Ltd.), sodium dihydrogen phosphate (manufactured by Kanto Chemical Co., Ltd.), sodium chloride (Japanese) Kojunyaku Kogyo Co., Ltd.), Sodium Azide (Wako Junyaku Kogyo Co., Ltd.), BCA Protein Assay Kit (Thermo Fisher Scientific Co., Ltd.), MES (Dojin Chemical Laboratory Co., Ltd.), Cow Serum Albumin (BSA; Oriental) Yeast Industry Co., Ltd.), Streptavidin-bound magnetic particles (Dynabeads MyOne Streptavidin C1; Thermo Fisher Scientific Co., Ltd.), Biotin Labeling Kit-NH ₂ (Donjin Chemical Laboratory Co., Ltd.), Anti-CD9 monoclonal antibody (clone A100-4) ; MES (manufactured by Dojin Chemical Research Institute), MES (manufactured by Dojin Chemical Research Institute), Alkaline Phosphace Labeling Kit-NH ₂ (manufactured by Dojin Chemical Research Institute), MOPS (manufactured by Dojin Chemical Research Institute), magnesium chloride 6 water Japanese products (manufactured by Wako Pure Chemical Industries, Ltd.), Tween20 (polyoxyethylene sorbitan monolaurate; manufactured by Dojin Chemical Research Institute, manufactured by Wako Pure Chemical Industries, Ltd.), 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-Methylacridan disodium salt (Lumigen ^TM APS-5; manufactured by Oriental Yeast Industries Co., Ltd.), Nymine L-202 [POE laurylamine (polyetheralkylamine); manufactured by Nichiyu Co., Ltd.], Nymeen DT-203 [ POE alkyl propylene diamine (polyether alkylamine); manufactured by Nichiyu Co., Ltd.], 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 ether with HLB value of 13 or less); manufactured by Nichiyu Co., Ltd.], Nonion HS-204.5 [POE octylphenyl ether (POE alkylphenyl ether with HLB value of 13 or less); Nichiyu , Triton X-100 [POE octylphenyl 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 fractions obtained when serum is subjected to gel filtration chromatography>
(1) Preparation of serum Whole blood collected from a healthy person belonging to Kyowa Medex Co., Ltd. was centrifuged at 2,000 rpm at 25 ° C. for 20 minutes to prepare serum.
(2) Separation of serum components by gel filtration chromatography The serum (500 μL) prepared in (1) above was applied to Superdex200HR 10/30, which is a column for gel filtration chromatography, and then sodium azide 0 was used as an eluate. PBS [phosphate buffered saline (10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2)] supplemented with .2 g / L was flowed at a flow rate of 0.5 mL / min. Fraction No. as the elution fraction. 1-80 (0.5 mL each) were obtained.
(3) Measurement of each fraction obtained in (2) above with the BCA protein assay kit Fraction No. obtained in (2) above. For each of the fractions 1 to 80, a reaction was carried out using the BCA protein assay kit, which is a kit for measuring protein mass, according to the instruction manual of the kit, and the absorbance (560 nm) was measured. The result is shown in FIG.

(4) Preparation of Measuring Reagents Used for Sandwich Method Using Antigen-Antibody Reaction A streptavidin-bound magnetic particle solution, a biotin-bound anti-CD9 monoclonal antibody solution, and an ALP (alkaline phosphatase) -labeled anti-CD9 monoclonal antibody solution were prepared.
-Streptavidin-bonded magnetic particle solution A streptavidin-bonded magnetic particle solution having the following composition was prepared using commercially available streptavidin-bonded magnetic particles (Dynabeads MyOne Streptavidin C1).
MES (pH 6.5) 0.05 mol / L
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Streptavidin-bound magnetic particles 0.225 mg / mL
-Biotin-binding anti-CD9 monoclonal antibody solution Using Biotin Labeling Kit-NH ₂ (manufactured by Dojin Chemical Research Institute), according to the instruction manual of the kit, anti-CD9 monoclonal antibody (clone A100-4; Medical Biology Research Institute) The product was labeled with biotin to prepare a biotin-binding anti-CD9 monoclonal antibody. Using the obtained biotin-binding anti-CD9 monoclonal antibody, a biotin-binding 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 0.1 mol / L
-ALP-labeled anti-CD9 monoclonal antibody solution Using Alkaline Phosphatase Labeling Kit-NH ₂ (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.), follow the instruction manual of the kit, and anti-CD9 monoclonal antibody (clone A100-4; Institute of Medical Biology). 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 0.1 mol / L

(5) Measurement of each fraction obtained in (2) above by a sandwich method using an antigen-antibody reaction Fraction No. obtained in (2) above. For each fraction (10 μL) of 1 to 80, the streptavidin-bound magnetic particle solution (30 μL) prepared in (4) above, the biotin-bound anti-CD9 monoclonal antibody solution (30 μL) prepared in (4) above, and The ALP-labeled anti-CD9 monoclonal antibody solution (30 μL) prepared in (4) above was added, stirred, and reacted at 37 ° C. for 10 minutes. Next, the magnetic particles were collected by magnetic force to remove the reaction solution other than the magnetic particles, and the collected magnetic particles were washed 5 times with the following cleaning solution.
・ Cleaning liquid MOPS (pH 7.3) 0.005 mol / L
Sodium chloride 0.3 mol / L
Magnesium chloride hexahydrate 0.2 mmol / L
Tween20 0.075% (w / v)
Then, the collected and washed magnetic particles emit light containing 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridane disodium salt (Lumigen ^TM APS-5) as a main component. 100 μL of the substrate solution was added and stirred, and the amount of luminescence (RLU) generated was measured. The result is shown in FIG.

(6) Identification of Fractions Containing Exosomes As is clear from FIG. 1 showing the results of (5) above, Fraction No. A large measurement signal was obtained for 18 and the fractions before and after it by the measurement by the sandwich method using an anti-CD9 monoclonal antibody. Therefore, the fraction No. It was found that 18 and the fractions before and after it contained exosomes having CD9 on their surface.
In addition to exosomes having CD9 on their surface, a single molecule of CD9 may be present in serum. However, the sandwich method of (5) above is 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. Since it is a sandwich method in which an antibody that recognizes the same Eptop is used for both of the antibodies, it is not possible to sandwich a single molecule of CD9. Therefore, the fact that the signal is obtained by the sandwich method in FIG. 1 means that only exosomes having CD9 on the surface thereof are detected, and the single molecule CD9 itself is not detected.

Further, as is clear from FIG. 1, the fraction No. In Nos. 20 to 40, a large measurement signal was obtained by measurement using the BCA protein assay kit. It was found that 20 to 40 contained a large amount of protein. In addition, as a result of subjecting these fractions to SDS-PAGE, these fractions contain a large amount of proteins corresponding to the molecular weights of albumin and gamma globulin (molecular weights of about 66 kDa and 150 kDa, respectively), which are the main proteins in serum. It was found that it was contained in.
Here, in gel filtration chromatography, the larger the molecular weight, the faster the elution. However, as is clear from FIG. 1, the fraction No. 1 showing the signal caused by CD9 (molecular weight of 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. indicating the signal caused by CD9. It was found that the fraction around 18 contained not a single molecule of CD9 but an exosome having CD9 on its surface.

<Preparation of reagents for exosome destruction>
[Example 1]
Each of the five surfactants shown in Table 3 below was added to PBS at a concentration of 10% (w / v) to prepare reagents A1 to A5 for destroying exosomes, respectively.
[Comparative Example 1]
The two surfactants shown in Table 3 below, that is, the surfactants of Triton X-100 and Nonidet P-40, were added to PBS at a rate of 10% (w / v) for exosome destruction. Reagents B1 and B2 were prepared, respectively.
[Comparative Example 2]
PBS was designated as control reagent B0.

[Example 2]
<Destroying exosomes using a reagent for destroying exosomes containing polyether alkylamine>
(1) Preparation of serum and treatment of serum with a 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, is collected at 2,000 rpm and 25 ° C. for 20 minutes. Serum was prepared by centrifugation.
To the prepared serum (900 μL), the exosome-destroying reagent A1 (100 μL) prepared in Example 1 above was added, and the mixture was allowed to stand for 10 minutes. Similarly, the exosome-destroying reagent A2 (100 μL) prepared in Example 1 above was added to the prepared serum (900 μL) and allowed to stand for 10 minutes.
(2) Separation of serum components by gel filtration chromatography The components of serum prepared and treated with a surfactant in (1) above using Superdex200HR 10/30 (manufactured by GE Healthcare), which is a column for gel filtration chromatography. Was separated. First, 500 μL of the surfactant-treated serum was applied to the column, and then, as an eluate, PBS containing 0.2 g / L of sodium azide [phosphate buffered saline (0.15 mol / L chloride). 10 mmol / L phosphate buffer containing sodium, pH 7.2)] was flowed at a flow rate of 0.5 mL / min, and fraction No. 1 was used as the elution fraction. 1-80 (0.5 mL each) were obtained.

(3) Preparation of measurement reagent used for sandwich method using antigen-antibody reaction Streptavidin-bound magnetic particle solution, biotin-bound anti-CD9 monoclonal antibody solution, and ALP labeling by the same method as (4) in Reference Example 1 above. An anti-CD9 monoclonal antibody solution was prepared.
(4) Measurement of each fraction obtained in (2) above by a sandwich method using an antigen-antibody reaction Fraction No. obtained in (2) above. Of 1 to 80, fraction No. For 15 to 25, each fraction was 10 μL, 30 μL of the streptavidin-bound magnetic particle solution prepared in (3) above, 30 μL of the biotin-bound anti-CD9 monoclonal antibody solution prepared in (3) above, and 30 μL of the biotin-bound anti-CD9 monoclonal antibody solution prepared in (3) above. 30 μL of the 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 to remove the reaction solution other than the magnetic particles, and the collected magnetic particles were washed 5 times with the cleaning solution used in (5) of Reference Example 1 above. Then, the collected and washed magnetic particles emit light containing 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridane disodium salt (Lumigen ^TM APS-5) as a main component. 100 μL of the substrate solution was added and stirred, and the amount of luminescence (RLU) generated was measured. The result is shown in FIG.

[Comparative Example 3]
(1) Preparation of serum and treatment of serum with control reagent B0 The control reagent B0 (100 μL) prepared in Comparative Example 2 above was added to the serum (900 μL) prepared in (1) of Example 2 above. Was added and allowed to stand for 10 minutes.
(2) Gel filtration chromatography-Measurement by sandwich method using antigen-antibody reaction (2)-(4) of Example 2 above, except that the serum obtained in (1) above is used as a sample for gel filtration chromatography. ), The separation operation of the serum component by gel filtration chromatography, and the fraction No. 1 eluted by the gel filtration chromatography. Measurements of 15 to 25 were performed by the sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Example 3]
Fraction No. obtained in (2) of Comparative Example 3 above. Fraction No. obtained from the serum treated with the exosome-destroying reagent A1 in (4) of Example 2 above when the amount of luminescence with respect to 18 was 100. The amount of light emitted relative to 18 was calculated. Similarly, Fraction No. obtained from the serum treated with the exosome-destroying reagent A2 obtained in (4) of Example 2 above. The amount of light emitted relative to 18 was calculated. The results are shown in Table 4.

As is clear from FIGS. 2 and 4, when serum is treated with an exosome-destroying reagent containing nymeen L-202 or nymeen DT-203 for 10 minutes, it is relative to the fraction containing exosomes (fraction No. 18). The amount of luminescence is significantly reduced as compared with the case where the serum is treated with the control reagent B0, and the addition of Nymin L-202 or Nymeen DT-203 to the serum destroys exosomes in a short time. It has been found. Therefore, it was clarified that the addition of polyether alkylamine to serum can destroy exosomes in serum easily and quickly without isolating exosomes in serum.

[Example 4]
<Exosome destruction using an exosome destruction reagent containing POE-POP alkyl ether>
(1) Preparation of serum and treatment of serum with a 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 collected at 2,000 rpm, 25. Serum was prepared by centrifugation at ° C. for 20 minutes.
To the prepared serum (900 μL), the exosome-destroying reagent A3 (100 μL) prepared in Example 1 above was added, and the mixture was allowed to stand for 10 minutes.
(2) Gel Filtration Chromatography-Measurement by Sandwich Method Using Antigen-Antibody Reaction (2)-(4) of Example 2 above, except that the serum obtained in (1) above is used as a sample for gel filtration chromatography. ), Separation of exosomes and other components by gel filtration chromatography, and fraction No. obtained by gel filtration chromatography. Exosomes in 15-25 were measured by the 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 The control reagent B0 (100 μL) prepared in Comparative Example 2 above was added to the serum (900 μL) prepared in (1) of Example 4 above. Was added and allowed to stand for 10 minutes.
(2) Gel Filtration Chromatography-Measurement by Sandwich Method Using Antigen-Antibody Reaction (2)-(4) of Example 2 above, except that the serum obtained in (1) above is used as a sample for gel filtration chromatography. ), Separation of exosomes and other components by gel filtration chromatography, and fraction No. obtained by gel filtration chromatography. Exosomes in 15-25 were measured by the sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Example 5]
Fraction No. obtained in (2) of Comparative Example 4 above. Fraction No. obtained from serum treated with the exosome-destroying reagent A3 in (2) of Example 4 above when the amount of luminescence with respect to 18 was 100. The amount of light emitted relative to 18 was calculated. The results are shown in Table 5.

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

[Example 6]
<Exosome destruction using an exosome destruction reagent containing a POE alkyl phenyl ether with an HLB value of 13 or less>
(1) Preparation of serum and treatment of serum with a 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, is 2 Serum was prepared by centrifugation at 000 rpm and 25 ° C. for 20 minutes.
To the prepared serum (900 μL), the exosome-destroying reagent A4 (100 μL) prepared in Example 1 above was added, and the mixture was allowed to stand for 10 minutes. Similarly, the exosome-destroying reagent A5 (100 μL) prepared in Example 1 above was added to the prepared serum (900 μL) and allowed to stand for 10 minutes.
(2) Gel Filtration Chromatography-Measurement by Sandwich Method Using Antigen-Antibody Reaction (2)-(4) of Example 2 above, except that the serum obtained in (1) above is used as a sample for gel filtration chromatography. ), Separation of exosomes and other components by gel filtration chromatography, and fraction No. obtained by gel filtration chromatography. Exosomes in 15-25 were measured by the sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Comparative Example 5]
<Exosome destruction using an exosome destruction reagent containing POE alkyl phenyl ether with an HLB value of more than 13>
(1) Preparation of serum and treatment of serum with a surfactant To the serum (900 μL) prepared in (1) of Example 6 above, the reagent B1 (100 μL) for destroying exosomes prepared in Comparative Example 1 above was added. ) Was added, and the mixture was allowed to stand for 10 minutes. Similarly, the exosome-destroying reagent B2 (100 μL) prepared in Comparative Example 1 was added to the serum (900 μL) prepared in (1) of Example 6 above, and the mixture was allowed to stand for 10 minutes.
(2) Gel Filtration Chromatography-Measurement by Sandwich Method Using Antigen-Antibody Reaction (2)-(4) of Example 2 above, except that the serum obtained in (1) above is used as a sample for gel filtration chromatography. ), Separation of exosomes and other components by gel filtration chromatography, and fraction No. obtained by gel filtration chromatography. Exosomes in 15-25 were measured by the 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 The control reagent B0 (100 μL) prepared in Comparative Example 2 above was added to the serum (900 μL) prepared in (1) of Example 6 above. Was added and allowed to stand for 10 minutes.
(2) Gel Filtration Chromatography-Measurement by Sandwich Method Using Antigen-Antibody Reaction (2)-(4) of Example 2 above, except that the serum obtained in (1) above is used as a sample for gel filtration chromatography. ), Separation of exosomes and other components by gel filtration chromatography, and fraction No. obtained by gel filtration chromatography. Exosomes in 15-25 were measured by the sandwich method using an antigen-antibody reaction. The result is shown in FIG.

[Example 7]
Fraction No. obtained in (2) of Comparative Example 6 above. Fraction No. obtained from serum treated with the exosome-destroying reagent A4 in (2) of Example 6 above when the amount of luminescence with respect to 18 was 100. The amount of light emitted relative to 18 was calculated. Similarly, Fraction No. obtained from the serum treated with the exosome-destroying reagent A5 obtained in (2) of Example 6 above. The amount of light emitted relative to 18 was calculated. The results are shown in Table 6.
In addition, the fraction No. obtained in (2) of Comparative Example 6 above. Fraction No. obtained from the serum treated with the exosome-destroying reagent B1 obtained in (2) of Comparative Example 5 above, where the amount of luminescence with respect to 18 was 100. The amount of light emitted relative to 18 was calculated. Similarly, Fraction No. obtained from the serum treated with the exosome-destroying reagent B2 obtained in (2) of Comparative Example 5 above. The amount of light emitted relative to 18 was calculated. The results are shown in Table 6.

As is clear from FIGS. 4 and 6, when serum is treated with an exosome-destroying reagent containing nonion NS-204.5 or nonion HS-204.5 for 10 minutes, the fraction containing exosomes (fraction No.) The amount of luminescence relative to .18) was significantly reduced as compared with 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 a significant decrease. It was found that exosomes were destroyed in a short time. Therefore, it was clarified that the addition of POE alkyl phenyl ether having an HLB value of 13 or less to the serum can easily and quickly destroy the exosomes in the serum without isolating the exosomes in the serum.
Further, as is clear from FIGS. 4 and 6, when serum is treated with an exosome-destroying reagent containing nonion NS-204.5 or nonion HS-204.5 for 10 minutes, a fraction containing exosomes (fractions). The amount of luminescence for image No. 18) is higher than that when serum is treated with an exosome-destroying reagent containing Triton X-100 or Nonidet P-40, which is a POE alkylphenyl ether having an HLB value of more than 13. It was significantly lower. Therefore, as compared to the addition of Triton X-100 or Nonidet P-40, which are POE alkyl phenyl ethers with an HLB value greater than 13, to the serum, Nonion NS-204.5 or Nonion HS-204.5 to the serum. The addition was found to significantly destroy exosomes. Therefore, it was clarified that the addition of POE alkyl phenyl ether having an HLB value of 13 or less to the serum can easily and quickly destroy the exosomes in the serum without isolating the exosomes in the serum.

[Example 8]
<Sample difference: serum and plasma>
(1) Preparation of serum and treatment of serum Whole blood collected from healthy person 1 belonging to Kyowa Medex Co., Ltd., which is different from the healthy person in Reference Example 1, Example 2, Example 4 and Example 6. Serum 1 was prepared by centrifugation at 2,000 rpm and 25 ° C. for 20 minutes.
The exosome-destroying reagent A4 (100 μL) prepared in Example 1 above was added to the prepared serum 1 (900 μL) and allowed to stand for 10 minutes to obtain _{serum 1 A4.}
Further, the control reagent B0 (100 μL) prepared in Comparative Example 2 above was added to the prepared serum 1 (900 μL) and allowed to stand for 10 minutes to obtain _{serum 1 B0.}
(2) Preparation of measurement reagent used for sandwich method using antigen-antibody reaction Streptavidin-bound magnetic particle solution, biotin-bound anti-CD9 monoclonal antibody solution, and ALP labeling by the same method as (4) in Reference Example 1 above. An anti-CD9 monoclonal antibody solution was prepared.
(3) Measurement by sandwich method using antigen-antibody reaction In _{each serum (10 μL) of serum 1 A4} and serum 1 _B0 prepared in (1) above, a streptavidin-bound magnetic particle solution (30 μL) prepared in (2) above was added. , The biotin-binding anti-CD9 monoclonal antibody solution (30 μL) prepared in (2) above and the ALP-labeled anti-CD9 monoclonal antibody solution (30 μL) prepared in (2) above were added, stirred, and reacted at 37 ° C. for 10 minutes. I let you. Next, the magnetic particles were collected by magnetic force to remove the reaction solution other than the magnetic particles, and the collected magnetic particles were washed 5 times with the cleaning solution used in (5) of Reference Example 1 above. Then, the collected and washed magnetic particles emit light containing 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium salt (Lumigen ^TM APS-5) as a main component. 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 amount in the sandwich method and calculation of S / N ratio The blank luminescence amount 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 and the light emission amount 1 _A4 and the light emission amount 1 _{B0 obtained in (3) above by the following formula.}

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 rate The relative value of _{the S / N ratio 1 A4} obtained in (4) above is calculated, assuming that the S / N ratio 1 _B0 obtained in (4) above is 100. Then, the S / N ratio residual ratio was set to 1. The results are shown in Table 7.
Here, the closer the S / N ratio survival rate is to 100, the more exosomes are not destroyed. That is, the closer the S / N ratio survival rate is to 0, the more exosomes are destroyed.
(6) Calculation of S / N Ratio Residual Rate in Plasma 1, Serum 2 and Plasma 2 Except for using EDTA plasma 1 obtained from healthy person 1 in (1) above as a sample, the above (1) to ( The S / N ratio residual rate 1'was calculated by the same method as in 5). The results are shown in Table 7.
In addition, S / S is performed by the same method as in (1) to (5) above, except that serum 2 or EDTA plasma 2 obtained from healthy person 2 different from healthy person 1 in (1) above is used as a sample. The N ratio residual rate 2 and the S / N ratio residual rate 2'were calculated respectively. The results are shown in Table 7.

As is clear from Table 7, it was found that the addition of Nonion NS-204.5 destroys exosomes in both serum and plasma samples obtained from healthy subjects 1 in a short time. did. In addition, it was found that the addition of Nonion NS-204.5 destroys exosomes in both serum and plasma samples obtained from healthy subjects 2 in a short time. Therefore, it was clarified that the exosome-destroying reagent of the present invention can easily and quickly destroy exosomes in both serum and plasma samples regardless of the difference between healthy subjects.

[Example 9]
<Preparation of reagents for exosome destruction>
Nonion NS-204.5 was added to PBS to the concentration shown in Table 8 below to prepare reagents A6 to A8 for exosome destruction, respectively.

[Example 10]
<Destroying exosomes in serum by reagents A6 to A8 for destroying exosomes>
(1) Preparation of serum and treatment of serum All 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 sera.
To the prepared serum (900 μL), each reagent (100 μL) of the exosome-destroying reagents _{A6 to A8} prepared in Example 9 above was added and allowed to stand for 10 minutes to obtain serum A6, serum _A7 and serum A8, respectively. It was.
Further, the control reagent B0 (100 μL) prepared in Comparative Example 2 above was added to the prepared serum (900 μL) and allowed to stand for 10 minutes to obtain _{serum B0.}
(2) Preparation of measurement reagent used for sandwich method using antigen-antibody reaction Streptavidin-bound magnetic particle solution, biotin-bound anti-CD9 monoclonal antibody solution, and ALP labeling by the same method as (4) in 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) above and the ALP-labeled anti-CD9 monoclonal antibody solution (30 μL) prepared in (2) above are added, stirred, and reacted at 37 ° C. for 10 minutes. It was. Next, the magnetic particles were collected by magnetic force to remove the reaction solution other than the magnetic particles, and the collected magnetic particles were washed 5 times with the cleaning solution used in (5) of Reference Example 1 above. Then, the collected and washed magnetic particles emit light containing 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridane disodium salt (Lumigen ^TM APS-5) as a main component. The substrate solution (100 μL) was added and stirred, and the luminescence amount _A6 , the luminescence amount _A7 , the luminescence amount _A8 and the luminescence amount _B0 were measured, respectively.

(4) Measurement of blank luminescence amount in the sandwich method and calculation of S / N ratio The blank luminescence amount 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 light emission amount of the light emission amount A6} , the light emission amount _A7 , the light emission amount _A8, and the light emission amount _B0 obtained in (3) above by the following formula.

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 Luminous amount

(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 ratios A6} and S / N obtained in (4) above are taken. Relative values of the ratio _A7 and the S / N ratio _A8 were calculated and used as the S / N ratio residual rate _A6 , the S / N ratio residual rate _A7, and the S / N ratio residual rate _A8 , respectively. The results are shown in Table 9. Here, the closer the S / N ratio survival rate is to 100, the more exosomes are not destroyed. That is, the closer the S / N ratio survival rate is to 0, the more exosomes are destroyed.

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

INDUSTRIAL APPLICABILITY The present invention provides a simple and rapid method for destroying exosomes in a sample and a reagent for destroying the exosome.

Claims (4)

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

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