WO2019159944A1

WO2019159944A1 - Method, reagent, and kit for measuring exosomes in sample

Info

Publication number: WO2019159944A1
Application number: PCT/JP2019/005026
Authority: WO
Inventors: 健太郎庄司; 小野　達也; 豪永井
Original assignee: 協和メデックス株式会社
Priority date: 2018-02-14
Filing date: 2019-02-13
Publication date: 2019-08-22
Also published as: JP7281092B2; JPWO2019159944A1

Abstract

Provided is a method for measuring exosomes in a sample by using an antigen-antibody reaction, the method being characterized in that the antigen-antibody reaction is carried out in the presence of a polyoxyethylene-based surfactant having an aromatic ring without destroying the exosomes. This method for measuring exosomes in a sample is useful for diagnosing cancer or the like.

Description

Method, reagent and kit for measuring exosomes in a sample

The present invention relates to a method for measuring exosomes in a sample, a measuring reagent, and a measuring kit.

Exosomes are vesicular granules having a lipid bilayer structure with a diameter of 30 to 200 nm that are secreted from animal cells. It is known that various membrane proteins exist on the exosome surface as well as general cell surfaces, and microRNA (miRNA) may be contained inside 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, such as cells of the immune system and various cancer cells, functioning as mediators of intercellular communication in vivo and related to physiological phenomena, Relevance to diseases such as cancer is drawing attention.

Until now, exosomes have been measured by centrifuging serum and other biological components to isolate exosomes, and measuring the isolated exosomes by Western blotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), etc. An exoscreen method using an immunoassay method using a first antibody that specifically binds to a first antigen possessed by an exosome and a second antibody that specifically binds to a second antigen possessed by an exosome (Patent Document 1), etc. It has been known.

In recent years, in an exosome separation method, a complex in which an exosome is brought into contact with a solid phase carrier bound with a ligand that recognizes a surface antigen present on the surface of the exosome to form a complex of the exosome and the solid phase carrier. A body forming step and a washing step for 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 that does not contain an aromatic group in the molecule. A method for separating exosomes characterized in that it is carried out has been reported (Patent Document 2).

International Publication No. 2013/094307 International Publication No. 2015/068772

Previous methods for measuring exosomes have been cumbersome and not sensitive enough. An object of the present invention is to provide a simple and sensitive method for measuring exosomes in a sample, a measuring reagent, and a measuring kit.

As a result of intensive studies to solve the above problems, the present inventors have a specific aromatic ring for the reaction between a sample containing exosomes and an antibody or an antibody fragment that binds to an antigen that exosomes have on its surface. The present invention was completed by finding that exosomes in a sample can be measured easily and with high sensitivity without destroying exosomes by performing in an aqueous medium containing a polyoxyethylene-based surfactant.

That is, the present invention relates to the following [1] to [17].
[1] A method for measuring an exosome in a sample using an antigen-antibody reaction, wherein the antigen-antibody reaction is performed in the presence of a polyoxyethylene-based surfactant having an aromatic ring that does not destroy the exosome. A method for measuring exosomes in a sample.
[2] Including the following steps (1) to (3), the reaction of step (1) and / or step (2) contains a polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes The method according to [1], which is performed in an aqueous medium.
(1) A sample is reacted with a first antibody or the antibody fragment that binds to a first antigen on the surface of the exosome in an aqueous medium, and the first antibody or the antibody fragment and the exosome Producing an immune complex 1 comprising:
(2) A second antibody or antibody fragment that binds to a second antigen on the surface of an exosome is reacted with the immune complex 1 produced in the step (1) in an aqueous medium, and the first antibody or Producing an immune complex 2 comprising the antibody fragment, the exosome, and the second antibody or the antibody fragment;
(3) A step of measuring the immune complex 2 produced in the step (2).
[3] The polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes is a polyoxyethylene alkylphenyl ether derivative or a polyoxyethylene polycyclic phenyl ether derivative [1] or [2] Method.
[4] The polyoxyethylene alkylphenyl ether derivative is a polyoxyethylene alkylphenyl ether or a polyoxyethylene alkylphenyl ether sulfate having an HLB (Hydrophilic-Lipophilic Balance) value of 14 or more and 20 or less [3] The method described.
[5] The method according to [3] or [4], wherein the polyoxyethylene polycyclic phenyl ether derivative is polyoxyethylene polycyclic phenyl ether or polyoxyethylene polycyclic phenyl ether sulfate ester salt.
[6] The method according to any one of [2] to [5], wherein at least one of the first antigen and the second antigen is an antigen selected from the group consisting of CD9, CD63 and CD81.

[7] The first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, the second antibody or the antibody fragment that binds to the second antigen that the exosome has on its surface, and the exosome does not destroy, A reagent for measuring exosomes in a sample, comprising a polyoxyethylene surfactant having an aromatic ring.
[8] The reagent according to [7], wherein the polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes is a polyoxyethylene alkylphenyl ether derivative or a polyoxyethylene polycyclic phenyl ether derivative.
[9] The reagent according to [8], wherein the polyoxyethylene alkylphenyl ether derivative is polyoxyethylene alkylphenyl ether having an HLB value of 14 or more and 20 or less, or polyoxyethylene alkylphenyl ether sulfate ester salt.
[10] The reagent according to [8] or [9], wherein the polyoxyethylene polycyclic phenyl ether derivative is polyoxyethylene polycyclic phenyl ether or polyoxyethylene polycyclic phenyl ether sulfate ester salt.
[11] The reagent according to any one of [7] to [10], wherein at least one of the first antigen and the second antigen is an antigen selected from the group consisting of CD9, CD63 and CD81.

[12] A first reagent containing the first antibody or antibody fragment that binds to the first antigen that the exosome has on its surface, and the second antibody or antibody fragment that binds to the second antigen that the exosome has on its surface Measurement of exosomes in a sample, wherein the polyoxyethylene-based surfactant having an aromatic ring is contained in the first reagent and at least one of the second reagents, and the second reagent contains a second reagent containing kit.
[13] A first reagent containing the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, the second reagent or the antibody fragment that binds to the second antigen that the exosome has on its surface And a third reagent containing a polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes.
[14] The polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes is a polyoxyethylene alkylphenyl ether derivative or a polyoxyethylene polycyclic phenyl ether derivative [12] or [13] kit.
[15] The kit according to [14], wherein the polyoxyethylene alkylphenyl ether derivative is polyoxyethylene alkylphenyl ether having an HLB value of 14 or more and 20 or less, or polyoxyethylene alkylphenyl ether sulfate ester salt.
[16] The kit according to [14] or [15], wherein the polyoxyethylene polycyclic phenyl ether derivative is polyoxyethylene polycyclic phenyl ether or polyoxyethylene polycyclic phenyl ether sulfate ester salt.
[17] The kit according to any one of [12] to [16], wherein at least one of the first antigen and the second antigen is an antigen selected from the group consisting of CD9, CD63, and CD81.

The present invention provides a method for measuring exosomes in a sample that is simple and highly sensitive, a measurement reagent, and a measurement kit.

1. Measurement method The method for measuring exosomes in a sample of the present invention is a method for measuring exosomes in a sample by using an antigen-antibody reaction, wherein the antigen-antibody reaction is polyoxyethylene having an aromatic ring that does not destroy exosomes. This method is characterized in that it is performed in the presence of a surfactant, and the antigen that the exosome has on its surface, that is, the exosome surface antigen, is measured using antigen-antibody reaction without destroying the exosome in the sample. It is a method to do. Here, “without destroying the exosome in the sample” means that the lipid bilayer structure peculiar to exosome is retained, and the exosome surface antigen is retained on the lipid bilayer membrane.

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

The sample in the present invention is not particularly limited as long as exosomes can be measured, and examples thereof include blood, urine, saliva, milk, nasal discharge, seminal plasma, cerebrospinal fluid, cell culture supernatant and the like. preferable. Examples of the blood include whole blood, serum, and plasma, and serum and plasma are preferable.

The method for measuring exosomes in the sample of the present invention is not particularly limited as long as it uses an antigen-antibody reaction, and examples thereof include a sandwich method and a competition method.

[Measurement method 1: Sandwich method]
The method for measuring exosomes in a sample of the present invention comprises the following steps (1A) to (3A), wherein the reaction of step (1A) and / or step (2A) does not destroy exosomes and has an aromatic ring. This is a method carried out in an aqueous medium containing an oxyethylene-based surfactant.
(1A) a first antibody or the antibody fragment that binds to the first antigen by reacting the sample with the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface; Producing an immune complex 1 comprising an exosome having the first antigen;
(2A) The second antibody or the antibody fragment that binds to the second antigen on the surface of the exosome is reacted with the immune complex 1 produced in the step (1A) in an aqueous medium, An immune complex 2 comprising a first antibody that binds or the antibody fragment, an exosome having the first antigen and the second antigen, and a second antibody or the antibody fragment that binds to the second antigen is generated. Process;
(3A) A step of measuring the immune complex 2 produced in the step (2A).

<Process (1A)>
Examples of the sample in the step (1A) include the above-described samples.
In the step (1A), the method of reacting the sample with the first antibody or the antibody fragment that binds to the first antigen that the exosome has on the surface thereof in an aqueous medium includes the exosome and the exosome that the exosome has on the surface. There is no particular limitation as long as it is a method for generating an immune complex 1 comprising the first antibody or antibody fragment that binds to one antigen.

The first antibody that binds to the first antigen on the surface of the exosome or the antibody fragment may or may not be immobilized on an insoluble carrier, but is preferably immobilized.
When the first antibody or the antibody fragment that binds to the first antigen on the surface of the exosome is immobilized on an insoluble carrier, the insoluble carrier on which the first antibody or the antibody fragment is immobilized is an antigen-antibody reaction. In this case, the first antibody or the antibody fragment to which one of the combination of affinity substances (A) is bound and the other of the combination of affinity substances ( The insoluble carrier to which a) is bound is reacted in the reaction solution of the antigen-antibody reaction, thereby generating an insoluble carrier in which the first antibody or the antibody fragment is immobilized in the reaction solution of the antigen-antibody reaction. Can do.

Using the first antibody or antibody fragment to which one (A) of a set of affinity substances is bound and an insoluble carrier to which the other (a) of the set of affinity substances is bound, Examples of the method for generating immune complex 1 in (1A) include the following methods.
A sample, the first antibody or the antibody fragment to which one (A) of a pair of affinity substances is bound, and an insoluble carrier to which the other (a) of the pair of affinity substances is bound A method of reacting simultaneously in an aqueous medium to produce immune complex 1;
-After reacting the sample and the first antibody or the antibody fragment to which one of the combination of affinity substances (A) is bound in an aqueous medium, A method of adding an insoluble carrier to which the other (a) of the combination of affinity substances is bound to produce immune complex 1;
An aqueous medium comprising the first antibody or the antibody fragment to which one (A) of a pair of affinity substances is bound and the insoluble carrier to which the other (a) of the pair of affinity substances is bound; A method in which the immune complex 1 is generated by adding a sample to the reaction solution after the reaction in the reaction.

Examples of combinations of Aa include the following combinations.
A combination of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
-Combinations of avidins (avidin, neutravidin, streptavidin, etc.) and biotin;
A combination of the Fc region of the first antibody that binds to the first antigen that the exosome has on its surface and the antibody that binds to the Fc region.

The insoluble carrier is not particularly limited as long as it is an insoluble carrier that immobilizes the first antibody that binds to the first antigen that the exosome has on its surface or the antibody fragment and enables the method of measuring exosomes of the present invention. Synthetic resin plates such as microtiter plates, glass or synthetic resin granules (beads), glass or synthetic resin spheres (balls), latex, magnetic particles, various membranes such as nitrocellulose membrane, synthesis Examples include resin test tubes. 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 antigen on the surface of the exosome to the insoluble carrier is not particularly limited as long as it allows the exosome measurement method of the present invention, Examples include physical adsorption and immobilization by chemical bonding. Examples of physical adsorption include electrostatic bonding, hydrogen bonding, and hydrophobic bonding. Examples of chemical bonds include covalent bonds and coordinate bonds.

The first antibody or the antibody fragment that binds to the first antigen on the surface of the exosome may be directly immobilized on an insoluble carrier using the above-described physical adsorption and / or chemical bond, or indirectly. It may be immobilized on an insoluble carrier. As an indirect immobilization method, for example, the first antibody that binds to the first antigen that the exosome has on its surface through specific binding between biotin and avidins (such as avidin, streptavidin, neutravidin, etc.) Examples thereof include a method of immobilizing an antibody fragment on an insoluble carrier. Further, the first antibody or the antibody fragment that binds to the first antigen that the exosome has on the surface thereof may be immobilized on an insoluble carrier by covalent bond via a linker.

Examples of the linker include a molecule capable of covalently binding both the functional group on the surface of the insoluble carrier and the functional group of the first antibody or antibody fragment that binds to the first antigen that the exosome has on its surface. The first reactive group capable of reacting with the functional group of the first antibody or antibody fragment that binds to the first antigen on the surface thereof, and the second reactive group capable of reacting with the functional group on the surface of the insoluble carrier Molecules having the same reactive active group, wherein the first reactive active group and the second reactive active group are different groups are preferably used. Examples of the functional group possessed by the first antibody or antibody fragment that binds to the first antigen on the surface of the exosome and the functional group retained on the surface of the insoluble carrier include a carboxyl group, an amino group, a glycidyl group, Examples thereof include a sulfhydryl group, a hydroxyl group, an amide group, an imino group, an N-hydroxysuccinimide group, and a maleimide group. Examples of the reactive group in the linker include aryl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, isothiocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, psoralen. And groups such as pyridyl disulfide and vinyl sulfone.

In the step (1A), the reaction temperature of the reaction between the sample and the first antibody or the antibody fragment that binds to the first antigen that the exosome has on the surface thereof is a temperature that enables the exosome measurement method of the present invention. There is no particular limitation, 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 exosome measurement method of the present invention, and is usually 1 minute to 24 hours, preferably 5 minutes to 3 hours, and 10 minutes to 2 hours. Is particularly preferred. The concentration of the first antibody or antibody fragment that binds to the first antigen on the surface of the exosome in the reaction solution is not particularly limited as long as it is a concentration that enables the exosome measurement method of the present invention. 100 μg / mL, preferably 0.03 to 20 μg / mL, particularly preferably 0.05 to 10 μg / mL. The concentration of the polyoxyethylene surfactant having an aromatic ring that does not destroy the exosome in the reaction solution is not particularly limited as long as it is a concentration that enables the exosome measurement method of the present invention, and is generally 0.00005 to 10.0% ( w / v), preferably 0.0001 to 5.0% (w / v), particularly preferably 0.0025 to 1.0% (w / v).

Alternatively, after the sample and an aqueous solution of a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes are mixed in advance, the obtained mixed solution may be subjected to an antigen-antibody reaction. An aqueous solution of a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes can be prepared by dissolving a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes in an aqueous medium described below. it can.

The temperature at which the sample and the aqueous solution of the polyoxyethylene-based surfactant having an aromatic ring that does not destroy the exosome is preliminarily mixed is not particularly limited as long as the temperature enables the exosome measurement method of the present invention. Usually, it is 0 to 50 ° C., preferably 4 to 45 ° C., and particularly preferably 15 to 40 ° C. The mixing time is not particularly limited as long as it enables the exosome measurement method of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, particularly preferably 30 minutes to 2 hours. . The concentration of the polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes in the mixed solution is not particularly limited as long as it is a concentration that enables the method for measuring exosomes of the present invention, and is usually 0.0005 to 30.0. % (w / v), preferably 0.001 to 20.0% (w / v), particularly preferably 0.025 to 10.0% (w / v).

≪Process (2A) ≫
In the step (2A), the immune complex 1 generated in the step (1A) may be reacted with the second antibody or the antibody fragment that binds to the second antigen that the exosome has on its surface, or the exosome May be reacted by adding the immune complex 1 produced in the step (1A) to the second antibody or antibody fragment that binds to the second antigen on the surface thereof.

In the step (2A), the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, the exosome, and the second antibody or the antibody fragment that binds to the second antigen that the exosome has on its surface; The reaction temperature of the reaction for producing the immune complex 2 is not particularly limited as long as it enables the method for measuring exosomes of the present invention, and is usually 0 to 50 ° C., and 4 to 45 ° C. A temperature of 15 to 40 ° C. is preferable. The reaction time of the reaction is not particularly limited as long as it enables the exosome measurement method of the present invention, and is 1 minute to 24 hours, preferably 5 minutes to 3 hours, and particularly 10 minutes to 2 hours. preferable. The concentration of the second antibody or antibody fragment that binds to the second antigen on the surface of the exosome in the reaction solution is not particularly limited as long as it is a concentration that enables the exosome measurement method of the present invention. 100 μg / mL, preferably 0.03 to 20 μg / mL, more preferably 0.05 to 10 μg / mL. The concentration of the polyoxyethylene surfactant having an aromatic ring that does not destroy the exosome in the reaction solution is not particularly limited as long as it is a concentration that enables the exosome measurement method of the present invention, and is generally 0.00005 to 10.0% ( w / v), preferably 0.0001 to 5.0% (w / v), particularly preferably 0.0025 to 1.0% (w / v).

Step (1A) and step (2A) may be performed sequentially or simultaneously.
When the step (1A) and the step (2A) are performed simultaneously, the sample, the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, and the second antigen that the exosome has on its surface The second antibody that binds to the antibody or the antibody fragment is reacted simultaneously in an aqueous medium, and the immune complex 2 comprising the first antibody or the antibody fragment, the exosome, and the second antibody or the antibody fragment And exosomes in the sample are measured by measuring the generated immune complex 2. When the step (1A) and the step (2A) are performed at the same time, the immune complex 2 is preferably generated on the above-described insoluble carrier. Examples of the method for generating the immune complex 2 on the insoluble carrier include the following methods.
(i) A sample comprising a first antibody or antibody fragment that binds to a first antigen that exosome has on its surface, a second antibody or antibody fragment that binds to a second antigen that exosome has on its surface, and an insoluble carrier And simultaneously reacting in an aqueous medium;
(ii) a sample, a first antibody that binds to a first antigen that the exosome has on its surface or an insoluble carrier on which the antibody fragment is immobilized, and a second antibody that binds to a second antigen that the exosome has on its surface Alternatively, a method of reacting the antibody fragment with an aqueous medium at the same time In the method (i), the first antibody or the antibody fragment to which one (A) of a pair of affinity substances is bound, It is preferable to use an insoluble carrier to which the other (a) of the combination of affinity substances is bound. Examples of the combination (A)-(a) include the combinations described above.

Moreover, you may add the process of wash | cleaning the insoluble support | carrier after reaction of a process (1A) between a process (1A) and a process (2A) as needed. The washing solution used for washing the insoluble carrier after the reaction in the step (1A) is not particularly limited as long as it is a washing solution that enables the exosome measurement method of the present invention. For example, phosphate buffered saline ( 10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2, hereinafter referred to as PBS), PBS containing a surfactant, and the like. Examples of the surfactant include nonionic surfactants such as Tween 20.
After the step (2A), a washing step may or may not be added, but it is preferable to add it. The washing solution used for washing the insoluble carrier after the step (2A) is not particularly limited as long as it is a washing solution that enables the exosome measurement method of the present invention, and examples thereof include the aforementioned washing solution.

The first antigen that the exosome in step (1A) has on its surface and the second antigen that the exosome in step (2) has on its surface may be different or the same. Examples of the combination of the first antigen and the second antigen include combinations shown in Table 1 described later.

≪Process (3A) ≫
In step (3A), the exosome concentration in the sample is determined by measuring immune complex 2 produced in step (2A) using the following method.

(I) When the second antibody or the antibody fragment that binds to the second antigen on the surface of the exosome is not labeled The exosome binds to the second antibody or the antibody fragment that binds to the second antigen on the surface The antibody (hereinafter referred to as the third antibody) or the labeled third antibody or the antibody fragment in which the label is bound to the antibody fragment of the third antibody is added to the immune complex 2 (the first antigen that the exosome has on its surface) The second antibody or the antibody in an immunocomplex comprising the first antibody or the antibody fragment to bind, the exosome, and the second antibody or the antibody fragment that binds to the second antigen that the exosome has on its surface) Reacting with a fragment, the first antibody or the antibody fragment, an exosome, the second antibody or the antibody fragment, a labeled third antibody or the antibody fragment, To form an immune complex 3 made, the label in the immune complex 3 by measuring by the method described later, it is possible to measure the amount of immune complex 2 produced in step (2A). Examples of the third antibody include an antibody that binds to the Fc region of the second antibody that binds to the second antigen that exosome has on its surface, or the antibody fragment. Examples of the label include a label described later.

The reaction temperature of the reaction between the labeled third antibody or the antibody fragment and the second antibody or the antibody fragment that binds to the second antigen on the surface of the exosome in the immune complex 2 is determined by the reaction temperature of the exosome of the present invention. The temperature is not particularly limited as long as it enables the measurement method, 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 enables the exosome measurement method of the present invention, and is usually 1 minute to 24 hours, preferably 5 minutes to 3 hours, and 10 minutes to 2 hours. Is particularly preferred. The concentration of the labeled third antibody or the antibody fragment in the reaction solution is not particularly limited as long as it allows the measurement method of exosomes of the present invention, and is usually 0.01 to 100 μg / mL, and 0.03 to 20 μg / mL. mL is preferable, and 0.05 to 10 μg / mL is preferable.

(Ii) When the second antibody or the antibody fragment that binds to the second antigen on the surface of the exosome is labeled The immunity generated in step (2A) by measuring the label in the immune complex 2 The complex 2 can be measured. That is, the immune complex produced in the step (2A) is measured by measuring the label in the immune complex 2 comprising the first antibody or the antibody fragment, the exosome, and the labeled second antibody or the antibody fragment. The body 2 can be measured. The labeled second antibody or the antibody fragment is a substance in which a label described later is bound to the second antibody or the antibody fragment, and can be prepared by a known method.

Examples of the label include an enzyme, a fluorescent substance, a luminescent substance, a radioisotope, biotin, digoxigenin, a polypeptide containing a tag sequence, metal colloid particles, and colored latex particles.

Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like.

Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), rhodamine B-isothiocyanate (RITC), and the like. Examples of other fluorescent substances include quantumquantdot (Science, 281, 2016-2018, 1998), phycobiliproteins such as phycoerythrin, GFP (Green fluorescent Protein), RFP (Red fluorescent Protein), YFP (Yellow fluorescent Protein) And fluorescent proteins such as BFP® (Blue® fluorescent® Protein).

Examples of the luminescent substance include acridinium esters and derivatives thereof, ruthenium complex compounds, and lophine. Examples of the ruthenium complex compound include ruthenium complex compounds shown in Clin. Chem. 37, 9, 1534-1539, 1991.

Examples of the radioisotope include ³ H, ¹⁴ C, ³⁵ S, ³² P, ¹²⁵ I, and ¹³¹ I. Examples of the polypeptide containing the tag sequence include FLAG. Examples of the metal colloid include gold colloid.

A first antibody or antibody fragment that binds to a first antigen on the surface of an exosome; an exosome; and a labeled second antibody or antibody fragment that binds to a second antigen on the surface of the exosome; Measurement of the label in the immune complex 2 comprising, and the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, the exosome, and the exosome that binds to the second antigen that the surface has The measurement of the label in the immune complex 3 composed of the second antibody or the antibody fragment and the labeled third antibody or the antibody fragment can be appropriately selected depending on the label used.

When the label is a coloring substance, that is, a substance that absorbs light of a certain wavelength, the absorbance can be measured using a spectrophotometer, a multiwell plate reader, or the like.

When the label is a fluorescent substance, the fluorescence intensity can be measured using a fluorometer, a fluorescent multiwell plate reader or the like.

When the label is a luminescent substance, the luminescence intensity can be measured using a luminescence photometer, a luminescence multiwell plate reader or the like.

When the label is a radioisotope, the radioactivity can be measured by a scintillation counter, a γ-well counter or the like.

When the label is an enzyme, the amount of label can be quantified by measuring the enzyme activity. For example, the amount of labeling can be measured by reacting a substrate of the enzyme with the enzyme and measuring the produced substance.

When the enzyme is peroxidase, the peroxidase activity can be measured by, for example, an absorbance method, a fluorescence method, or the like. As a method for measuring peroxidase activity by the absorbance method, for example, peroxidase is reacted with a combination of hydrogen peroxide and an oxidative coloring chromogen as a substrate, and the absorbance of the reaction solution is measured with a spectrophotometer or a multiwell plate reader. The method of measuring by etc. is mentioned. Examples of the oxidative coloring chromogen include a leuco chromogen and an oxidative coupling coloring chromogen.

The leuco chromogen is a substance that is converted into a pigment by itself 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] An amine (BCMA) etc. are mentioned.

The oxidative coupling chromogen is a substance that forms 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 and an aniline (Trinder reagent), a combination of a coupler and 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-Dimethylani 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 can be mentioned.

Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.

As a method for measuring peroxidase activity by a fluorescence method, for example, the peroxidase is reacted with a combination of hydrogen peroxide and a fluorescent substance as a substrate, and the intensity of fluorescence generated by a fluorometer, a fluorescent multiwell plate reader or the like is measured. The measuring method etc. are mentioned. Examples of the fluorescent substance include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, and coumarin.

As a method for measuring peroxidase activity by a luminescence method, for example, a peroxidase is reacted with a combination of hydrogen peroxide and a luminescent substance as a substrate, and the intensity of luminescence generated by a luminescence intensity meter or a luminescence multiwell plate reader is measured. The measuring method etc. are mentioned. Examples of the luminescent substance include a luminol compound and a lucigenin compound.

When the enzyme is alkaline phosphatase, the alkaline phosphatase activity can be measured by, for example, a luminescence method. Examples of the method for measuring alkaline phosphatase activity by a luminescence method include a method in which alkaline phosphatase and its substrate are reacted and the luminescence intensity of the produced luminescence is measured with a luminescence intensity meter, a luminescence multiwell plate reader, or the like.

Examples of alkaline phosphatase substrates include 3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), 2-chloro- 5- {4-methoxyspiro [1,2-dioxetane-3,2 '-(5'-chloro) tricyclo [3.3.1.1 ^3.7 ] decane] -4-yl} phenyl phosphate disodium salt (CDP-Star ^TM ), 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-methylacridane disodium, 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium (Lumigen ^™ APS-5) and the like.

When the enzyme is β-D-galactosidase, β-D-galactosidase activity can be measured by, for example, an absorbance method (colorimetric method), a luminescence method, or a fluorescence method. As a method of measuring β-D-galactosidase activity by absorbance method (colorimetric method), β-D-galactosidase and its substrate are reacted, and the absorbance of the reaction solution is measured with a spectrophotometer or a multiwell plate reader. And the like. Examples of the substrate for β-D-galactosidase in the method for measuring β-D-galactosidase activity by an absorbance method (colorimetric method) include o-nitrophenyl-β-D-galactopyranoside. As a method for measuring β-D-galactosidase activity by a luminescence method, for example, a method in which β-D-galactosidase and its substrate are reacted and the luminescence intensity of the reaction solution is measured with a luminescence intensity meter, a luminescence multiwell plate reader, etc. Etc. Examples of the substrate for β-D-galactosidase in the method for measuring β-D-galactosidase activity by the luminescence method include galacton-plus (Galacton-Plus, manufactured by Applied Biosystems) and similar compounds. Can be mentioned. As a method of measuring β-D-galactosidase activity by a fluorescence method, for example, a method of reacting β-D-galactosidase and its substrate and measuring the fluorescence of the reaction solution with a fluorometer, a fluorescent multiwell plate reader, etc. Etc. Examples of the substrate for β-D-galactosidase in the method for measuring β-D-galactosidase activity by a fluorescence method include 4-methylumbelliferyl-β-D-galactopyranoside.

When the enzyme is luciferase, the luciferase activity can be measured by, for example, a luminescence method. Examples of the method for measuring luciferase activity by the luminescence method include a method of reacting luciferase with its substrate and measuring the luminescence intensity of the reaction solution with a luminescence intensity meter, a luminescence multiwell plate reader, or the like. Examples of the luciferase substrate include luciferin and coelenterazine.

When the label (label 1) is other than a fluorescent substance, a luminescent substance, a radioisotope, and an enzyme, a substance that specifically binds to the label (the label 1) is selected from a fluorescent substance, a luminescent substance, a radioisotope, an enzyme, etc. The labeled body labeled with the label (label 2) is reacted with the label in the immune complex 2 (label 1) or the label in the immune complex 3 (label 1), and the labeled body and the immune complex A label (label 1) in the body 2 or a complex with the label (label 1) in the immune complex 3 is generated, and the label (label 2) in the complex, that is, a fluorescent substance or a luminescent substance The label can be measured by measuring a radioisotope or an enzyme by the above-described method. Examples of the substance that specifically binds to the label include an avidin and the like in addition to an antibody that specifically binds to the label and when the label is biotin.

The concentration of exosome in the sample can be determined by performing the following step (4A) and step (5A) after step (3A).
(4A) Steps (1A) to (3A) are performed using a known concentration of exosome as a sample, and a calibration curve representing the relationship between the exosome concentration and the measured value of the label is created;
(5A) A step of determining the concentration of exosome in the sample from the calibration curve prepared in step (4A) and the measured value of the label measured in step (2A).

[Measurement method 2: competitive method]
As another embodiment of the method for measuring exosomes of the present invention, there is a competition method using a first antibody that binds to a first antigen that exosome has on its surface, or an antibody fragment thereof, and a competitive substance. In the competition method, a labeled competitor substance, or a labeled first antibody or the antibody fragment that binds to a first antigen that exosome has on its surface can be used.
A labeled competitor is a substance in which the aforementioned label is bound to a competitor, and can be prepared by a known method. The labeled first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface is the labeled first antibody or the antibody fragment that binds to the first antigen that the exosome has on the surface. It is a bound substance and can be prepared by known methods.
The competitive substance means a substance that binds to the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, and that the binding is competitive with the exosome that is the measurement target substance. In addition, the exosome itself as the measurement target substance and the first antigen are also included. The competitor is preferably a substance having the same structure as the epitope in the exosome recognized by the first antibody or the antibody fragment, and the binding strength to the first antibody or the antibody fragment Those having the same strength as the binding of the exosome to one antibody or the antibody fragment are more preferable, and specific examples include the exosome itself as the measurement target substance and the first antigen.

In the present invention, one embodiment of the competition method (competition method 1) includes, for example, a method including the following steps.
(1B) A polyoxyethylene-based surfactant having an aromatic ring that does not destroy the exosome, the sample, the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, and the labeled competitor To produce an immune complex 4 of the first antibody or the antibody fragment and an exosome, and an immune complex 4 of the first antibody or the antibody fragment and a labeled competitor The step of causing;
(2B) a step of measuring the label in the immune complex 4 of the first antibody or the antibody fragment produced in step (1B) and a labeled competitor;
(3B) performing the steps (1B) and (2B) using a known concentration of exosome instead of the sample, and creating a calibration curve representing the relationship between the exosome concentration and the measured value of the label;
(4B) A step of determining the exosome concentration in the sample from the calibration curve created in step (3B) and the measured value of the label measured in step (2B).

The first antibody or antibody fragment that binds to the first antigen on the surface of the exosome may or may not be immobilized on an insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

In the step (1B), after diluting the sample with an aqueous solution of a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes, the obtained diluted sample is diluted with the first antibody or the antibody fragment, And you may make it react with a labeled competitor. An aqueous solution of a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes can be prepared by dissolving a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes in an aqueous medium described below. it can.

When the first antibody or the antibody fragment is immobilized on an insoluble carrier, the insoluble carrier on which the first antibody or the antibody fragment is immobilized may be produced in a reaction solution for antigen-antibody reaction. In this case, the first antibody or the antibody fragment to which one (B) of a pair of affinity substances is bound and the insoluble carrier to which the other (b) of the pair of affinity substances is bound are antigen antibodies. By reacting in the reaction solution, an insoluble carrier having the first antibody or the antibody fragment immobilized thereon can be produced in the antigen-antibody reaction solution. Examples of the combination of BB include the following combinations.
A combination of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
-Combinations of avidins (avidin, neutravidin, streptavidin, etc.) and biotin;
A combination of the Fc region of the first antibody and an antibody that binds to the Fc region.

The reaction temperature of the reaction between the sample and the first antibody or the antibody fragment in the step (1B) of the competition method 1 is not particularly limited as long as it is a temperature that enables measurement of the exosome of the present invention. 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 allows measurement of the exosome of the present invention, and is usually 1 minute to 24 hours, preferably 5 minutes to 3 hours, and preferably 10 minutes to 2 hours. Particularly preferred. The concentration of the first antibody or the antibody fragment in the reaction solution in the reaction is not particularly limited as long as it enables the method for measuring exosomes of the present invention, and is usually 0.01 to 100 μg / mL, 0.03 to 20 μg / mL is preferable, and 0.05 to 10 μg / mL is preferable. In the reaction, the concentration of the polyoxyethylene-based surfactant having an aromatic ring that does not destroy the exosome is not particularly limited as long as it is a concentration that enables the measurement method of the exosome of the present invention, and is generally 0.00005 to 10.0% ( w / v), preferably 0.0001 to 5.0% (w / v), particularly preferably 0.0025 to 1.0% (w / v).

The method for measuring the label in the step (2B) of the competition method 1 is not particularly limited as long as it is a method that enables measurement of the exosome of the present invention, and examples thereof include the method described above.

As another aspect (competition method 2) of the competition method in this invention, the method including the following processes is mentioned, for example.
(1C) A polyoxyethylene system having an aromatic ring that does not destroy the exosome, the sample, the labeled first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, and the competitor An immunocomplex of the labeled first antibody or the antibody fragment and exosome by reacting in an aqueous medium containing a surfactant, and the labeled first antibody or the antibody fragment and a competitor Generating an immune complex 5 with:
(2C) a step of measuring the label in the immune complex 5 of the labeled first antibody or antibody fragment produced in step (1C) and the competitor;
(3C) performing the steps (1C) and (2C) using a known concentration of exosome instead of the sample, and creating a calibration curve representing the relationship between the exosome concentration and the measured value of the label;
(4C) A step of determining the exosome concentration in the sample from the calibration curve created in step (3C) and the measured value of the label measured in step (2C).

The competitor is preferably immobilized on an insoluble carrier. Examples of the insoluble carrier include the aforementioned insoluble carrier. As a label | marker, the above-mentioned label | marker etc. are mentioned, for example.

In step (1C), the sample is diluted with an aqueous solution of a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes, and the diluted sample obtained is labeled with the first antibody. Or you may make it react with this antibody fragment and a competitor. An aqueous solution of a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes can be prepared by dissolving a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes in an aqueous medium described below. it can.

When the competitive substance is immobilized on the insoluble carrier, the insoluble carrier on which the competitive substance is immobilized may be produced in the reaction solution of the antigen-antibody reaction. In this case, one of the combinations of the affinity substances is used. The competitive substance was immobilized by reacting the competitive substance bound with (C) with the insoluble carrier bound with the other (c) of the pair of affinity substances in the reaction solution of the antigen-antibody reaction. An insoluble carrier can be produced in the reaction solution of the antigen-antibody reaction. Examples of the combination of Cc include the following combinations.
A combination of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
A combination of avidin (such as avidin, neutravidin, streptavidin, etc.) and biotin.

The reaction of the sample in the step (1C) of the competition method 2 with the labeled first antibody or the antibody fragment, and the reaction between the competitor and the labeled first antibody or the antibody fragment The reaction temperature is not particularly limited as long as it enables the measurement of 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 allows measurement of the exosome of the present invention, and is usually 1 minute to 24 hours, preferably 5 minutes to 3 hours, and preferably 10 minutes to 2 hours. Particularly preferred. The concentration of the labeled first antibody or antibody fragment in the reaction solution is not particularly limited as long as it allows the measurement method of exosome of the present invention, and is usually 0.01 to 100 μg / mL, 0.03 -20 μg / mL is preferable, and 0.05-10 μg / mL is preferable. In the reaction, the concentration of the polyoxyethylene-based surfactant having an aromatic ring that does not destroy the exosome is not particularly limited as long as it is a concentration that enables the measurement method of the exosome of the present invention, and is generally 0.00005 to 10.0% ( w / v), preferably 0.0001 to 5.0% (w / v), particularly preferably 0.0025 to 1.0% (w / v).

The method for measuring the label in the step (2C) of the competition method 2 is not particularly limited as long as it is a method that enables measurement of the exosome of the present invention, and examples thereof include the method described above.

In the present invention, the first antigen that the exosome has on its surface and the second antigen that the exosome has on its surface are not particularly limited as long as they are antigens that enable the exosome measurement method of the present invention. CD11, CD13, CD37, CD53, CD63, CD81, CD82, CD86, CD147, ICAM-1 (intercellular adhesion molecule-1: intercellular adhesion molecule-1), EpCAM (epithelial cell adhesion molecule: epithelial cell adhesion molecule), Rab5, Annexin V, or LAMP1 (lyssome-associated membrane protein 1: lysosomal membrane protein 1), GPRC5C (G-pro tein coupled Receptor familyC group 5 member C: G protein coupled receptor, family C, group 5, member C) and the like.

The first antigen that the exosome has on its surface and the second antigen that the exosome has on its surface may be different or the same. Examples of the combination of the first antigen and the second antigen include the combinations described in Table 1 below.

In the present invention, as the first antibody that binds to the first antigen that the exosome has on its surface and the second antibody that binds to the second antigen that the exosome has on its surface, the exosome measuring method of the present invention can be used. For example, Purifed であれば Mouse 抗体 Anti-Human CD9 (manufactured by Becton Dickinson of Japan, Purifed Mouse Anti-Human CD11a (manufactured by Becton of Dickinson, clone: 27 / CD11a), Purifed Mouse-Mouse-CD11a) Human CD11b / Mac-1 (Nippon Becton Dickinson Co., Ltd., clone: ICRF44), Purified Mouse Anti-Human 日本 CD11c (Nippon Becton Dickinson) Manufactured, clone: 3.9), Purified Mouse ｔｉ Anti-Human CD13 (manufactured by Becton Dickinson Japan, clone: WM15), Purified Mouse Anti-Human CD37 (manufactured by Becton Dickinson Japan, clone: M-B371 ed) Mouse Anti-Human CD53 (Nippon Becton Dickinson, Clone: HI29), Clone: M-L13), Purifed Mouse Anti-HumanCD63 (Nippon Becton, Dickinson, Purified Mouse Anti-Humanton, CD81) Manufactured, clone: JS-81), clone: H5C6), Anti-CD82 antibo y [C33] (manufactured by Abcam), Purified MouseAnti-Human CD86 (Nippon Becton Dickinson, clone: 2331 (FUN-1)), anti-CD147 antibody [MEM-M6 / 1] (manufactured by Novus biologics) Anti -ICAM1 antibody [HM1] (manufactured by Abcam), Anti-EpCAM antibody [323 / A3] (manufactured by Abcam), Purified Mouse Anti-Rab5 (manufactured by Becton Dickinson Japan, clone: 1 / Rab5), Anti-Annexin V antibody [EPR3979] (manufactured by Abcam), Purified Mouse Anti-Human Lamp-1 (Nippon Becton Dickinson, clone: 25 / Lamp-1), Anti-GPRC5C antibody-C-terminal (ab137482) (manufactured by Abcam), Rabbit Anti-Human GPRC5C (Extracellular Domain) [119-10086] (manufactured by RayBiotech), and the like.

Examples of the combination of the first antibody and the second antibody include the combinations described in Table 2 below.

In the present invention, the antibody fragment of the first antibody that binds to the first antigen on the surface of the exosome is particularly an antibody fragment that binds to the first antigen and enables the exosome measurement method of the present invention. There is no limitation, for example, Fab obtained by papain treatment of an antibody, F (ab ′) ₂ obtained by pepsin treatment of an antibody, Fab ′ obtained by pepsin treatment-reduction treatment of an antibody, etc. Examples thereof include antibody fragments from which the Fc portion has been removed, antibody fragments from which the Fc portion has been removed by genetic engineering techniques, and the like.
In the present invention, the antibody fragment of the second antibody that binds to the second antigen on the surface of the exosome is particularly an antibody fragment that binds to the second antigen and enables the exosome measurement method of the present invention. There is no limitation, for example, Fab obtained by papain treatment of an antibody, F (ab ′) ₂ obtained by pepsin treatment of an antibody, Fab ′ obtained by pepsin treatment-reduction treatment of an antibody, etc. Examples thereof include antibody fragments from which the Fc portion has been removed, antibody fragments 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 antigen that the exosome has on its surface is not particularly limited as long as it is an antibody that binds to the first antigen and enables the exosome measurement method of the present invention. Either a polyclonal antibody or a monoclonal antibody can be used.
In the present invention, the second antibody that binds to the second antigen that the exosome has on its surface is not particularly limited as long as it binds to the second antigen and enables the exosome measurement method of the present invention, Either a polyclonal antibody or a monoclonal antibody can be used.

In the present invention, the polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes is a polyoxyethylene surfactant having a function not to destroy exosomes and having an aromatic ring in the molecule. Examples of the aromatic ring in the polyoxyethylene surfactant having an aromatic ring that does not destroy the exosome include a benzene ring. Examples of the polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes in the present invention include polyoxyethylene alkylphenyl ether derivatives and polyoxyethylene polycyclic phenyl ether derivatives.

The polyoxyethylene alkylphenyl ether derivative is not particularly limited as long as it is a polyoxyethylene alkylphenyl ether derivative that has a function of not destroying exosomes and enables the exosome measurement method of the present invention. For example, the HLB value is 14 Examples thereof include polyoxyethylene alkylphenyl ethers and polyoxyethylene alkylphenyl ether sulfates that are 20 or less.

Examples of the alkyl in the polyoxyethylene alkylphenyl ether having an HLB value of 14 to 20 and the polyoxyethylene alkylphenyl ether sulfate ester include alkyl having 8 to 24 carbon atoms, and those having 10 to 20 carbon atoms. Alkyl is preferred. 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. 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, icosyl and the like. Can be mentioned.

Examples of commercially available polyoxyethylene alkylphenyl ethers having an HLB value of 14 or more and 20 or less include nonionic HS-215 (HLB value: 15.0), nonionic HS-220 (HLB value: 16.2), and nonionic HS. -240 (HLB value: 17.9), Nonion NS-215 (HLB value: 15.0), Nonion NS-220 (HLB value: 16.0), Nonion HS-240 (HLB value: 17.8) ( As mentioned above, Triton X-405 (HLB value: 17.9), Triton X-705 (HLB value: 18.4) (above, Sigma-Aldrich) and the like can be mentioned.

Examples of the salt in the polyoxyethylene alkylphenyl ether sulfate ester salt include lithium salt, sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, monoethanolamine salt and the like. Commercially available polyoxyethylene alkyl ether sulfate salts include, for example, Trax N-300, Trax N-700B (manufactured by NOF Corporation) Hightenol N-07, Hightenol N-08, Hightenol N-17 ( As mentioned above, Dai-ichi Kogyo Seiyaku Co., Ltd.) etc. are mentioned.

The polyoxyethylene polycyclic phenyl ether derivative is not particularly limited as long as it is a polyoxyethylene polycyclic phenyl ether derivative having a function of not destroying exosomes and enabling the exosome measurement method of the present invention. Examples thereof include ethylene polycyclic phenyl ether and polyoxyethylene polycyclic phenyl ether sulfate.

The polyoxyethylene polycyclic phenyl ether and the polycyclic phenyl in the polyoxyethylene polycyclic phenyl ether sulfate salt are a phenyl group in which two or more groups having one aromatic ring (substituent) in the group are substituted, Examples thereof include a phenyl group in which one or more groups (substituents) having two or more aromatic rings in the group are substituted. Examples of the group having one aromatic ring in the group include benzyl and 1- (phenyl) ethyl. Examples of the group having two or more aromatic rings in the group include naphthyl. Examples of commercially available products of polyoxyethylene polycyclic phenyl ether include New Coal 704, New Coal 706, New Coal 707, New Coal 708, New Coal 709, New Coal 710, New Coal 712, New Coal 714, New Coal 610, New Coal 2607, New Coal 2609, New Coal 2614 (manufactured by Nippon Emulsifier Co., Ltd.) and the like.

Examples of the salt in the polyoxyethylene polycyclic phenyl ether sulfate ester salt include lithium salt, sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, monoethanolamine salt and the like. Examples of commercially available polyoxyethylene polycyclic phenyl ether sulfates include Newcor 707-SF, Newcor 707-SFC, Newcoal 707-SN, Newcoal 714-SF, Newcoal 714-SN (and above, Japan) Emulsifiers).

In the method for measuring exosomes of the present invention, a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes, selected from the group consisting of polyoxyethylene alkylphenyl ether derivatives and polyoxyethylene polycyclic phenyl ether derivatives May be used in combination of two or more.

The aqueous medium in the present invention is not particularly limited as long as it is an aqueous medium that enables the exosome measurement method 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 set pH. The buffer used for preparing the buffer solution is not particularly limited as long as it has a buffering capacity. For example, a lactate buffer, a citrate buffer, an acetate buffer, a succinate buffer, a phthalate buffer, a phosphorus Acid buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, carbonate buffer, Good buffering agents and the like can be mentioned.

Examples of the good buffer 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) methyl] amino} ethanesulfonic 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) methylglycine] (Tricine) buffer, [N, N-bis (2-hydroxyethyl) glycine] (B icine) buffer, 3- [N-tris (hydroxymethyl) methyl] aminopropanesulfonic acid (TAPS) buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexyl) Amino) -2-hydroxypropanesulfonic acid (CAPSO) buffer, 3- (N-cyclohexylamino) propanesulfonic acid (CAPS) buffer, and the like.

The aqueous medium may contain salts, metal ions, sugars, preservatives, proteins, protein stabilizers 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, ammonium bromide and the like. . Examples of the metal ion include magnesium ion, manganese ion, zinc ion and the like. Examples of the saccharide include mannitol and sorbitol. Examples of the preservative include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), Bioace, Procrine 300, Proxel GXL, and the like. Examples of the protein include bovine serum albumin (hereinafter referred to as BSA). Examples of the protein stabilizer include peroxidase stabilization buffer (Peroxidase Stabilizing Buffer, manufactured by DakoCytomation).

2. Measurement Reagent The exosome measurement reagent of the present invention is a reagent used in the exosome measurement method of the present invention. Specific embodiments of the exosome measuring reagent of the present invention are described below.

・ Measurement reagent 1
A first antibody or antibody fragment that binds to the first antigen that the exosome has on its surface, a second antibody or antibody fragment that binds to the second antigen that the exosome has on its surface, and an aromatic ring that does not destroy the exosome A reagent containing a polyoxyethylene-based surfactant having the first antibody or the antibody fragment may or may not be immobilized on an insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

・ Measurement reagent 2
A reagent comprising a first antibody that binds to a first antigen that an exosome has on its surface, or an antibody fragment thereof, a labeled competitor, and a polyoxyethylene-based surfactant having an aromatic ring that does not destroy an exosome The antibody or the antibody fragment may or may not be immobilized on an insoluble carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier. Examples of labels and competitors include the aforementioned labels and competitors, respectively.

In the above-described measurement reagent 1 and measurement reagent 2, the insoluble carrier on which the first antibody or the antibody fragment that binds to the first antigen on the surface of the exosome is immobilized is the sample, the first antibody or the antibody fragment. It may be produced in the reaction solution of the reaction.
In this case, in the measurement reagent of the present invention, instead of the insoluble carrier on which the first antibody or the antibody fragment is immobilized, the first antibody to which one of a pair of affinity substances (D) is bound. Alternatively, the antibody fragment and an insoluble carrier to which the other (d) of the combination of a pair of affinity substances is bound are included. Examples of the combination of one set of affinity substances, that is, the combination of D and d include the following combinations.
-A combination of biotin and avidins (avidin, newtraavidin, streptavidin, etc.);
A combination of avidin (such as avidin, newtraavidin, streptavidin, etc.) and biotin;
A combination of the Fc region of the first antibody that binds to the first antigen that the exosome has on its surface and the antibody that binds to the Fc region.

・ Measurement reagent 3
Reagent containing a labeled polyoxyethylene-based surfactant having an aromatic ring that does not destroy the first antibody or the antibody fragment, the competitor, and the exosome that bind to the first antigen that the exosome has on its surface The competitor is preferably immobilized on an insoluble carrier. Examples of the insoluble carrier include the aforementioned insoluble carrier. Examples of labels and competitors include the aforementioned labels and competitors, respectively.

In the measurement reagent 3 described above, the insoluble carrier on which the competitive substance is immobilized may be generated in the reaction solution of the reaction between the sample and the competitive substance.
In this case, in the measurement reagent of the present invention, instead of the insoluble carrier on which the competitive substance is immobilized, the competitive substance bound with one (E) of the pair of affinity substances and the set of affinity substances And an insoluble carrier to which the other (e) of the combination is bound. Examples of a combination of affinity substances, that is, a combination of E and e, include the following combinations.
-A combination of biotin and avidins (avidin, newtraavidin, streptavidin, etc.);
A combination of avidins (such as avidin, newtraavidin, streptavidin, etc.) and biotin.

The measurement reagent of the present invention may be lyophilized or liquid. When a measurement reagent in a lyophilized state is used, it is dissolved in an aqueous medium before measurement and made into a liquid state for measurement. In the liquid measurement reagent, a polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes, the first antibody or the antibody fragment that binds to the first antigen that exosome has on its surface, the labeled first One antibody or the antibody fragment, the second antibody or the antibody fragment that binds to the second antigen on the surface of the exosome, the competitive substance, and the labeled competitive substance are dissolved in an aqueous medium. As an aqueous medium, the above-mentioned aqueous medium is mentioned, for example. The aqueous medium may contain the aforementioned salts, metal ions, sugars, preservatives, proteins, protein stabilizers, and the like.

In the measurement reagent of the present invention, the first antigen that exosome has on its surface and the second antigen that exosome has on its surface are the first antigen that exosome has on its surface and exosome on its surface. Each of the second antigens has. In the measurement reagent of the present invention, the combination of the first antigen that the exosome has on its surface and the second antigen that the exosome has on its surface includes the combination of the first antigen and the second antigen described above.

Examples of the polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes in the measurement reagent of the present invention include the aforementioned polyoxyethylene-based surfactants that have an aromatic ring and do not destroy exosomes. In the measurement reagent of the present invention, the content of the polyoxyethylene surfactant having an aromatic ring that does not destroy the exosome is not particularly limited as long as it is a content that enables the measurement method of the exosome of the present invention. In the aforementioned aqueous medium or in a state dissolved in the aforementioned aqueous medium, the content is 0.00005 to 10.0% (w / v), preferably 0.0001 to 5.0% (w / v), 0.0025 A content of ˜1.0% (w / v) is particularly preferred.
In the measurement reagent of the present invention, a combination of two or more polyoxyethylene surfactants having an aromatic ring that does not destroy exosomes may be contained.

In the measurement reagent of the present invention, the first antibody or antibody fragment that binds to the first antigen that exosome has on its surface, and the second antibody or antibody fragment that binds to the second antigen that exosome has on its surface include Examples include the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, and the second antibody or the antibody fragment that binds to the second antigen that the exosome has on its surface, respectively. In the measurement reagent of the present invention, the combination of the first antibody that binds to the first antigen that the exosome has on its surface and the second antibody that binds to the second antigen that the exosome has on its surface includes the above-mentioned first antibody And a combination with the second antibody. The content of the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface and the second antibody or the antibody fragment that binds to the second antigen that the exosome has on the surface in the measurement reagent of the present invention There is no particular limitation as long as it is a content that enables the method for measuring exosomes of the present invention, and it is usually 0.01 to 100 μg / mL in the aforementioned aqueous medium or dissolved in the aforementioned aqueous medium. Yes, 0.03 to 20 μg / mL is preferable, and 0.05 to 10 μg / mL is particularly preferable.

Examples of the label and the competitive substance in the measurement reagent of the present invention include the aforementioned label and the competitive substance, respectively.

3. Measurement Kit The exosome measurement reagent of the present invention can be in the form of a kit from the viewpoint of storage, transportation, distribution and the like. The measurement kit of the present invention is used in the exosome measurement method of the present invention. Specific embodiments of the exosome measurement kit of the present invention are described below.

・ Measurement kit 1
A first reagent containing the first antibody or antibody fragment that binds to the first antigen that the exosome has on its surface, and a second reagent that contains the second antibody or antibody fragment that binds to the second antigen that the exosome has on its surface And a polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes and is contained in at least one of the first reagent and the second reagent, the kit The first antibody or the antibody fragment is insoluble It may or may not be immobilized on the carrier, but is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

・ Measurement kit 2
A first reagent containing the first antibody or antibody fragment that binds to the first antigen that the exosome has on its surface, and a second reagent that contains the second antibody or antibody fragment that binds to the second antigen that the exosome has on its surface A kit comprising two reagents and a third reagent containing a polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes. Even if the first antibody or the antibody fragment is immobilized on an insoluble carrier, the kit is immobilized. However, it is preferable to be immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

In the measurement kit 1 and the measurement kit 2 described above, the insoluble carrier on which the first antibody or the antibody fragment that binds to the first antigen of the exosome is immobilized is a sample, the first antibody or the antibody fragment. It may be produced in the reaction solution of the reaction.
In this case, the first antibody to which one (F) of a pair of affinity substances is bound to the measurement reagent of the present invention instead of the insoluble carrier on which the first antibody or the antibody fragment is immobilized. Alternatively, the antibody fragment and an insoluble carrier to which the other (f) of the pair of affinity substances is bound are included. Examples of the combination of one set of affinity substances, that is, the combination of F and f include the following combinations.
-A combination of biotin and avidins (avidin, newtraavidin, streptavidin, etc.);
A combination of avidin (such as avidin, newtraavidin, streptavidin, etc.) and biotin;
A combination of the Fc region of the first antibody that binds to the first antigen that the exosome has on its surface and the antibody that binds to the Fc region.

The first antibody or antibody fragment to which one (F) of a pair of affinity substances is bound and the insoluble carrier to which the other (f) of the pair of affinity substances is bound are the same reagent. Although it may be contained in separate reagents, it is preferably contained in separate reagents.
The first antibody or antibody fragment to which one (F) of a set of affinity substances is bound and the insoluble carrier to which the other (f) of the set of affinity substances is bound are separate reagents. The following kits included in the kit, that is, the measurement kits 3 to 5 are also included in the kit of the present invention.

・ Measurement kit 3
A first reagent containing the first antibody or antibody fragment that binds to the first antigen of the exosome bound to one of the pair of affinity substances (F), and a set of affinity substances An insoluble carrier to which the other (f) of the combination is bound, and a second reagent that binds to a second antigen that the exosome has on its surface, or a second reagent that includes the antibody fragment, and does not destroy the exosome, an aromatic ring A kit comprising a polyoxyethylene-based surfactant having at least one of the first reagent and the second reagent

・ Measurement kit 4
A first reagent containing the first antibody or the antibody fragment, which binds to the first antigen that the exosome has on its surface, to which one of the combination of affinity substances (F) is bound, and the first reagent that the exosome has on its surface A second reagent containing a second antibody or antibody fragment that binds to two antigens and a third reagent containing an insoluble carrier to which the other (f) of a set of affinity substances is bound, and does not destroy exosomes A kit comprising an aromatic ring-containing polyoxyethylene-based surfactant in at least one of the first to third reagents

・ Measurement kit 5
A first reagent containing the first antibody or the antibody fragment, which binds to the first antigen that the exosome has on its surface, to which one of the combination of affinity substances (F) is bound, and the first reagent that the exosome has on its surface A second reagent containing a second antibody that binds to two antigens or an antibody fragment thereof, a third reagent containing an insoluble carrier to which the other (f) of a pair of affinity substances is bound, and an aroma that does not destroy exosomes And a fourth reagent containing a polyoxyethylene-based surfactant having a ring

Moreover, the following measurement kits 6 and 7 are mentioned as a measurement kit used for the competition method 1.
・ Measurement kit 6
Polysiloxane having an aromatic ring, which contains a first reagent containing a first antibody or antibody fragment that binds to a first antigen on the surface of an exosome and a second reagent containing a labeled competitor, and does not destroy the exosome An oxyethylene-based surfactant is contained in at least one of the first reagent and the second reagent. The kit, whether the first antibody or the antibody fragment is immobilized on an insoluble carrier Although it is good, it is preferably immobilized. Examples of the insoluble carrier include the aforementioned insoluble carrier.

・ Measurement kit 7
A polyoxyethylene system having an aromatic ring that does not destroy exosomes, a first reagent that contains a first antibody or antibody fragment that binds to a first antigen that exosome has on its surface, a second reagent that contains a labeled competitor A kit comprising a third reagent containing a surfactant The first antibody or the antibody fragment may or may not be immobilized on an insoluble carrier, but is preferably immobilized. . Examples of the insoluble carrier include the aforementioned insoluble carrier.

In the measurement kit 6 and the measurement kit 7 described above, the insoluble carrier on which the first antibody or the antibody fragment that binds to the first antigen of the exosome is immobilized is a sample, the first antibody, or the antibody fragment. It may be produced in the reaction solution of the reaction.
In this case, the first antibody to which one (G) of a pair of affinity substances is bound to the measurement reagent of the present invention instead of the insoluble carrier on which the first antibody or the antibody fragment is immobilized. Alternatively, the antibody fragment and an insoluble carrier to which the other (g) of a pair of affinity substances is bound are included. Examples of the combination of one affinity substance, that is, the combination of G and g, include the following combinations.
-A combination of biotin and avidins (avidin, newtraavidin, streptavidin, etc.);
A combination of avidin (such as avidin, newtraavidin, streptavidin, etc.) and biotin;
A combination of the Fc region of the first antibody that binds to the first antigen that the exosome has on its surface and the antibody that binds to the Fc region.

The first antibody or antibody fragment to which one (G) of a pair of affinity substances is bound and the insoluble carrier to which the other (g) of the pair of affinity substances is bound are the same reagent. Although it may be contained in separate reagents, it is preferably contained in separate reagents.
The first antibody or the antibody fragment to which one (G) of a set of affinity substances is bound and the insoluble carrier to which the other (g) of the set of affinity substances is bound are separate reagents. Also included in the kit of the present invention are the following kits, that is, measurement kits 8 to 10 included in the kit.

・ Measurement kit 8
A first reagent containing the first antibody or antibody fragment that binds to the first antigen that the exosome has on its surface, to which one (G) of a set of affinity substances is bound, and a set of affinity substances A polyoxyethylene-based surfactant having an aromatic ring, which contains an insoluble carrier to which the other (g) of the combination is bound and a second reagent containing a labeled competitor, and does not destroy exosomes. Kit included in at least one of the reagents

・ Measurement kit 9
A first reagent containing a first antibody or an antibody fragment thereof, which binds to a first antigen on the surface of an exosome, to which one of a pair of affinity substances (G) is bound, and a label containing a labeled competitor A polyoxyethylene-based surfactant having an aromatic ring, which contains two reagents and a third reagent containing an insoluble carrier to which the other (g) of the pair of affinity substances is bound, and does not destroy the exosome. ~ Kit included in at least one of the three reagents

・ Measurement kit 10
A first reagent containing a first antibody or an antibody fragment thereof, which binds to a first antigen on the surface of an exosome, to which one of a pair of affinity substances (G) is bound, and a label containing a labeled competitor A third reagent comprising an insoluble carrier to which the other (g) of the combination of two reagents and a pair of affinity substances is bound, and a fourth reagent comprising a polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes Including, kit

Furthermore, the following measurement kits 11 and 12 are listed as measurement kits used in the competition method 2.
・ Measurement kit 11
A labeled aromatic ring containing a first reagent containing a first antibody or antibody fragment that binds to a first antigen on the surface of an exosome and a second reagent containing a competitor, and does not destroy the exosome It is preferable that the kit competitor is immobilized on an insoluble carrier, in which at least one of the first reagent and the second reagent contains a polyoxyethylene-based surfactant having Examples of the insoluble carrier include the aforementioned insoluble carrier.

・ Measurement kit 12
A labeled first reagent containing a first antibody or antibody fragment that binds to a first antigen on the surface of an exosome, a second reagent containing a competitor, and a polycyclic aromatic ring that does not destroy the exosome The kit competitor containing the third reagent containing an oxyethylene surfactant is preferably immobilized on an insoluble carrier. Examples of the insoluble carrier include the aforementioned insoluble carrier.

In the measurement kit 11 and the measurement kit 12, the insoluble carrier on which the competitive substance is immobilized may be generated in a reaction solution of the reaction between the sample and the competitive substance.
In this case, in the measurement reagent of the present invention, instead of the insoluble carrier on which the competitive substance is immobilized, the competitive substance bound with one (H) of a set of affinity substances and the set of affinity substances And an insoluble carrier to which the other (h) of the combination is bound. Examples of a combination of affinity substances, that is, a combination of H and h include the following combinations.
-A combination of biotin and avidins (avidin, newtraavidin, streptavidin, etc.);
A combination of avidins (such as avidin, newtraavidin, streptavidin, etc.) and biotin.

A competing substance to which one (H) of a set of affinity substances is bound and an insoluble carrier to which the other (h) of a set of affinity substances are bound are contained in the same reagent, but are separated from each other. Although it may be contained in the reagent of this, it is preferable to be contained in a separate reagent.
A competing substance to which one (H) of a set of affinity substances is bound and an insoluble carrier to which the other (h) of the set of affinity substances is bound are included in separate reagents. These kits, that is, measurement kits 13 to 15 are also included in the kit of the present invention.

・ Measurement kit 13
A first reagent containing a competitor to which one of a set of affinity substances (H) is bound, an insoluble carrier to which the other (h) of the set of affinity substances is bound, and labeled And a second reagent containing the first antibody or the antibody fragment, and a polyoxyethylene-based surfactant having an aromatic ring that does not destroy the exosome is contained in at least one of the first reagent and the second reagent ,kit

・ Measurement kit 14
A first reagent containing a competitor bound with one (H) of a set of affinity substances and a second reagent containing an insoluble carrier bound with the other (h) of the set of affinity substances; A labeled third reagent containing the first antibody or the antibody fragment, and a polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes as at least one of the first to third reagents Included, kit

・ Measurement kit 15
A first reagent containing a competitor bound with one (H) of a set of affinity substances and a second reagent containing an insoluble carrier bound with the other (h) of the set of affinity substances; A kit comprising a labeled third reagent containing the first antibody or antibody fragment and a fourth reagent containing a polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes

The constituent reagents of the measurement kit of the present invention may be lyophilized or liquid. When a lyophilized component reagent is used, it is dissolved in an aqueous medium and measured before use for measurement. In a liquid constituent reagent, a polyoxyethylene-based surfactant having an aromatic ring that does not destroy an exosome, a first antibody or the antibody fragment that binds to a first antigen that the exosome has on its surface, and the labeled first antibody One antibody or the antibody fragment, the second antibody or the antibody fragment that binds to the second antigen on the surface of the exosome, the competitive substance, and the labeled competitive substance are dissolved in an aqueous medium. As an aqueous medium, the above-mentioned aqueous medium is mentioned, for example. The aqueous medium may contain the aforementioned salts, metal ions, sugars, preservatives, proteins, protein stabilizers, and the like.

In the measurement kit of the present invention, the first antigen that the exosome has on its surface and the second antigen that the exosome has on its surface are the aforementioned first antigen that the exosome has on its surface and the exosome that has its surface. And the second antigen respectively. In the measurement kit of the present invention, examples of the combination of the first antigen that the exosome has on its surface and the second antigen that the exosome has on its surface include the combination of the aforementioned first antigen and second antigen.

In the measurement kit of the present invention, the first antibody or the antibody fragment that binds to the first antigen that exosome has on its surface, and the second antibody or the antibody fragment that binds to the second antigen that exosome has on its surface include The first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, and the second antibody or the antibody fragment that binds to the second antigen that the exosome has on its surface, respectively, can be mentioned. In the measurement kit of the present invention, the combination of the first antibody that binds to the first antigen that the exosome has on its surface and the second antibody that binds to the second antigen that the exosome has on its surface includes the aforementioned first antibody And a combination with the second antibody.

The content of the first antibody or the antibody fragment that binds to the first antigen that the exosome has on the surface of the measurement kit of the present invention is not particularly limited as long as it allows the measurement method of the exosome of the present invention. Usually, it is usually 0.01 to 100 μg / mL, preferably 0.03 to 20 μg / mL, particularly preferably 0.05 to 10 μg / mL in the aforementioned aqueous medium or in a state dissolved in the aforementioned aqueous medium.
The content of the second antibody or antibody fragment that binds to the second antigen that the exosome has on the surface of the measurement kit of the present invention is not particularly limited as long as it allows the exosome measurement method of the present invention. Usually, it is usually 0.01 to 100 μg / mL, preferably 0.03 to 20 μg / mL, particularly preferably 0.05 to 10 μg / mL in the aforementioned aqueous medium or in a state dissolved in the aforementioned aqueous medium.

In the measurement kit of the present invention, examples of the label and the competitive substance include the aforementioned label and the competitive substance.

Examples of the polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes in the measurement kit of the present invention include the polyoxyethylene surfactants having an aromatic ring that do not destroy exosomes.
The content of the polyoxyethylene surfactant having an aromatic ring that does not destroy the exosome in the constituent reagent of the measurement kit of the present invention is not particularly limited as long as it is a content that enables the measurement method of the exosome of the present invention. In general, the content is 0.00005 to 30.0% (w / v) in the above-mentioned aqueous medium or dissolved in the above-mentioned aqueous medium, and preferably 0.0001 to 20.0% (w / v). A content of 0.0025 to 10.0% (w / v) is particularly preferable.
The measurement kit of the present invention may contain a combination of two or more polyoxyethylene surfactants having an aromatic ring that does not destroy exosomes.

EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to this Example.
<Material>
Disodium hydrogen phosphate (phosphate buffer; manufactured by Kanto Chemical Co., Inc.), sodium dihydrogen phosphate (phosphate buffer; manufactured by Kanto Chemical Co., Ltd.), sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.), magnesium chloride (Wako Pure) Yakuhin Kogyo Co., Ltd., MES (Dojindo Laboratories), MOPS (Dojindo Laboratories), Tween 20 (Kanto Chemical Co.), Streptavidin-binding magnetic particles (Dynabeads MyOne Streptavidin C1; Dynal) Nonionic HS-240 (HLB value: 17.9), Nonionic NS-240 (HLB value: 17.8), Nonionic HS-220 (HLB value: 16.2), Nonionic NS-220 (HLB value: 16. 0), nonionic HS-215 (HLB value: 15.0), nonionic NS-215 (HLB value: 15.0) (the polyoxyethylene alkylphenyl ester having an HLB value of 14 to 20) -Tel; manufactured by NOF Corporation), New Coal 610, New Coal 714, New Coal 723, New Coal 740, New Coal 2609, New Coal 2614 (above, polyoxyethylene polycyclic phenyl ether; manufactured by Nippon Emulsifier Co., Ltd.), New Coal 740SF, New Coal 723SF (above, polyoxyethylene polycyclic phenyl ether sulfate ester; manufactured by Nippon Emulsifier Co., Ltd.), Trax N-700B (polyoxyethylene alkylphenyl ether sulfate ester salt; manufactured by NOF Corporation), Triton X-100 [Polyoxyethylene alkylphenyl ether (HLB value: 13.5); manufactured by Sigma-Aldrich], Nonidet P-40 [Polyoxyethylene alkylphenyl ether (HLB value: 13.1); manufactured by Sigma-Aldrich), BSA Tal Yeast Co., Ltd.), anti-human CD9 monoclonal antibody (clone: A100-4) (manufactured by Medical and Biological Research Institute), anti-human CD63 monoclonal antibody (clone: C047-1) (manufactured by Medical and Biological Research Institute) Anti-human CD81 monoclonal antibody (clone: A103-10) (manufactured by Medical and Biological Research Institute).

Using a combination of the first antibody and the second antibody shown in Table 3 below, it comprises a streptavidin-conjugated magnetic particle solution, a biotin-conjugated first antibody solution, and an alkaline phosphatase (ALP) -labeled second antibody solution. Exosome measurement kits were prepared (Tables 4 to 9).
Further, as a comparative example 1 using a combination of the first antibody and the second antibody shown in Table 3 below, Triton X in the streptavidin-conjugated magnetic particle solution, the biotin-conjugated first antibody solution, and the ALP-labeled second antibody solution was used. Measurement kit containing -100 (measurement kit 1X-6X), measurement kit containing nonidet P-40 (measurement kit 1Y-6Y), PBS instead of surfactant [phosphate buffered saline (0.15 mol / L Measurement kits (measurement kits 1Z to 6Z) containing 10 mmol / L phosphate buffer containing sodium chloride, pH 7.2) were also prepared (Tables 4 to 9).

<Streptavidin-bonded magnetic particle solution>
A commercially available streptavidin-coupled magnetic particle (Dynabeads MyOne Streptavidin C1) was used to prepare a streptavidin-coupled magnetic particle solution having the following composition.
MES (pH6.5) 0.05 mol / L
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Streptavidin-bound magnetic particles 0.225 mg / mL
Surfactant (Concentration of surfactant described in Tables 4 to 9)

<Biotin-conjugated first antibody solution>
Using Biotin Labeling Kit-NH ₂ (manufactured by Dojindo Laboratories), label the anti-CD9 monoclonal antibody with biotin according to the instruction manual of the kit, and attach the biotin-conjugated anti-CD9 monoclonal antibody (biotin-conjugated first antibody). Prepared. Using a similar method, biotin-conjugated anti-CD63 monoclonal antibody (biotin-conjugated first antibody) and biotin-conjugated anti-CD81 monoclonal antibody (biotin-conjugated first antibody) were prepared.
Using the obtained biotin-conjugated first antibody, a biotin-conjugated anti-CD9 monoclonal antibody solution, biotin-conjugated anti-CD63 monoclonal antibody solution, and biotin-conjugated anti-CD81 monoclonal antibody solution having the following composition were prepared.
・ Biotin-conjugated anti-CD9 monoclonal antibody solution 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
Surfactant (Concentration of surfactant described in Tables 4 to 9)
・ Biotin-conjugated anti-CD63 monoclonal antibody solution MES (pH 6.5) 0.05 mol / L
Biotin-conjugated anti-CD63 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Surfactant (Concentration of surfactant described in Tables 4 to 9)
・ Biotin-conjugated anti-CD81 monoclonal antibody solution MES (pH6.5) 0.05 mol / L
Biotin-conjugated anti-CD81 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Surfactant (Concentration of surfactant described in Tables 4 to 9)

<ALP-labeled second antibody solution>
Using Alkaline Phosphatase Labeling Kit-NH ₂ (Dojindo Laboratories), label the anti-CD9 monoclonal antibody with ALP according to the instruction manual of the kit, and ALP-labeled anti-CD9 monoclonal antibody (ALP-labeled second antibody) Was made. Using the same method, an ALP-labeled anti-CD63 monoclonal antibody (ALP-labeled second antibody) and an ALP-labeled anti-CD81 monoclonal antibody (ALP-labeled second antibody) were prepared. Using the obtained ALP-labeled second antibody, an ALP-labeled anti-CD9 monoclonal antibody solution, an ALP-labeled anti-CD63 monoclonal antibody solution, and an ALP-labeled anti-CD81 monoclonal antibody solution having the following composition were prepared.
・ ALP-labeled anti-CD9 monoclonal antibody solution 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
Surfactant (Concentration of surfactant described in Tables 4 to 9)
・ ALP-labeled anti-CD63 monoclonal antibody solution MES (pH6.5) 0.05 mol / L
ALP-labeled anti-CD63 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Surfactant (Concentration of surfactant described in Tables 4 to 9)
・ ALP-labeled anti-CD81 monoclonal antibody solution MES (pH 6.5) 0.05 mol / L
ALP-labeled anti-CD81 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Surfactant (Concentration of surfactant described in Tables 4 to 9)

(1) Exosome measurement method Peripheral blood is collected from healthy volunteers belonging to Kyowa Medex Co., and serum (hereinafter referred to as healthy human serum) is prepared from the peripheral blood by centrifugation (2000 rpm, 20 minutes, 25 ° C). did.
Using each kit described in Tables 4 to 9, exosomes in the samples were measured by the following procedure. Healthy human serum (10 μL) or PBS (0 concentration sample) (10 μL), streptavidin-conjugated magnetic particle solution (30 μL), biotin-conjugated first antibody solution (30 μL), and ALP-labeled second antibody solution (30 μL) was added, and the mixture was stirred and reacted at 37 ° C. for 10 minutes. The magnetic particles are collected magnetically to remove the reaction solution other than the magnetic particles, and a 0.005 mol / L MOPS buffer solution containing 0.07% Tween 20, 0.2 mmol / L magnesium chloride, 0.3 mol / L sodium chloride ( The magnetic particles were washed 5 times with pH 7.3). Then, add a luminescent substrate solution (100 μL) containing 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium salt (Lumigen ^TM APS-5) as the main component and stir. The amount of light emitted (RLU) was measured.

(2) Comparison of measurement sensitivity in each measurement kit Calculate the S / N ratio using the amount of luminescence in each measurement kit obtained in (1) above (luminescence of healthy human serum; luminescence of 0 concentration sample) did. The S / N ratio is one of the indexes for evaluating measurement sensitivity in immunoassay, and is obtained when measuring a sample with a known concentration against the signal (noise) obtained when measuring a zero concentration sample. It shows the ratio of the signal that is generated. The S / N ratio can be calculated by the following formula.

S / N ratio = [Luminescence of healthy human serum] / [Luminescence of 0 concentration sample]

Thereafter, in order to compare the S / N ratio in each measurement kit, a measurement kit (measurement kits 1Z to 6Z) containing PBS in a streptavidin-conjugated magnetic particle solution, a biotin-conjugated first antibody solution, and an ALP-labeled second antibody solution is used. The S / N ratio in each measurement kit was calculated using the S / N ratio at the time of measurement as a reference (100%), and was taken as the relative value of measurement sensitivity (%). The results are shown in Tables 4 to 9. Here, the larger the measurement sensitivity relative value (%), the higher the measurement sensitivity. When the measurement sensitivity relative value (%) exceeds 100%, the measurement sensitivity is higher than that of the measurement kit without surfactant. It is high.

As is apparent from Tables 4 to 9, a measurement kit (measurement kit 1A to 1P, measurement kit 2A to 2M, measurement kit) containing a polyoxyethylene surfactant having an aromatic ring that does not destroy the exosome of Example 1 Kit 3A-3L, measurement kit 4A-4M, measurement kit 5A-5M, measurement kit 6A-6M) when measuring exosomes in serum, that is, when the surfactant is present in an antigen-antibody reaction, When measuring the exosome in serum using the measurement kit of the comparative example 1, it turned out that the measurement sensitivity of exosome is high compared with the case where the said surfactant does not exist in an antigen antibody reaction.

Exosome measurement kits 1a to 1g comprising the following streptavidin-conjugated magnetic particle solution, biotin-conjugated anti-CD9 monoclonal antibody solution, and ALP-labeled anti-CD9 monoclonal antibody solution were prepared.

<Streptavidin-bonded magnetic particle solution>
Using the same magnetic particles (Dynabeads MyOne Streptavidin C1) as in Example 1, a streptavidin-bound magnetic particle solution having the following composition was prepared.
MES (pH6.5) 0.05 mol / L
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Streptavidin-bound magnetic particles 0.225 mg / mL
Nonion NS-240 Concentrations listed in Table 10

<Biotin-conjugated anti-CD9 monoclonal antibody solution>
A biotin-conjugated anti-CD9 monoclonal antibody solution having the following composition was prepared using the biotin-conjugated anti-CD9 monoclonal antibody prepared in Example 1.
MES (pH6.5) 0.05 mol / L
Biotin-conjugated anti-CD9 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Nonion NS-240 Concentrations listed in Table 10

<ALP-labeled anti-CD9 monoclonal antibody solution>
Using the ALP-labeled anti-CD9 monoclonal antibody prepared in Example 1, an ALP-labeled anti-CD9 monoclonal antibody solution having the following composition was prepared.
MES (pH6.5) 0.05 mol / L
ALP-labeled anti-CD9 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Nonion NS-240 Concentrations listed in Table 10

Using the respective measurement kits of the measurement kits 1a to 1g of Example 3 and the measurement kit 1Z of Comparative Example 1 in the same manner as in Example 2, in the serum of healthy humans diluted 2-fold with PBS, and PBS The exosome in (0 concentration sample) was measured, and the concentration of nonion NS-240 which is polyoxyethylene alkylphenyl ether in antigen-antibody reaction was examined. From the obtained luminescence amount in each measurement kit, the measurement sensitivity relative value (%) was calculated according to the method of Example 2 (2). The results are shown in Table 10.

As is apparent from Table 10, when measuring exosomes in serum using a measurement kit containing nonion NS-240 in a streptavidin-conjugated magnetic particle solution, biotin-conjugated anti-CD9 monoclonal antibody solution, and ALP-labeled anti-CD9 monoclonal antibody solution That is, when nonion NS-240 is present at 0.0025 to 1.0% in the antigen-antibody reaction, when exosomes in serum are measured using the measurement kit 1Z of Comparative Example 1, that is, nonion NS-240 in the antigen-antibody reaction. It was found that the measurement sensitivity of exosome was higher than that in the absence of.

Exosome measurement kits 2a to 2f comprising the following streptavidin-conjugated magnetic particle solution, biotin-conjugated anti-CD9 monoclonal antibody solution, and ALP-labeled anti-CD9 monoclonal antibody solution were prepared.

<Streptavidin-bonded magnetic particle solution>
Using the same magnetic particles (Dynabeads MyOne Streptavidin C1) as in Example 1, a streptavidin-bound magnetic particle solution having the following composition was prepared.
MES (pH6.5) 0.05 mol / L
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Streptavidin-bound magnetic particles 0.225 mg / mL
Trax N-700B Concentrations listed in Table 11

<Biotin-conjugated anti-CD9 monoclonal antibody solution>
A biotin-conjugated anti-CD9 monoclonal antibody solution having the following composition was prepared using the biotin-conjugated anti-CD9 monoclonal antibody prepared in Example 1.
MES (pH6.5) 0.05 mol / L
Biotin-conjugated anti-CD9 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Trax N-700B Concentrations listed in Table 11

<ALP-labeled anti-CD9 monoclonal antibody solution>
Using the ALP-labeled anti-CD9 monoclonal antibody prepared in Example 1, an ALP-labeled anti-CD9 monoclonal antibody solution having the following composition was prepared.
MES (pH6.5) 0.05 mol / L
ALP-labeled anti-CD9 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Trax N-700B Concentrations listed in Table 11

Using the respective kits of the measurement kits 2a to 2f of Example 5 and the measurement kit 1Z of Comparative Example 1, in the same manner as in Example 2, in the serum of healthy humans diluted 2-fold with PBS, and PBS ( The exosome in the 0 concentration sample was measured, and the concentration of Trax N-700B, which is a polyoxyethylene alkylphenyl ether sulfate salt, in the antigen-antibody reaction was examined. From the obtained luminescence amount in each measurement kit, the measurement sensitivity relative value (%) was calculated according to the method of Example 2 (2). The results are shown in Table 11.

As is clear from Table 11, when exosomes in serum are measured using a measurement kit containing Trax N-700B in a streptavidin-conjugated magnetic particle solution, a biotin-conjugated anti-CD9 monoclonal antibody solution, and an ALP-labeled anti-CD9 monoclonal antibody solution That is, when Trax N-700B is present at 0.005 to 1.0% in the antigen-antibody reaction, when exosomes in serum are measured using the measurement kit 1Z of Comparative Example 1, that is, Trax N-700B in the antigen-antibody reaction. It was found that the measurement sensitivity of exosome was higher than that in the absence of.

Exosome measurement kits 3a to 3g comprising the following streptavidin-conjugated magnetic particle solution, biotin-conjugated anti-CD9 monoclonal antibody solution, and ALP-labeled anti-CD9 monoclonal antibody solution were prepared.

<Streptavidin-bonded magnetic particle solution>
Using the same magnetic particles (Dynabeads MyOne Streptavidin C1) as in Example 1, a streptavidin-bound magnetic particle solution having the following composition was prepared.
MES (pH6.5) 0.05 mol / L
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
Streptavidin-bound magnetic particles 0.225 mg / mL
New Coal 740 Concentrations listed in Table 12

<Biotin-conjugated anti-CD9 monoclonal antibody solution>
A biotin-conjugated anti-CD9 monoclonal antibody solution having the following composition was prepared using the biotin-conjugated anti-CD9 monoclonal antibody prepared in Example 1.
MES (pH6.5) 0.05 mol / L
Biotin-conjugated anti-CD9 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
New Coal 740 Concentrations listed in Table 12

<ALP-labeled anti-CD9 monoclonal antibody solution>
Using the ALP-labeled anti-CD9 monoclonal antibody prepared in Example 1, an ALP-labeled anti-CD9 monoclonal antibody solution having the following composition was prepared.
MES (pH6.5) 0.05 mol / L
ALP-labeled anti-CD9 monoclonal antibody 75 ng / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
New Coal 740 Concentrations listed in Table 12

Using the kits of measurement kits 3a to 3g of Example 7 and measurement kit 1Z of Comparative Example 1 in the same manner as in Example 2, in the serum of healthy humans diluted 2-fold with PBS, and PBS ( The exosome in the 0 concentration sample) was measured, and the concentration of Newcor 740, which is polyoxyethylene polycyclic phenyl ether in the antigen-antibody reaction, was examined. From the obtained luminescence amount in each measurement kit, the measurement sensitivity relative value (%) was calculated according to the method of Example 2 (2). The results are shown in Table 12.

As is clear from Table 12, when exosomes in serum were measured using a measurement kit containing Neucor 740 in a streptavidin-conjugated magnetic particle solution, a biotin-conjugated anti-CD9 monoclonal antibody solution, and an ALP-labeled anti-CD9 monoclonal antibody solution, That is, in the antigen-antibody reaction, when Newcol 740 is present at 0.0025 to 1.0%, when the exosome in serum is measured using the measurement kit 1Z of Comparative Example 1, that is, Newcol 740 is not present in the antigen-antibody reaction. Compared to the case, it was found that the measurement sensitivity of exosome was higher.

According to the present invention, there are provided a method, a reagent and a measurement kit for measuring exosomes in a sample that are effective for clinical diagnosis of cancer and the like, simple and highly sensitive.

Claims

A method for measuring an exosome in a sample using an antigen-antibody reaction, wherein the antigen-antibody reaction is performed in the presence of a polyoxyethylene-based surfactant having an aromatic ring that does not destroy the exosome. And a method for measuring exosomes in a sample.
In an aqueous medium containing a polyoxyethylene-based surfactant having an aromatic ring, which comprises the following steps (1) to (3), and wherein the reaction of step (1) and / or step (2) does not destroy exosomes The method of claim 1, wherein
(1) A sample is reacted with a first antibody or the antibody fragment that binds to a first antigen on the surface of the exosome in an aqueous medium, and the first antibody or the antibody fragment and the exosome Producing an immune complex 1 comprising:
(2) A second antibody or antibody fragment that binds to a second antigen on the surface of an exosome is reacted with the immune complex 1 produced in the step (1) in an aqueous medium, and the first antibody or Producing an immune complex 2 comprising the antibody fragment, the exosome, and the second antibody or the antibody fragment;
(3) A step of measuring the immune complex 2 produced in the step (2).
3. The method according to claim 1, wherein the polyoxyethylene-based surfactant having an aromatic ring that does not destroy exosomes is a polyoxyethylene alkylphenyl ether derivative or a polyoxyethylene polycyclic phenyl ether derivative.
The method according to claim 3, wherein the polyoxyethylene alkylphenyl ether derivative is polyoxyethylene alkylphenyl ether or polyoxyethylene alkylphenyl ether sulfate having an HLB value of 14 or more and 20 or less.
The method according to claim 3 or 4, wherein the polyoxyethylene polycyclic phenyl ether derivative is polyoxyethylene polycyclic phenyl ether or polyoxyethylene polycyclic phenyl ether sulfate salt.
The method according to any one of claims 2 to 5, wherein at least one of the first antigen and the second antigen is an antigen selected from the group consisting of CD9, CD63 and CD81.
The first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, the second antibody or the antibody fragment that binds to the second antigen that the exosome has on its surface, and the aromatic ring that does not destroy the exosome A reagent for measuring exosomes in a sample, comprising a polyoxyethylene-based surfactant.
The reagent according to claim 7, wherein the polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes is a polyoxyethylene alkylphenyl ether derivative or a polyoxyethylene polycyclic phenyl ether derivative.
The reagent according to claim 8, wherein the polyoxyethylene alkylphenyl ether derivative is polyoxyethylene alkylphenyl ether having an HLB value of 14 or more and 20 or less, or polyoxyethylene alkylphenyl ether sulfate ester salt.
The reagent according to claim 8 or 9, wherein the polyoxyethylene polycyclic phenyl ether derivative is polyoxyethylene polycyclic phenyl ether or polyoxyethylene polycyclic phenyl ether sulfate ester salt.
The reagent according to any one of claims 7 to 10, wherein at least one of the first antigen and the second antigen is an antigen selected from the group consisting of CD9, CD63 and CD81.
A first reagent containing the first antibody or antibody fragment that binds to the first antigen that the exosome has on its surface, and a second antibody or antibody fragment that binds to the second antigen that the exosome has on its surface A kit for measuring exosomes in a sample, wherein the polyoxyethylene-based surfactant having an aromatic ring, which contains a second reagent and does not destroy exosomes, is contained in at least one of the first reagent and the second reagent.
A first reagent containing the first antibody or the antibody fragment that binds to the first antigen that the exosome has on its surface, a second reagent that contains the second antibody or the antibody fragment that binds to the second antigen that the exosome has on the surface A kit for measuring an exosome in a sample, comprising a reagent and a third reagent containing a polyoxyethylene-based surfactant having an aromatic ring that does not destroy the exosome.
The kit according to claim 12 or 13, wherein the polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes is a polyoxyethylene alkylphenyl ether derivative or a polyoxyethylene polycyclic phenyl ether derivative.
The kit according to claim 14, wherein the polyoxyethylene alkylphenyl ether derivative is polyoxyethylene alkylphenyl ether having an HLB value of 14 or more and 20 or less, or polyoxyethylene alkylphenyl ether sulfate ester salt.
The kit according to claim 14 or 15, wherein the polyoxyethylene polycyclic phenyl ether derivative is polyoxyethylene polycyclic phenyl ether or polyoxyethylene polycyclic phenyl ether sulfate ester salt.
The kit according to any one of claims 12 to 16, wherein at least one of the first antigen and the second antigen is an antigen selected from the group consisting of CD9, CD63 and CD81.