WO2019159944A1 - Procédé de mesure, réactif de mesure, et kit de mesure d'exosomes dans un échantillon - Google Patents

Procédé de mesure, réactif de mesure, et kit de mesure d'exosomes dans un échantillon Download PDF

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Publication number
WO2019159944A1
WO2019159944A1 PCT/JP2019/005026 JP2019005026W WO2019159944A1 WO 2019159944 A1 WO2019159944 A1 WO 2019159944A1 JP 2019005026 W JP2019005026 W JP 2019005026W WO 2019159944 A1 WO2019159944 A1 WO 2019159944A1
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antibody
exosome
antigen
polyoxyethylene
binds
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PCT/JP2019/005026
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English (en)
Japanese (ja)
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健太郎 庄司
小野　達也
豪 永井
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協和メデックス株式会社
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Priority to JP2020500506A priority Critical patent/JP7281092B2/ja
Publication of WO2019159944A1 publication Critical patent/WO2019159944A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Definitions

  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • [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.
  • 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).
  • 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.
  • 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.
  • polyoxyethylene polycyclic phenyl ether derivative is polyoxyethylene polycyclic phenyl ether or polyoxyethylene polycyclic phenyl ether sulfate ester salt.
  • polyoxyethylene polycyclic phenyl ether derivative is polyoxyethylene polycyclic phenyl ether or polyoxyethylene polycyclic phenyl ether sulfate ester salt.
  • 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 polyoxyethylene surfactant having an aromatic ring that does not destroy exosomes is a polyoxyethylene alkylphenyl ether derivative or a polyoxyethylene polycyclic phenyl ether derivative.
  • 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • (3A) A step of measuring the immune complex 2 produced in the step (2A).
  • the sample in the step (1A) examples include the above-described samples.
  • 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.
  • 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.
  • the insoluble carrier on which the first antibody or the antibody fragment is immobilized is an antigen-antibody reaction.
  • 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.
  • 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.
  • 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
  • combinations of Aa include the following combinations.
  • a combination of biotin and avidins avidin, neutravidin, streptavidin, etc.
  • -Combinations of avidins avidin, neutravidin, streptavidin, etc.
  • biotin avidin, neutravidin, streptavidin, etc.
  • biotin avidin, neutravidin, streptavidin, etc.
  • biotin avidin, neutravidin, streptavidin, etc.
  • biotin avidin, neutravidin, streptavidin, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • linker examples 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.
  • 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.
  • 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 ⁇ g / mL, preferably 0.03 to 20 ⁇ g / mL, particularly preferably 0.05 to 10 ⁇ g / 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).
  • 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).
  • 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.
  • 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 ⁇ g / mL, preferably 0.03 to 20 ⁇ g / mL, more preferably 0.05 to 10 ⁇ g / 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.
  • 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.
  • 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.
  • 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)-
  • 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.
  • phosphate buffered saline 10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2, hereinafter referred to as PBS
  • PBS PBS containing a surfactant
  • the surfactant include nonionic surfactants such as Tween 20.
  • 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.
  • step (3A) the exosome concentration in the sample is determined by measuring immune complex 2 produced in step (2A) using the following method.
  • the exosome binds to the second antibody or the antibody fragment that binds to the second antigen on the surface of the exosome.
  • the antibody hereinafter referred to as the third antibody
  • 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
  • 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
  • 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 ⁇ g / mL, and 0.03 to 20 ⁇ g / mL. mL is preferable, and 0.05 to 10 ⁇ g / mL is preferable.
  • 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.
  • fluorescent substance examples include fluorescein isothiocyanate (FITC), rhodamine B-isothiocyanate (RITC), and the like.
  • 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.
  • radioisotope examples include 3 H, 14 C, 35 S, 32 P, 125 I, and 131 I.
  • polypeptide containing the tag sequence examples include FLAG.
  • metal colloid examples include gold colloid.
  • 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.
  • the fluorescence intensity can be measured using a fluorometer, a fluorescent multiwell plate reader or the like.
  • the luminescence intensity can be measured using a luminescence photometer, a luminescence multiwell plate reader or the like.
  • the radioactivity can be measured by a scintillation counter, a ⁇ -well counter or the like.
  • the amount of label can be quantified by measuring the enzyme activity.
  • the amount of labeling can be measured by reacting a substrate of the enzyme with the enzyme and measuring the produced substance.
  • the peroxidase activity can be measured by, for example, an absorbance method, a fluorescence method, or the like.
  • 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.
  • 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.
  • a peroxide active substance such as hydrogen peroxide and peroxidase.
  • CCAP tetramethylbenzidine, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine
  • CCAP 10-N-methylcarbamoyl-3,7- Bis (dimethylamino) -10H-phenothiazine
  • MCDP 10-N-methylcarbamoyl-3,7- Bis (dimethylamino) -10H-phenothiazine
  • DA-64 N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine sodium salt
  • DA-67 10-N-carboxy
  • 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.
  • a peroxide active substance such as hydrogen peroxide and peroxidase.
  • 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.
  • coupler examples include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine.
  • 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,
  • phenols examples include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.
  • 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.
  • the fluorescent substance include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, and coumarin.
  • 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.
  • the luminescent substance include a luminol compound and a lucigenin compound.
  • the alkaline phosphatase activity can be measured by, for example, a luminescence method.
  • a luminescence method examples 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.
  • alkaline phosphatase substrates examples 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-
  • ⁇ -D-galactosidase activity can be measured by, for example, an absorbance method (colorimetric method), a luminescence method, or a fluorescence method.
  • an absorbance method colorimetric method
  • a luminescence method luminescence method
  • a fluorescence method As a method of measuring ⁇ -D-galactosidase activity by absorbance method (colorimetric method), ⁇ -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 ⁇ -D-galactosidase in the method for measuring ⁇ -D-galactosidase activity by an absorbance method include o-nitrophenyl- ⁇ -D-galactopyranoside.
  • a method for measuring ⁇ -D-galactosidase activity by a luminescence method for example, a method in which ⁇ -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 ⁇ -D-galactosidase in the method for measuring ⁇ -D-galactosidase activity by the luminescence method include galacton-plus (Galacton-Plus, manufactured by Applied Biosystems) and similar compounds. Can be mentioned.
  • a method of measuring ⁇ -D-galactosidase activity by a fluorescence method for example, a method of reacting ⁇ -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 ⁇ -D-galactosidase in the method for measuring ⁇ -D-galactosidase activity by a fluorescence method include 4-methylumbelliferyl- ⁇ -D-galactopyranoside.
  • the luciferase activity can be measured by, for example, a luminescence method.
  • 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.
  • the luciferase substrate include luciferin and coelenterazine.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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.
  • one embodiment of the competition method includes, for example, a method including the following steps.
  • 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.
  • the insoluble carrier include the aforementioned insoluble carrier.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g / mL, 0.03 to 20 ⁇ g / mL is preferable, and 0.05 to 10 ⁇ g / mL is preferable.
  • 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.
  • (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
  • the competitor is preferably immobilized on an insoluble carrier.
  • the insoluble carrier include the aforementioned insoluble carrier.
  • marker the above-mentioned label
  • 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.
  • the insoluble carrier on which the competitive substance is immobilized may be produced in the reaction solution of the antigen-antibody reaction.
  • 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 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 ⁇ g / mL, 0.03 -20 ⁇ g / mL is preferable, and 0.05-10 ⁇ g / mL is preferable.
  • 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.
  • 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.
  • the exosome measuring method of the present invention can be used 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.
  • Examples of the combination of the first antibody and the second antibody include the combinations described in Table 2 below.
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the aromatic ring in the polyoxyethylene surfactant having an aromatic ring that does not destroy the exosome include a benzene ring.
  • 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.
  • the HLB value is 14 Examples thereof include polyoxyethylene alkylphenyl ethers and polyoxyethylene alkylphenyl ether sulfates that are 20 or less.
  • alkyl in the polyoxyethylene alkylphenyl ether having an HLB value of 14 to 20 and the polyoxyethylene alkylphenyl ether sulfate ester examples include alkyl having 8 to 24 carbon atoms, and those having 10 to 20 carbon atoms. Alkyl is preferred.
  • alkyl having 8 to 24 carbon atoms examples 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.
  • alkyl having 10 to 20 carbon atoms examples 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).
  • 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.
  • 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.
  • a lactate buffer 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.
  • good buffer examples include 2-morpholinoethanesulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, and tris (hydroxymethyl) aminomethane (Tris).
  • MES 2-morpholinoethanesulfonic acid
  • Bis-Tris bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane
  • Tris tris (hydroxymethyl) aminomethane
  • 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) Bu
  • the aqueous medium may contain salts, metal ions, sugars, preservatives, proteins, protein stabilizers and the like.
  • 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.
  • the metal ion include magnesium ion, manganese ion, zinc ion and the like.
  • 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).
  • 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.
  • 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.
  • the insoluble carrier include the aforementioned insoluble carrier.
  • 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.
  • the insoluble carrier include the aforementioned insoluble carrier.
  • labels and competitors include the aforementioned labels and competitors, respectively.
  • 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.
  • the insoluble carrier on which the first antibody or the antibody fragment is immobilized 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.
  • 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.
  • biotin and avidins avidin, newtraavidin, streptavidin, etc.
  • avidin such as avidin, newtraavidin, streptavidin, etc.
  • 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.
  • the insoluble carrier include the aforementioned insoluble carrier.
  • labels and competitors include the aforementioned labels and competitors, respectively.
  • 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.
  • 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.
  • 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.
  • a measurement reagent in a lyophilized state it is dissolved in an aqueous medium before measurement and made into a liquid state for measurement.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a combination of two or more polyoxyethylene surfactants having an aromatic ring that does not destroy exosomes may be contained.
  • 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.
  • 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 ⁇ g / mL in the aforementioned aqueous medium or dissolved in the aforementioned aqueous medium. Yes, 0.03 to 20 ⁇ g / mL is preferable, and 0.05 to 10 ⁇ g / 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.
  • 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.
  • the insoluble carrier include the aforementioned insoluble carrier.
  • 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.
  • 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.
  • 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.
  • biotin and avidins avidin, newtraavidin, streptavidin, etc.
  • avidin such as avidin, newtraavidin, streptavidin, etc.
  • 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 kit comprising an aromatic ring-containing polyoxyethylene-based surfactant in at least one of the first to third reagents
  • 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
  • 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.
  • the insoluble carrier include the aforementioned insoluble carrier.
  • 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.
  • 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.
  • 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.
  • biotin and avidins avidin, newtraavidin, streptavidin, etc.
  • avidin such as avidin, newtraavidin, streptavidin, etc.
  • 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
  • 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
  • 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
  • a fourth reagent comprising a polyoxy
  • 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.
  • the insoluble carrier include the aforementioned insoluble carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • a lyophilized component reagent When a lyophilized component reagent is used, it is dissolved in an aqueous medium and measured before use for measurement.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g / mL, preferably 0.03 to 20 ⁇ g / mL, particularly preferably 0.05 to 10 ⁇ g / 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.
  • it is usually 0.01 to 100 ⁇ g / mL, preferably 0.03 to 20 ⁇ g / mL, particularly preferably 0.05 to 10 ⁇ g / mL in the aforementioned aqueous medium or in a state dissolved in the aforementioned aqueous medium.
  • 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.
  • nonionic HS-215 HLB value of 14 to 20
  • nonionic NS-215 HLB value of 14 to 20
  • New Coal 610 New Coal 714, New Coal 723, New Coal 740, New Coal 2609, New Coal 2614
  • New Coal 740SF New Coal 723SF
  • 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-Ald
  • the first antibody and the second antibody shown in Table 3 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).
  • Triton X in the streptavidin-conjugated magnetic particle solution, the biotin-conjugated first antibody solution, and the ALP-labeled second antibody solution was used as a comparative example 1 using a combination of the first antibody and the second antibody shown in Table 3 below.
  • 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%
  • 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).
  • biotin-conjugated anti-CD63 monoclonal antibody biotin-conjugated first antibody
  • biotin-conjugated anti-CD81 monoclonal antibody biotin-conjugated first antibody
  • biotin-conjugated anti-CD9 monoclonal antibody solution 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.
  • 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.
  • 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.
  • 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 ⁇ L) or PBS (0 concentration sample) (10 ⁇ L), streptavidin-conjugated magnetic particle solution (30 ⁇ L), biotin-conjugated first antibody solution (30 ⁇ L), and ALP-labeled second antibody solution (30 ⁇ L) 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 ⁇ L) 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.
  • 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]
  • 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.
  • the larger the measurement sensitivity relative value (%) the higher the measurement sensitivity.
  • the measurement sensitivity relative value (%) exceeds 100%, the measurement sensitivity is higher than that of the measurement kit without surfactant. It is high.
  • 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,
  • 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.
  • 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
  • Biotin-conjugated anti-CD9 monoclonal antibody 75 ng / mL
  • BSA 0.1%
  • Sodium chloride 0.1 mol / L
  • 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%
  • Sodium chloride 0.1 mol / L Nonion NS-240 Concentrations listed in Table 10
  • Example 2 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.
  • 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.
  • 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
  • Biotin-conjugated anti-CD9 monoclonal antibody 75 ng / mL
  • BSA 0.1%
  • 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
  • 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.
  • 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
  • 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
  • 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
  • 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.

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Abstract

L'invention concerne un procédé de mesure d'exosomes dans un échantillon qui est destiné à mesurer des exosomes dans un échantillon à l'aide d'une réaction antigène-anticorps, et qui est caractéristique en ce que la réaction antigène-anticorps a lieu en présence d'un tensio-actif à base de polyoxyéthylène ne détruisant pas les exosomes, et possédant un cycle aromatique. Le procédé de mesure d'exosomes dans un échantillon de l'invention, est utile dans le cadre d'un diagnostic du cancer, ou similaire.
PCT/JP2019/005026 2018-02-14 2019-02-13 Procédé de mesure, réactif de mesure, et kit de mesure d'exosomes dans un échantillon WO2019159944A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021132213A1 (fr) * 2019-12-23 2021-07-01 富士レビオ株式会社 Procédé de récupération de vésicule extracellulaire et tube de collecte de sang

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012039395A1 (fr) * 2010-09-24 2012-03-29 和光純薬工業株式会社 Procédé pour l'acquisition de petit arn
WO2013094307A1 (fr) * 2011-12-22 2013-06-27 Ochiya Takahiro Procédé d'analyse d'exosome, réactif pour l'utilisation dans l'analyse d'exosome, et appareil d'analyse d'exosome
JP2014526032A (ja) * 2011-06-07 2014-10-02 カリス ライフ サイエンシズ ルクセンブルク ホールディングス エス．アー．エール．エル． 癌に対する循環バイオマーカー
WO2015068772A1 (fr) * 2013-11-06 2015-05-14 Jsr株式会社 Procédé de séparation, procédé de détection, procédé de mesure de signal, procédé pour déterminer une maladie, procédé pour évaluer une efficacité de médicament de traitement de maladie, trousse et composition liquide
JP2018130085A (ja) * 2017-02-17 2018-08-23 Jsr株式会社 保存安定化剤、安定化方法、保存方法、添加剤、単離方法、及び回収効率改善剤

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012039395A1 (fr) * 2010-09-24 2012-03-29 和光純薬工業株式会社 Procédé pour l'acquisition de petit arn
JP2014526032A (ja) * 2011-06-07 2014-10-02 カリス ライフ サイエンシズ ルクセンブルク ホールディングス エス．アー．エール．エル． 癌に対する循環バイオマーカー
WO2013094307A1 (fr) * 2011-12-22 2013-06-27 Ochiya Takahiro Procédé d'analyse d'exosome, réactif pour l'utilisation dans l'analyse d'exosome, et appareil d'analyse d'exosome
WO2015068772A1 (fr) * 2013-11-06 2015-05-14 Jsr株式会社 Procédé de séparation, procédé de détection, procédé de mesure de signal, procédé pour déterminer une maladie, procédé pour évaluer une efficacité de médicament de traitement de maladie, trousse et composition liquide
JP2018130085A (ja) * 2017-02-17 2018-08-23 Jsr株式会社 保存安定化剤、安定化方法、保存方法、添加剤、単離方法、及び回収効率改善剤

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021132213A1 (fr) * 2019-12-23 2021-07-01 富士レビオ株式会社 Procédé de récupération de vésicule extracellulaire et tube de collecte de sang

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