WO2016121821A1 - Separation method, detection method, signal measurement method, method for determining disease, method for evaluating drug efficacy, kit, liquid composition, and analyte diluent - Google Patents

Abstract

The purpose of the present invention is to provide a method by which non-specific adsorption to a solid-phase carrier can be reduced, and vesicles having lipid bilayer membranes can be separated selectively and efficiently. Provided is a method for separating vesicles having lipid bilayer membranes, characterized by including a complex formation step for bringing a biological specimen that includes vesicles having lipid bilayer membranes, and a solid-phase carrier to which are bonded ligands that recognize a surface antigen present on the surfaces of the vesicles, into contact, causing a complex of the vesicles and the solid-phase carrier to form, and a washing step for washing the complex, the complex formation step and/or the washing step being carried out in the presence of an inorganic salt or organic salt at a final concentration of 0.15-2 M.

Description

Separation method, detection method, signal measurement method, disease determination method, drug efficacy evaluation method, kit, liquid composition, and specimen diluent

The present invention relates to a separation method, a detection method, a signal measurement method, a disease determination method, a drug efficacy evaluation method, a kit, a liquid composition, and a specimen diluent. More specifically, a method for separating vesicles having a lipid bilayer such as exosome, a method for detecting nucleic acid or protein using the method, a signal measuring method, a method for determining a disease, a method for evaluating the efficacy of a disease therapeutic agent, a disease The present invention relates to a determination or medicinal effect evaluation kit, a liquid composition used in the separation method, and a specimen diluent.

Vesicles have a structure encased in a lipid bilayer, and exosomes are known as vesicle granules existing in body fluids in the body among such vesicles. It is known that various membrane proteins exist on the surface of exosomes as well as general cell surfaces. On the other hand, in addition to various proteins such as cytokines, microRNA (miRNA) is present inside exosomes. Has also been found to be included.

In addition, it is known that exosomes are secreted from various cells such as cells of the immune system and various cancer cells. The function of exosomes as a mediator of intercellular communication in vivo, exosomes and physiological phenomena, cancer, etc. Relevance to other diseases has attracted attention and is being investigated. For example, when using an antibody of EpCAM, which is a tumor marker, exosomes are isolated from circulating blood of ovarian cancer patients, and there is already a relationship between the expression level of exosome-derived miRNA and the progression of ovarian cancer. It has been reported (Non-Patent Document 1).

Further, there are CD9, CD63, and CD81 belonging to the tetraspanin family as four-transmembrane membrane proteins expressed on exosomes. Non-patent document 2 discloses that the amount of exosomes in the plasma of melanoma patients is normal. It has been reported that this can be detected and quantified with an antibody against CD63 and an antibody against tumor-related marker Caveolin-1. In addition, a signal derived from an exosome in a cancer patient is quantified and analyzed by reacting the plasma sample after centrifugation with a combination of an anti-CD63 antibody and antibodies against various membrane proteins. Patent Document 1).

As described above, attempts have been made to specifically capture exosomes from clinical specimens such as serum, plasma, blood, etc., and use proteins and miRNAs contained in exosomes as new biomarkers for early diagnosis of disease and discovery of recurrence. ing.

International Publication No. 2010/065968

However, since the above clinical specimen contains high-concentration proteins such as albumin and globulin, when using exosomes separated from the clinical specimen for disease determination, it is caused by nonspecific adsorption to the solid phase carrier or the inner wall of the reaction vessel. Thus, it is a serious problem that a specific reaction such as an antigen-antibody reaction is inhibited by a sample component, or a sample to be determined to be negative is erroneously determined to be positive (hereinafter false positive). This problem is particularly noticeable when the clinical specimen is serum or plasma.
In order to reduce this false positive, methods such as adding a surfactant during the reaction or overcoating a solid phase carrier with a protein or polymer have been used. There was a limit to reducing false positives caused by. Decreasing false positives becomes an increasingly important issue in the determination of diseases using vesicles such as exosomes and the evaluation of drug efficacy.
In addition, prior to exosome separation, it has been proposed to perform a pretreatment such as removing a sample component that can inhibit the antigen-antibody reaction and concentrating the exosome. As such a pretreatment method, polyethylene glycol (PEG) is proposed. Although the isolation method using a precipitation method, the isolation method using an ultracentrifuge, etc. are generally performed, it is difficult to sufficiently reduce nonspecific adsorption even if such pretreatment is performed, There was also a problem that it took time and effort.
The problem to be solved by the present invention is to provide a method capable of reducing non-specific adsorption to a solid support and selectively and efficiently separating vesicles having lipid bilayer membranes.

Thus, as a result of intensive studies, the present inventors contacted a biological sample containing a vesicle having a lipid bilayer membrane with a solid phase carrier bound with a ligand that recognizes a surface antigen present on the surface of the vesicle. In the method of separating a vesicle having a lipid bilayer membrane, comprising a complex forming step of forming a complex of the vesicle and the solid phase carrier, and a washing step of washing the complex. By performing at least one of the step and the washing step in the presence of an inorganic salt or organic salt having a final concentration of 0.15 to 2M, nonspecific adsorption to the solid support can be reduced, and a lipid bilayer membrane is provided. The inventors have found that vesicles can be selectively and efficiently separated, thereby completing the present invention.

That is, the present invention provides the following <1> to <10>.

<1> A biological sample containing a vesicle having a lipid bilayer membrane is contacted with a solid phase carrier bound with a ligand that recognizes a surface antigen present on the surface of the vesicle, and the vesicle and the solid phase carrier are contacted A complex forming step for forming a complex with the complex, and a washing step for washing the complex, wherein at least one of the complex forming step and the washing step includes an inorganic salt having a final concentration of 0.15 to 2M or A method for separating vesicles having a lipid bilayer membrane (hereinafter, also referred to as a first separation method), which is performed in the presence of an organic salt.

<2> A method for detecting a nucleic acid in a vesicle, further comprising a nucleic acid detection step for detecting the nucleic acid in the vesicle after the separation method of <1> above.

<3> A method for detecting a protein derived from a vesicle, further comprising a protein detection step of detecting a protein present on at least one of the inside and the surface of the vesicle after the separation method of <1>.

<4> A method for measuring a signal derived from a vesicle, further comprising a signal measurement step of measuring a signal intensity derived from a vesicle formed with the complex after the separation method of <1>.

<5> A method for determining whether or not a subject has developed a disease, wherein the complex is formed by the signal measurement method of <4> above using a biological sample derived from the subject. A method for determining a disease, comprising a step of measuring a signal intensity derived from a prepared vesicle.

<6> A method for evaluating the efficacy of a drug for treating a disease, wherein the complex is formed by the signal measurement method of <4> above using a biological sample derived from a subject before and after administration of the drug for treating a disease. A method for evaluating the efficacy of a therapeutic agent for a disease, comprising a step of measuring a signal intensity derived from a vesicle.

<7> A solid phase carrier to which a ligand that recognizes a surface antigen existing on the surface of a vesicle having a lipid bilayer is bound, and an inorganic salt or an organic salt, and the content of the inorganic salt or organic salt is within the system And a liquid composition in an amount capable of adjusting the final concentration to 0.15 to 2M.

<8> A biological sample containing vesicles having a lipid bilayer membrane is contacted with a solid phase carrier bound with a ligand that recognizes a surface antigen present on the surface of the vesicle, and the vesicle and the solid phase carrier are contacted The complex forming step and the washing step used in a method for separating a vesicle having a lipid bilayer membrane, comprising: a complex forming step for forming a complex with the complex; and a washing step for washing the complex. A liquid composition to be added to at least one of the above, wherein an inorganic salt or an organic salt is contained, and the content of the inorganic salt or organic salt can adjust the final concentration in the system to 0.15 to 2M A liquid composition, characterized in that

<9> A complex of the vesicle and the insoluble carrier used in a method of separating a vesicle having a lipid bilayer membrane from a biological sample containing the vesicle having a lipid bilayer membrane using an insoluble carrier. A specimen diluent for formation, comprising an inorganic salt or an organic salt, wherein the content of the inorganic salt or organic salt is such that the final concentration in the system can be adjusted to 0.15 to 2M. Sample dilution liquid.

<10> A method for separating vesicles having a lipid bilayer membrane from a biological sample containing vesicles having a lipid bilayer membrane using an insoluble carrier, the inorganic salt or organic salt containing the inorganic salt or organic salt A method for separating a vesicle having a lipid bilayer, wherein a specimen diluent having a salt content such that the final concentration in the system can be adjusted to 0.15 to 2M is added to the system ( Hereinafter, it is also referred to as a second separation method).

According to the method for separating vesicles having a lipid bilayer membrane of the present invention, non-specific adsorption to a solid phase carrier can be reduced, and vesicles having a lipid bilayer membrane can be selectively and efficiently separated.
Therefore, according to the present invention, nucleic acids in vesicles, proteins derived from vesicles, and signals derived from vesicles can be detected and measured easily and accurately. Moreover, the disease determination method and drug efficacy evaluation method of the present invention are unlikely to be false positives. In addition, by using the kit, liquid composition, and specimen diluent of the present invention, nonspecific adsorption to the solid phase carrier can be reduced, and vesicles having lipid bilayer membranes can be selectively and efficiently separated.

FIG. 5 is a view showing an effect of reducing nonspecific adsorption by setting the final concentration of buffer-derived sodium chloride to 0.15 to 0.35 M (Examples 1 to 5) (clinical sample: serum). FIG. 5 is a view showing a nonspecific adsorption reducing action by setting the final concentration of buffer-derived sodium chloride to 0.15 to 0.35 M (Examples 1 to 5) (clinical specimen: citrated plasma). FIG. 5 is a view showing a reactivity improving effect by setting the final concentration of buffer-derived sodium chloride at a reaction temperature of 25 ° C. to 0.15 to 0.35 M (Examples 1 to 5) (clinical sample: serum). It is a figure which shows the reactivity improvement effect by making final concentration of the sodium chloride derived from the buffer in reaction temperature 4 degreeC into 0.25M (Example 3) (clinical sample: serum). It is the figure which confirmed the nonspecific adsorption | suction reduction effect | action at the time of using various clinical specimens. It is the figure which confirmed the reactivity improvement effect at the time of using various clinical specimens. It is the figure which confirmed the reactivity improvement effect at the time of using an anti-CD63 antibody coupling | bonding magnetic particle as a solid-phase carrier. It is the figure which confirmed the reactivity improvement effect at the time of using an anti-CD81 antibody coupling | bonding magnetic particle as a solid-phase carrier. It is the figure which confirmed the reactivity improvement effect at the time of using an anti- EpCAM antibody coupling | bonding magnetic particle as a solid-phase carrier. It is a figure which shows the detection result of the nucleic acid in a vesicle at the time of making it react with human healthy subject serum using an anti-CD9 antibody coupling | bonding magnetic particle as a solid-phase carrier. It is a figure which shows the detection result of the nucleic acid in the vesicle derived from a human healthy subject serum. It is a figure which shows the detection result of the nucleic acid in the vesicle derived from human healthy subject heparin plasma. It is a figure which shows the detection result of the protein of the vesicle surface originating in human healthy subject serum. It is a figure which shows the detection result of the protein of the vesicle surface originating in human healthy subject heparin plasma. It is a figure which shows the detection result of the protein which maintained the three-dimensional structure of the native form of the vesicle surface derived from a human healthy subject serum. It is a figure which shows the detection result of the protein which maintained the three-dimensional structure of the native form of the vesicle surface derived from human healthy person heparin plasma. Results of detection of proteins on the surface of vesicles from a pseudo-disease-derived biological sample in which exosome-containing culture supernatant prepared from human colon cancer HT29 cells was added to the serum of healthy human subjects while maintaining the native structure of the native form FIG. The exosome-containing culture supernatant prepared from human colon cancer HT29 cells was added to heparin plasma of healthy human subjects, and the protein on the vesicle surface was detected while maintaining the native form of the three-dimensional structure. It is a figure which shows a result. It is a figure which shows the result of having detected the protein of the vesicle surface from the pseudo-disease origin biological sample which added the exosome containing culture supernatant prepared from the human colon cancer HT29 cell to human healthy subject serum. It is a figure which shows the result of having detected the protein of the vesicle surface from the pseudo-disease origin biological sample which added the exosome containing culture supernatant prepared from the human colon cancer HT29 cell to human healthy person heparin plasma.

[Separation method of vesicles having lipid bilayer membrane (first separation method)]
In the first separation method of the present invention, a biological sample containing vesicles having a lipid bilayer membrane is contacted with a solid phase carrier to which a ligand recognizing a surface antigen present on the surface of the vesicle is bound. A complex forming step of forming a complex of the solid phase carrier and a washing step of washing the complex, and at least one of the complex forming step and the washing step is performed with an inorganic salt having a final concentration of 0.15 to 2M Alternatively, it is carried out in the presence of an organic salt. Here, in this specification, the final concentration (final concentration) is a concentration of an inorganic salt derived from a biological sample (usually about 0.14M), an inorganic salt that does not contain an organic salt, or an organic salt, It refers to the final concentration of inorganic or organic salts in the system that exist artificially during the washing process. The final concentration of the inorganic salt or organic salt can be adjusted by any method using a solid composition such as powder or granules or a liquid composition such as a specimen diluent. Further, the final concentration may be measured using various known measuring methods and measuring instruments.

(Composite formation process)
In the complex formation step, a biological sample containing a vesicle having a lipid bilayer membrane is contacted with a solid phase carrier to which a ligand that recognizes a surface antigen present on the surface of the vesicle is bound, and the vesicle and the solid phase carrier are contacted. And forming a complex. By the contact, the vesicle is captured by the ligand, and a complex of the vesicle and the solid phase carrier is formed. In addition to the ligand bound to the solid phase carrier, a ligand that recognizes a surface antigen present on the surface of a vesicle having a lipid bilayer may coexist in the reaction system of the complex formation step.

The biological sample is not particularly limited as long as it contains vesicles having lipid bilayer membranes, such as body fluid, fungal fluid, cell culture medium, cell culture supernatant, tissue cell disruption fluid, etc. Various liquids are mentioned. Among these, a body fluid and a cell culture supernatant are preferable, and a body fluid is more preferable. Body fluids include whole blood, serum, plasma, blood components, various blood cells, blood clots, platelets, and other blood composition components, as well as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymph fluid, bone marrow fluid, tissue fluid , Saliva, gastric juice, joint fluid, pleural effusion, bile, ascites, amniotic fluid, etc., preferably blood composition components. Even if such a clinical specimen is used as a biological sample, the first separation method of the present invention has low non-specific adsorption to the solid phase carrier and high reactivity of the capture reaction. According to the first separation method, vesicles can be selectively and efficiently separated from a wide variety of biological samples. For example, nonspecific adsorption hardly occurs even when plasma is used as the biological sample, and the reactivity of the capture reaction is high even when serum is used.
The blood composition component may be treated with an anticoagulant such as citric acid, heparin, EDTA or the like.

The biological sample may be pre-treated by adding it to a buffer composition containing an inorganic salt or an organic salt in a final concentration of 0.15 to 2M. You can also use it as it is. That is, according to the first separation method of the present invention, it is possible to easily and selectively perform separation without pretreatment by a PEG precipitation method, an isolation method using an ultracentrifuge, or the like.

Examples of the vesicle having the lipid bilayer include vesicles such as cells and exosomes released from the cells to the outside. In the first separation method of the present invention, the vesicles are exosomes. In some cases, it is particularly preferably used.
The surface antigen present on the surface of the vesicle is not particularly limited as long as it is a substance present on the surface of the vesicle and is antigenic. Examples of exosome surface antigens include tetraspanins such as CD9, CD63, and CD81; antigen presentation-related proteins such as MHCI and MHCII; adhesion molecules such as integrin, ICAM-1, and EpCAM; cytokines such as EGFRvIII and TGF-β / Cytokine receptors, enzymes and the like. Among these, antigenic proteins present on the exosome surface are preferable.

The solid phase carrier used in the first separation method of the present invention is not particularly limited as long as a ligand that recognizes a surface antigen present on the surface of the vesicle is bound thereto.
As the ligand, an antibody that recognizes a surface antigen present on the surface of a vesicle is preferable, and an antibody that recognizes a surface antigen present on the surface of an exosome is more preferable. Moreover, although it may be a monoclonal antibody or a polyclonal antibody, it is preferably a monoclonal antibody.
The monoclonal antibody is not particularly limited, and a known method such as K.I. Watanabe et al. , Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis, J. et al. Clin. Invest. 114 (2004), 898-907. Monoclonal antibodies that recognize tetraspanins such as exosome surface antigens CD9, CD63, and CD81 can be prepared with reference to International Publication No. 2013/099925.
The antibody class includes IgG and IgM, and IgG is preferred. Moreover, you may use the fragment which reduced these molecules. For example, F (ab ′) 2, Fab ′, Fab and the like can be mentioned.

Examples of the material of the solid phase carrier to which the ligand is bonded include, for example, polystyrenes, polyethylenes, polypropylenes, polyesters, poly (meth) acrylonitriles, styrene-butadiene copolymers, poly (meth) acrylic esters, Polymer compounds such as fluororesin, crosslinked dextran, and polysaccharides; glass; metal; magnetic substance; resin composition containing magnetic substance; and combinations thereof.
The shape of the solid phase carrier is not particularly limited, and examples thereof include a tray shape, a spherical shape, a particle shape, a fiber shape, a rod shape, a disc shape, a container shape, a cell, a microplate, and a test tube.
In the present invention, magnetic particles are preferred from the viewpoint of solid-liquid separation and ease of washing.

Examples of the magnetic particles include metals such as triiron tetroxide (Fe ₃ O ₄ ), iron sesquioxide (γ-Fe ₂ O ₃ ), various ferrites, iron, manganese, nickel, cobalt, and chromium; cobalt, nickel And magnetic fine particles made of an alloy such as manganese; magnetic particles containing these magnetic substances in a resin. Examples of the resin include a hydrophobic polymer and a hydrophilic polymer.
Among these, magnetic particles containing a magnetic substance in a resin are preferable, and those in which a polymer layer is formed on the surface of mother particles containing superparamagnetic fine particles are more preferable. For example, a hydrophobic first polymer layer is formed on the surface of a mother particle containing superparamagnetic fine particles described in JP-A-2008-32411, and at least the surface has a glycidyl group on the first polymer layer. Examples include magnetic particles in which a second polymer layer is formed and a polar group is introduced by chemically modifying the glycidyl group.

Here, as the superparamagnetic fine particles, iron oxide-based fine particles having a particle size of 20 nm or less (preferably a particle size of 5 to 20 nm) are typical, and XFe ₂ O ₄ (X = Mn, Co, Ni, Mg, Cu). ferrite represented by Li _0.5 Fe _0.5, etc.), magnetite represented by Fe ₃ O _4, include γ-Fe ₂ O _3, strong saturation magnetization, and residual magnetization is small in terms, gamma-Fe ₂ It is preferable that either one of O ₃ and Fe ₃ O ₄ is included.

The monomers for forming the hydrophobic first polymer layer are roughly classified into monofunctional monomers and crosslinkable monomers.
Examples of the monofunctional monomer include aromatic vinyl monomers such as styrene, α-methylstyrene, and halogenated styrene; methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, stearyl acrylate, stearyl methacrylate, cyclohexyl acrylate, and cyclohexyl methacrylate. And ethylenically unsaturated carboxylic acid alkyl ester monomers such as isobornyl acrylate and isobornyl methacrylate. In addition, as the crosslinkable monomer, ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, dipentaerythritol hexa Examples include polyfunctional (meth) acrylates such as methacrylate; conjugated diolefins such as butadiene and isoprene, as well as divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate.

The monomer for forming the second polymer layer is mainly intended to introduce a functional group to the particle surface and includes a glycidyl group-containing monomer. As content of a glycidyl group containing monomer, 20 mass% or more is preferable in the monomer for forming a 2nd polymer layer. Here, examples of the copolymerizable monomer containing a glycidyl group include glycidyl acrylate, glycidyl methacrylate, and allyl glycidyl ether.

The polar group introduced by chemically modifying the glycidyl group of the second polymer layer is preferably a functional group capable of reacting with a ligand, and at least selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom Those containing one or more of one kind of atom are more preferable. Of these, an amino group, an aldehyde group, a carboxy group, and an active ester group are more preferable. In particular, when the second polymer layer of the magnetic particle has the polar group and 2,3-dihydroxypropyl group, the binding property to the ligand is good.
In addition, a solid phase carrier such as magnetic particles may have a substance having a biotin binding site immobilized on the surface thereof. Examples of the substance having a biotin binding site include one or more compounds selected from avidin, streptavidin, tamavidin, and derivatives thereof. Furthermore, in this case, the solid phase carrier may be bound with an antibody bound with biotins via a substance having a biotins binding site. Immobilization of a substance having a biotin-binding site on the surface of a solid phase carrier may be performed by a known method such as the method described in Japanese Patent No. 4716034.

As a method for binding a ligand to a solid phase carrier, a chemical binding method such as a physical adsorption method, a covalent bond method, or an ion bond method is used. Examples of the physical adsorption method include a method of directly immobilizing a ligand on a solid phase carrier, a method of chemically binding to another protein such as albumin, and then adsorbing and immobilizing. As a chemical bonding method, a method of directly bonding on a solid phase carrier using a functional group capable of reacting with a ligand introduced on the surface of the solid phase carrier, or a spacer molecule between the solid phase carrier and the ligand. Examples include a method of binding after introducing a carbodiimide compound (such as a carbodiimide compound), a method of binding a ligand to another protein such as albumin, and then chemically binding the protein to a solid phase carrier.

Further, a commercially available product may be used as the solid phase carrier to which a ligand that recognizes a surface antigen present on the surface of the vesicle is bound. For example, Exosome-Human CD9 Isolation Reagent (from cell culture), Exosome-Human CD63 Isolation / Detection Reagent (from cellCultureCulture Media) (both from Lifetech Corporation CD) CD81 Exo-Flow Capture kit (both manufactured by SBI).

The amount of the solid phase carrier to which the ligand is bound is preferably 0.00001 to 0.1 mass times, more preferably 0.0001 to 0.01 mass times with respect to the biological sample.

In addition to the above components, the complex formation step may be performed using a surfactant; a protein such as albumin; a nucleic acid; a saccharide such as sucrose as necessary.
Among these, from the viewpoint of reducing non-specific adsorption and from the viewpoint of suppressing damage to vesicles, surfactants are preferable, nonionic surfactants are more preferable, and polyethylene glycol type nonionic surfactants are particularly preferable. preferable. Further, the surfactant preferably does not contain an aromatic group in the molecule.
For example, higher alcohol ethylene oxide adduct, fatty acid ethylene oxide adduct, polyhydric alcohol fatty acid ester ethylene oxide adduct, higher alkylamine ethylene oxide adduct, fatty acid amide ethylene oxide adduct, fat and oil ethylene oxide adduct, polyalkylene glycol Examples include ethylene oxide adducts. Among these, polyalkylene glycol ethylene oxide adduct is preferable. Specific examples include polyoxyethylene (160) polyoxypropylene (30) glycol and polyoxyethylene (196) polyoxypropylene (67) glycol.
The amount of the surfactant used in the final concentration is preferably 0.005 to 10% (w / v), more preferably 0.015 to 3.5% (w / v).

In addition, the reaction temperature in the complex forming step is preferably in the range of 2 to 42 ° C, more preferably in the range of 15 to 40 ° C, and particularly preferably in the range of 20 to 37 ° C. The complex formation step in the first separation method of the present invention is very simple because the reaction proceeds selectively and efficiently even at or near room temperature. The reaction time is usually 1 minute to 48 hours, preferably 5 minutes to 24 hours, more preferably 10 minutes to 10 hours, and particularly preferably 10 minutes to 5 hours.

In the complex formation step, the pH in the system is not particularly limited, but is preferably in the range of pH 5 to 10, more preferably in the range of pH 6 to 8. In order to maintain the target pH, a buffer solution is usually used, and examples thereof include a phosphate buffer solution, a tris (hydroxymethyl) aminomethane buffer solution, a HEPES buffer solution, and a MES buffer solution.

(Washing process)
The washing step is a step of washing the complex of the vesicle formed in the complex forming step and the solid phase carrier. Thereby, unreacted components, unreacted labeling substances and the like are removed. You may wash | clean the system containing the composite_body | complex in a composite_body | complex formation process as it is.

The washing step is usually divided into two types depending on the shape of the solid support. In the case where the solid phase carrier is in the form of particles, such as magnetic particles, for example, there is a method in which the magnetic particles are dispersed in a cleaning solution for washing, while the solid phase carrier is in the form of a microplate. Can be cleaned by bringing a cleaning liquid into contact with the surface. In any embodiment, in the present invention, it is preferable to use a cleaning liquid containing a surfactant as the cleaning liquid. As the surfactant, those similar to those that may be used in the complex forming step are preferable.

When the solid phase carrier is a magnetic particle, the washing step includes collecting the magnetic particles by a magnetic force to separate the magnetic particles from the liquid phase, and separating the magnetic particles separated in the collecting step in the washing liquid. It is preferable to include a dispersion step of dispersing in the water. As a result, unreacted substances and contaminants in the biological sample can be more efficiently washed and separated and removed from the surface of the magnetic particles.
Specifically, a magnetic field was applied to the reaction vessel, and the magnetic particles were collected by adhering to the reaction vessel wall, and then the reaction supernatant was removed, and a washing solution was added as necessary to apply the magnetic field in the same manner. What is necessary is just to repeat the operation of removing the post-supernatant. As the cleaning liquid, a cleaning liquid containing the surfactant and buffer mentioned in the complex forming step is preferable.

(Inorganic salt or organic salt)
In the first separation method of the present invention, at least one of the complex formation step and the washing step is performed in the presence of an inorganic salt or organic salt having a final concentration of 0.15 to 2M. From the viewpoint of properties, the complex formation step is preferably performed in the presence of an inorganic salt or organic salt having a final concentration of 0.15 to 2M. Among inorganic salts and organic salts, inorganic salts are preferred from the viewpoint of enhancing the desired effect of the present invention.

Examples of the inorganic salt include alkali metal halides; salts of inorganic acids such as carbonates and bicarbonates, alkali metal halides are preferable, and alkali metal chlorides are more preferable. Specific examples of the inorganic salt include sodium chloride, potassium chloride, calcium chloride, sodium carbonate, sodium bicarbonate and the like, and one of these may be used alone or two or more may be used in combination. Good. Among these, sodium chloride, potassium chloride, and calcium chloride are preferable, sodium chloride and potassium chloride are more preferable, and sodium chloride is particularly preferable.

The organic salt is preferably an organic acid salt, more preferably an organic acid alkali metal salt, still more preferably a carboxylic acid alkali metal salt, and particularly preferably a carboxylic acid sodium salt. Specific examples of the organic salt include sodium acetate, sodium citrate, sodium tartrate, sodium malate, sodium succinate, sodium lactate and the like. You may use it in combination.

Also, the final concentration of inorganic salt or organic salt in the system is 0.15 to 2M. When the final concentration defined in the present specification is less than 0.15 M or higher than 2 M, nonspecific adsorption may increase or the reactivity of the capture reaction may decrease, and the osmotic pressure difference between the inside and outside of the vesicle may occur. There is also concern that the vesicles will be destroyed. The final concentration is preferably 0.2M or more, more preferably 0.25M or more, preferably 1M or less, more preferably 0.5M or less, still more preferably 0.45M or less, and particularly preferably 0.35M. It is as follows.

(Dissociation process)
The separation method of the present invention may have a dissociation step of dissociating the captured vesicle from the ligand after the washing step.
As conditions for releasing specific binding due to antigen-antibody reaction when the ligand is a specific antibody, various conditions are known by affinity chromatography or the like (for example, “Affinity Chromatography principles & methods” Pharmacia). See LKB Biotechnology). The dissociation step may be performed according to this. That is, as the dissociation solution used in the present invention, acidic solutions such as hydrochloric acid, sulfuric acid, propionic acid, acetic acid, glycine / hydrochloric acid buffer; alkaline solutions such as sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, ammonia aqueous solution, diethylamine; High ionic strength solution such as 3M sodium chloride aqueous solution and 4.5M magnesium chloride aqueous solution; Surfactant-containing solution such as SDS, Triton X-100, Tween 20; Buffer containing a substance that lowers the polarity such as dioxane, ethylene glycol; Trichloro; In addition to chaotropic ions such as acetic acid and thiocyanic acid, buffer solutions containing urea, guanidine hydrochloride and the like can be mentioned.

[Nucleic acid detection method and protein detection method]
The method for detecting a nucleic acid in a vesicle of the present invention further comprises a nucleic acid detection step for detecting the nucleic acid in the vesicle after the first separation method.
The method for detecting a protein derived from a vesicle of the present invention further comprises a protein detection step of detecting a protein present on at least one of the inside and the surface of the vesicle after the first separation method.
These detection methods may be performed according to a conventional method except that the first separation method is performed. When the vesicles having the lipid bilayer are exosomes, nucleic acids and proteins are collected from the collected exosomes according to known methods such as PCR, electrophoresis, Western blotting, immunochemical methods, flow cytometry, etc. What is necessary is just to detect. Among these, flow cytometry is preferable because it can be easily detected with high sensitivity and the three-dimensional structure of the native form can be maintained. Examples of the nucleic acid include miRNA and mRNA.
In particular, since exosomes are secreted from various cells such as cells of the immune system and various cancer cells, the nucleic acid detection method of the present invention detects exosome-derived nucleic acids (miRNA, etc.) By analyzing, it becomes possible to determine physiological phenomena and various diseases.

[Signal measurement method]
The vesicle-derived signal measurement method of the present invention further comprises a signal measurement step of measuring the signal intensity derived from the vesicles forming the complex after the first separation method.
The signal intensity may be measured by immunoassay, for example, enzyme immunoassay (EIA), enzyme immunometric assay (ELISA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence. Examples include immunoassay, immunoblotting, western blotting, flow cytometry, and the like. Among these, flow cytometry is preferable because it can be easily detected with high sensitivity and the three-dimensional structure of the native form can be maintained. The ELISA method is preferable from the viewpoint that the antibody can be easily detected with high sensitivity. Examples of the ELISA method include a competitive method and a sandwich method.
Here, an example of the separation method in the case of using the sandwich ELISA method is shown. First, a ligand that recognizes a surface antigen present on the surface of a vesicle is bound to a solid phase carrier, and then a biological sample containing a vesicle having a lipid bilayer is contacted to form a complex, followed by washing. To this, a monoclonal antibody or an antibody fragment thereof or a labeled antibody obtained by modifying a disease-specific membrane protein antibody or an antibody fragment thereof is added to form a further complex. Then, by detecting the amount of label in the formed complex, the amount of signal derived from vesicles contained in the biological sample and the amount of disease-specific vesicles contained in the biological sample can be measured.

[Disease determination method]
A method for determining whether or not a subject of the present invention has developed a disease is a signal derived from a vesicle formed with the complex by the above signal measurement method using a biological sample derived from the subject. It includes a step of measuring strength (hereinafter also referred to as step (I)).
Then, when the signal intensity measured in the step (I) is compared with the signal intensity of the biological sample derived from the control person, the signal intensity in the subject is recognized to be stronger than the signal intensity in the control person. What is necessary is just to determine with the examiner developing the disease (henceforth process (II)).
Diseases that can be determined by the disease determination method of the present invention include cancer diseases (colon cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer, lung cancer, renal cancer, skin cancer. ), Inflammatory diseases (rheumatic, osteoarthritis, nephropathy, pancreatitis, hepatitis, allergy, etc.), neurodegenerative diseases such as Alzheimer, brain diseases, diseases related to immunodeficiency, infertility, depression, autism And psychiatric diseases such as Parkinson's disease, autoimmune diseases, cardiovascular diseases, blood diseases, gastrointestinal diseases, diseases associated with aging, infectious diseases and the like. Among these, it is useful for determination of cancer diseases and immune system diseases, and particularly useful for determination of cancer diseases.
Moreover, if it applies to the parameter | index of the immune activity of cytotoxic T cell (CTL), it can apply also to the effect determination with respect to the cancer disease of a cancer vaccine.

Step (I) may be performed according to a conventional method except that measurement is performed by the above signal measurement method.
In step (II), the signal intensity measured in step (I) is compared based on a statistical analysis based on the signal of the biological sample derived from the control person. The analysis method is not particularly limited, and a known method can be used. Further, in the subsequent determination, for example, when the signal of the biological sample derived from the subject is stronger than the signal of the control person, it is determined that the possibility of developing the disease is high. In the present invention, the control person means the average of the same age and the same person as the subject who has not developed the disease, the signal intensity in the control person may be measured together with the signal intensity in the subject, A statistical value obtained from a separately measured value may be used.

[Methods for evaluating the efficacy of drugs for treating diseases]
The method for evaluating the efficacy of a therapeutic agent for a disease according to the present invention is a signal intensity derived from a vesicle formed with a complex by a signal measurement method using a biological sample derived from a subject before and after administration of a therapeutic agent for a disease. It includes a step of measuring (hereinafter also referred to as step (A)).
Then, it is recognized that the signal intensity derived from the complex in the biological sample derived from the subject after administration of the disease therapeutic agent is weaker than the signal intensity derived from the complex in the biological sample derived from the subject before administration of the disease therapeutic agent. In such a case, it may be determined that there is a high possibility that the therapeutic agent for the disease has a medicinal effect (hereinafter also referred to as step (B)).
Examples of the disease therapeutic drug include drugs for treating a disease that can be determined by the determination method. Preferred specific examples are anticancer drugs and anti-immune disease drugs.

Step (A) may be performed according to a conventional method except that measurement is performed by the above signal measurement method.
In the step (B), the signal intensity measured in the step (A) is compared based on a statistical analysis based on the signal in the biological sample before administration of the disease therapeutic agent. The analysis method is not particularly limited, and a known method can be used. Further, in the subsequent determination, for example, when the amount of signal in the biological sample after administration of the disease therapeutic drug is small compared to the amount of signal in the biological sample before administration, the drug may have an effect of suppressing the disease. It is judged that the nature is high.
In particular, since exosomes are secreted from various cells such as cells of the immune system and various cancer cells, changes in blood exosomes before and after administration of disease therapeutic agents (in addition to increase / decrease in abundance, membrane proteins) It is considered possible to evaluate the drug efficacy in patients. In addition, for example, by combining a ligand for a membrane protein specific for cancer cells and a ligand for a surface antigen of exosomes, it can be expected to improve the specificity of cancer diagnosis, identify the type of cancer, etc. It is considered possible to develop new diagnostic agents.

〔kit〕
The kit of the present invention comprises a solid phase carrier bound with a ligand that recognizes a surface antigen present on the surface of a vesicle having a lipid bilayer membrane, and an inorganic salt or an organic salt, and the content of the inorganic salt or organic salt is And a liquid composition in an amount capable of adjusting the final concentration in the system to 0.15 to 2M. Such a kit is useful for the first separation method, determination of the disease, and evaluation of the efficacy of the therapeutic agent for the disease.
Examples of the inorganic salt or organic salt contained in the solid phase carrier and the liquid composition include the same salts as those used in the first separation method.
The final concentration is preferably 0.2 M or more, more preferably 0.25 M or more, and preferably 1 M or less, more preferably 0.5 M or less, still more preferably 0.45 M or less, particularly preferably 0. 35M or less.
Specifically, the content of the inorganic salt or organic salt in the liquid composition is preferably 0.3M or more, more preferably 0.4M or more, still more preferably 0.5M or more, and preferably 4M. Below, more preferably 2M or less, still more preferably 1M or less, still more preferably 0.9M or less, and particularly preferably 0.7M or less.
Moreover, the liquid composition may contain the same surfactant as that in the first separation method. The pH of the liquid composition is not particularly limited, but is preferably in the range of pH 5 to 10, more preferably in the range of pH 6 to 8. In order to maintain the target pH, a buffer solution is usually used, and examples thereof include a phosphate buffer solution, a tris (hydroxymethyl) aminomethane buffer solution, a HEPES buffer solution, and a MES buffer solution.

[Liquid composition, specimen diluent and second separation method]
The liquid composition of the present invention comprises contacting a biological sample containing vesicles having a lipid bilayer with a solid phase carrier bound with a ligand that recognizes a surface antigen present on the surface of the vesicle, Formation of a complex used in a method for separating a vesicle having a lipid bilayer membrane, comprising: a complex forming step of forming a complex between the solid phase carrier and the solid phase carrier; and a washing step of washing the complex. A liquid composition to be added to at least one of the step and the washing step, comprising an inorganic salt or an organic salt, the content of the inorganic salt or organic salt being 0.15 to the final concentration in the system The amount can be adjusted to 2M.
In addition, the specimen diluent of the present invention is used in a method for separating a vesicle having a lipid bilayer from a biological sample containing a vesicle having a lipid bilayer using an insoluble carrier, Sample diluent for forming a complex with an insoluble carrier, containing an inorganic salt or organic salt, and the content of the inorganic salt or organic salt is adjusted to a final concentration in the system of 0.15 to 2M It is a quantity that can be produced.
The second separation method of the present invention is a method for separating vesicles having a lipid bilayer membrane from a biological sample containing vesicles having a lipid bilayer membrane using an insoluble carrier, which comprises an inorganic salt or an organic salt. A specimen diluent containing a salt and having an inorganic salt or organic salt content that can adjust the final concentration in the system to 0.15 to 2M is added to the system.
The composition and pH of the liquid composition and specimen diluent of the present invention are the same as those of the liquid composition provided in the kit of the present invention. In addition, the sample diluent of the present invention is used for vesicle separation using an insoluble carrier (for example, a gel filtration carrier or a filter) other than a solid phase carrier bound with a ligand that recognizes a surface antigen present on the surface of the vesicle. It may be used. Therefore, the specimen diluent of the present invention can be used for vesicle separation using various carriers.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

(Test Example 1)
Negative control particles that bind anti-mouse IgG antibodies that do not react with exosomes to magnetic particles, clinical specimens containing exosomes, and reaction buffer are mixed, washed, and CD81 expressed on the exosome membrane is recognized. By detecting exosomes that were non-specifically adsorbed to the negative control particles by ELISA using the antibody to be detected, the non-specific adsorption reducing action of the reaction buffer was confirmed. The specific procedure is shown below.

First, the following reaction buffers were prepared as the reaction buffers of Reference Example 1 and Examples 1 to 5.
Reaction buffer of Reference Example 1 and Examples 1 to 5: Tris-buffered saline (TBS (10 × TBS manufactured by Sigma, 10 times diluted, the same shall apply hereinafter)) and polyoxyethylene (160 ) Polyoxypropylene (30) glycol (Gibco's Pluronic F-68, hereinafter the same) was added to a concentration of 0.03% (w / v), and sodium chloride (Wako) was added as an inorganic salt. , Reagent special grade) added to the concentrations shown in Table 1

Next, the following buffers were prepared as control buffers.
Control buffer: TBS added with polyoxyethylene (160) polyoxypropylene (30) glycol as a nonionic surfactant to a concentration of 0.03% (w / v). The following specimens were prepared as 5.
Specimen 1 (HT29 sup. In Control Buffer): HT29 cell culture supernatant 100-fold concentrated solution added to control buffer as a 20-fold dilution Specimen 2 (Serum): Pool serum (manufactured by Kojin Bio) )
Specimen 3 (HT29 sup. In Serum): A HT29 cell culture supernatant 100-fold concentrated solution added to a pooled serum (manufactured by Kojin Bio Inc.) as a spike so as to be diluted 20-fold Specimen 4 (Plasma): Pool quenched Acid plasma (manufactured by Kojin Bio)
Specimen 5 (HT29 sup. In Plasma): a citrate plasma (manufactured by Kojin Bio Inc.) added with a 100-fold concentrated HT29 cell culture supernatant as a spike so as to be diluted 20-fold. Next, negative control particles As described above, an anti-mouse IgG antibody (manufactured by JSR Life Sciences, the same hereinafter) was prepared by binding to magnetic particles (MS300 / Carboxyl, manufactured by JSR Life Sciences, the same hereinafter).

Next, 0.0125 mg of negative control particles were added to a 96-well white plate (Corning # 2371007), and 25 μL of control buffer and samples 1 to 5 were added to each well, one type per well. The reaction buffer of Reference Example 1 was added so that the final concentration of sodium chloride was 0.07 M, and the mixture was reacted at 25 ° C. for 20 minutes (pH: 7.4). Further, the same reaction was carried out using the same amount of each of the reaction buffers of Examples 1 to 5 instead of the reaction buffer of Reference Example 1 (Example 1: final concentration 0.15M, Example 2: final concentration 0). 2M, Example 3: final concentration of 0.25M, Example 4: final concentration of 0.3M, Example 5: final concentration of 0.35M (Examples 1-5: pH 7.4)).
Next, the negative control particles after the reaction were washed with a washing buffer (TBS containing 0.1% by mass of Tween 20), and diluted with TBS containing 1% by mass of BSA. An alkaline phosphatase (hereinafter, ALP) -labeled anti-CD81 antibody 50 μL of (0.1 μg / mL) was added and reacted at 25 ° C. for 20 minutes. After washing with the same washing buffer as described above, 50 μL of a luminescent substrate (Fujirebio Class III series Lumipulse substrate solution) was added, and after 5 minutes, the luminescence intensity was measured using a luminescence measuring device (Promega GloMax). . The results are shown in FIGS. 1-1 and 1-2.
As shown in FIGS. 1-1 and 1-2, when the final concentration of sodium chloride was 0.07 M (Reference Example 1), a background signal that was thought to be due to nonspecific adsorption of the analyte components was detected. When the final concentration of sodium chloride was 0.15 to 0.35 M (Examples 1 to 5), nonspecific adsorption was reduced and the background signal was reduced.

(Test Example 2-1)
An anti-CD9 antibody (abcam manufactured by Abcam, ab2215, hereinafter the same) prepared by binding to magnetic particles (hereinafter also referred to as anti-CD9 antibody-binding magnetic particles) is prepared, and a clinical specimen containing the anti-CD9 antibody-binding magnetic particles and exosomes Are mixed with the reaction buffers of Reference Example 1 and Examples 1 to 5, and after washing, the exosomes captured by ELISA are detected in the same manner as in Test Example 1, so that Reference Example 1 and Examples 1 to 5 are detected. The reactivity when each reaction buffer was used was evaluated. The specific procedure is shown below.
First, the following specimens were prepared as specimens 6 to 8.
Specimen 6 (HT29 sup. In Control Buffer): A control buffer prepared in Test Example 1 added with a HT29 cell culture supernatant 100-fold concentrated solution as a spike so as to be diluted 100-fold Specimen 7 (Serum): Pool Serum (manufactured by Kojin Bio)
Specimen 8 (HT29 sup. In Serum): HT29 cell culture supernatant 100-fold concentrated solution added to pooled serum (manufactured by Kojin Bio) as a 100-fold dilution

Next, 0.1 mg of anti-CD9 antibody-binding magnetic particles was added to 2 mL of Protein LoBind tube (manufactured by Eppendorf, # 0030108132), and samples 6 to 8 were added to each tube, one sample per sample (sample 6: 0.1 mL, specimen 7: 0.1 mL, specimen 8: 0.1 mL). The reaction buffer of Reference Example 1 was added so that the final concentration of sodium chloride was 0.07 M, and the mixture was reacted at 25 ° C. for 5 hours (pH: 7.4). Further, the same reaction was carried out using the same amount of each of the reaction buffers of Examples 1 to 5 instead of the reaction buffer of Reference Example 1 (Example 1: final concentration 0.15M, Example 2: final concentration 0). 2M, Example 3: final concentration of 0.25M, Example 4: final concentration of 0.3M, Example 5: final concentration of 0.35M (Examples 1-5: pH 7.4)).
Next, after washing with 0.5 mL of Washing / Dilution Buffer (manufactured by JSR Life Sciences), it was dispensed to a 96-well white plate (# 23711007, manufactured by Corning) at 0.0125 mg / well. To this, 50 μL of ALP-labeled anti-CD81 antibody (0.1 μg / mL) diluted with TBS containing 1% by mass of BSA was added and reacted at 25 ° C. for 20 minutes. The particles after the reaction were washed using a washing buffer (TBS containing 0.1% by weight of Tween 20), and 50 μL of a luminescent substrate (Class III series Lumipulse substrate solution manufactured by Fujirebio Inc.) was added. Luminescence intensity was measured using GloMax (Promega). The results are shown in FIG.
As shown in FIG. 2-1, it was found that when the final concentration of sodium chloride was 0.15 to 0.35 M (Examples 1 to 5), the reactivity of the capture reaction in serum was improved.

(Test Example 2-2)
The same test as in Test Example 2-1 was performed by changing the reaction temperature of the capture reaction from 25 ° C. to 4 ° C. The specific procedure is shown below.
0.1 mg of anti-CD9 antibody-binding magnetic particles was added to 2 mL of Protein LoBind tube (Eppendorf, # 0030108132), and samples 6 to 8 were added to each tube, one sample per sample (sample 6: 0. 1 mL, specimen 7: 0.1 mL, specimen 8: 0.1 mL). The reaction buffer of Reference Example 1 was added so that the final concentration of sodium chloride was 0.07 M, and the mixture was reacted at 4 ° C. for 5 hours (pH: 7.4). In addition, an equivalent amount of the reaction buffer of Example 3 was used instead of the reaction buffer of Reference Example 1, and a similar reaction was performed (Example 3: final concentration of 0.25 M (pH: 7.4)).
Next, after washing with 0.5 mL of a washing buffer (TBS containing 0.1% by weight of Tween 20), it was dispensed to a 96-well white plate (Corning # 2371007) at 0.0125 mg / well. To this, 50 μL of ALP-labeled anti-CD81 antibody (0.1 μg / mL) diluted with TBS containing 1% by mass of BSA was added and reacted at 25 ° C. for 20 minutes. The particles after the reaction were washed using a washing buffer (TBS containing 0.1% by weight of Tween 20), and 50 μL of a luminescent substrate (Class III series Lumipulse substrate solution manufactured by Fujirebio Inc.) was added. Luminescence intensity was measured using GloMax (Promega). The results are shown in Fig. 2-2. FIG. 2-2 also shows the results of tests conducted at a reaction temperature of 25 ° C.
From the results of Test Example 2-2 (FIG. 2-2), when the final concentration of sodium chloride is 0.25 M (Example 3), the serum concentration is not limited regardless of whether the reaction temperature is 4 ° C. or 25 ° C. It has been found that the reactivity of the capture reaction at is improved.

(Test Example 3)
The nonspecific adsorption reducing action of the reaction buffer of Example 3 was confirmed by changing the sample. The specific procedure is shown below.
First, the following specimens were prepared as specimens 9-17.
Specimen 9 (HT29 sup. Spike Control Buffer): HT29 cell culture supernatant 100-fold concentrated solution added as a spike to the control buffer prepared in Test Example 1 Specimen 10 (Serum): Pool Serum (manufactured by Kojin Bio)
Specimen 11 (HT29 sup. Spike Serum): Pool serum (manufactured by Kojin Bio Inc.) spiked with 100-fold concentrated HT29 cell culture supernatant as a spike Specimen 12 (Plasma (Heparin)) : Pooled heparin plasma (manufactured by Kojin Bio)
Specimen 13 (HT29 sup. Spike Plasma (Heparin)): Pooled heparin plasma (manufactured by Kojin Bio Inc.), spiked with 100-fold concentrated HT29 cell culture supernatant as a spike Specimen 14 (Plasma (EDTA)): Pooled EDTA plasma (manufactured by Kojin Bio)
Specimen 15 (HT29 sup. Spike Plasma (EDTA)): A pooled EDTA plasma (manufactured by Kojin Bio Inc.) added with HT29 cell culture supernatant 100-fold concentrated solution as a spike at a dilution of 100-fold Specimen 16 (Plasma (Citrate): Pooled citrate plasma (manufactured by Kojin Bio)
Specimen 17 (HT29 sup. Spike Plasma (Citrate)): Pool citrate plasma (manufactured by Kojin Bio Inc.), spiked with HT29 cell culture supernatant 100-fold concentrated solution diluted 100-fold

Next, 0.1 mg of the negative control particles prepared in Test Example 1 were added to 2 mL of Protein LoBind tube (manufactured by Eppendorf, # 0030108132), and 0.1 mL of specimens 9 to 17 were added to each tube, one type per tube. Added. The reaction buffer of Reference Example 1 was added so that the final concentration of sodium chloride was 0.07 M, and the mixture was reacted at 25 ° C. for 5 hours (pH: 7.4). In addition, an equivalent amount of the reaction buffer of Example 3 was used instead of the reaction buffer of Reference Example 1, and a similar reaction was performed (Example 3: final concentration of 0.25 M (pH: 7.4)).
Next, after washing with 0.5 mL of a washing buffer (TBS containing 0.1% by weight of Tween 20), it was dispensed to a 96-well white plate (Corning # 2371007) at 0.0125 mg / well. To this, 50 μL of ALP-labeled anti-CD81 antibody (0.1 μg / mL) diluted with TBS containing 1% by mass of BSA was added and reacted at 25 ° C. for 20 minutes. The particles after the reaction were washed using a washing buffer (TBS containing 0.1% by weight of Tween 20), and 50 μL of a luminescent substrate (Class III series Lumipulse substrate solution manufactured by Fujirebio Inc.) was added. Luminescence intensity was measured using GloMax (Promega).
As shown in FIG. 3, when the final concentration of sodium chloride was 0.25 M (Example 3), the background signal was small regardless of the type of specimen.

(Test Example 4)
The reactivity improvement effect of the reaction buffer of Example 3 was confirmed by changing the sample. The specific procedure is shown below.
To 2 mL of Protein LoBind tube (Eppendorf, # 0030108132), 0.1 mg of anti-CD9 antibody-bound magnetic particles was added, and 0.1 mL of each of Samples 9 to 13 shown in Test Example 3 was added to each tube. Seed added. The reaction buffer of Reference Example 1 was added so that the final concentration of sodium chloride was 0.07 M, and the mixture was reacted at 25 ° C. for 5 hours (pH: 7.4). In addition, an equivalent amount of the reaction buffer of Example 3 was used instead of the reaction buffer of Reference Example 1, and a similar reaction was performed (Example 3: final concentration of 0.25 M (pH: 7.4)).
Next, after washing with 0.5 mL of a washing buffer (TBS containing 0.1% by weight of Tween 20), it was dispensed to a 96-well white plate (Corning # 2371007) at 0.0125 mg / well. To this, 50 μL of ALP-labeled anti-CD81 antibody (0.1 μg / mL) diluted with TBS containing 1% by mass of BSA was added and reacted at 25 ° C. for 20 minutes. The particles after the reaction were washed using a washing buffer (TBS containing 0.1% by weight of Tween 20), and 50 μL of a luminescent substrate (Class III series Lumipulse substrate solution manufactured by Fujirebio Inc.) was added. Luminescence intensity was measured using GloMax (Promega). The results are shown in FIG.
As shown in FIG. 4, when the final concentration of sodium chloride was 0.25 M (Example 3), the reactivity of the capture reaction was high regardless of the type of specimen.

(Test Example 5)
Anti-CD63 antibody (manufactured by JSR Life Sciences) bound to magnetic particles (hereinafter referred to as anti-CD63 antibody-bound magnetic particles), anti-CD81 antibody (Clone M38, Abnova MAB6435) bound to magnetic particles (hereinafter referred to as “anti-CD63 antibody-bound magnetic particles”) , Anti-CD81 antibody-binding magnetic particles) and anti-EpCAM antibody (manufactured by JSR Life Sciences) bonded to magnetic particles (hereinafter referred to as anti-EpCAM antibody-binding magnetic particles) were prepared.
The test was performed except that the anti-CD9 antibody-bound magnetic particles were changed to anti-CD63 antibody-bound magnetic particles, anti-CD81 antibody-bound magnetic particles, and anti-EpCAM antibody-bound magnetic particles, and the samples 10 to 13 shown in Test Example 3 were used as samples. In the same manner as in Example 4, the reactivity improvement effect of the reaction buffer of Example 3 was examined. The results are shown in FIG. 5-1 (anti-CD63 antibody-bound magnetic particles), FIG. 5-2 (anti-CD81 antibody-bound magnetic particles), and FIG. 5-3 (anti-EpCAM antibody-bound magnetic particles).

(Test Example 6 Detection of nucleic acids in vesicles)
Twelve 2 ml of Protein LoBind tubes (Eppendorf, # 0030108132) were prepared and numbered with tubes 1-12. Tubes 1 to 4 each contain 0.1 mg of anti-CD9 antibody-binding magnetic particles, tubes 5 to 8 each contain 0.5 mg of anti-CD9 antibody-binding magnetic particles, and tubes 9 to 12 contain 1 mg of anti-CD9 antibody-binding magnetic particles. Each was added.
Next, 1 mL of the reaction buffer of Example 3 was added to tubes 1 to 12 so that the final concentration of sodium chloride was 0.25 M, and 1 mL of the specimen 10 shown in Test Example 3 was further added. 1 hour at 25 ° C for tubes 1, 5, 9; 5 hours at 25 ° C for tubes 2, 6, 10; 24 hours at 25 ° C for tubes 3, 7, 11; The reaction was performed at 4 ° C. for 24 hours.
Next, each particle after the reaction was washed three times with 0.5 mL of a washing buffer (TBS containing 0.1% by weight of Tween 20), and this suspension was added to 2 mL of a new Protein LoBind tube (Eppendorf). Product, # 0030108132). After collecting antibody-bound magnetic particles using a magnetic flux collecting rack, 0.4 mL of the supernatant was extracted, and microRNA was recovered from the remaining 0.1 mL of the antibody-bound particle solution and quantified. The results are shown in FIG.
In addition, ExoCap ^™ Nucleic Acid Elution Buffer (manufactured by MBL, # MEX-E) was used for microRNA recovery. In addition, for quantification of microRNA (miR-21), TaqMan MicroRNA Reverse Transcription Kit (manufactured by Life Technologies, # 4365597), TaqMan MicroRNA Assays (manufactured by Life Technologies, # 1867948384), TaqMan UniMinGunUrGianUNG Using Life Technologies, # 4440040), the recommended protocol was followed for operation.

As shown in FIG. 6, nucleic acids in vesicles derived from human healthy human serum could be detected by reacting in the presence of sodium chloride having a final concentration of 0.25M. Furthermore, it was confirmed that the amount of nucleic acid detected increased depending on the amount of antibody-bound particles, temperature, and time.

(Test Example 7 Detection of nucleic acids in vesicles)
Eight 8 ml of Protein LoBind tubes (Eppendorf, # 0030108132) were prepared and numbered with tubes 13-20. Tubes 13-14 contain 1 mg of anti-CD9 antibody-coupled magnetic particles, tubes 15-16 contain 1 mg of anti-CD63 antibody-coupled magnetic particles, tubes 17-18 contain 1 mg of anti-CD81 antibody-coupled magnetic particles, tube 19 -20 to Composite antibody-bound magnetic particles (mixed anti-CD9 antibody-bound magnetic particles, anti-CD63 antibody-bound magnetic particles, anti-CD81 antibody-bound magnetic particles and anti-EpCAM antibody-bound magnetic particles in a 1: 1: 1: 1 ratio) 1 mg each was added.
Next, 0.3 mL of the reaction buffer of Example 3 was added to tubes 13 to 20 so that the final concentration of sodium chloride was 0.25M. The sample 10 shown in Test Example 3 was added to the odd-numbered tube, and 0.3 mL each of the sample 12 shown in Test Example 3 was added to the even-numbered tube and reacted at 25 ° C. for 24 hours.
Next, each particle after the reaction was washed three times with 0.5 mL of a washing buffer (TBS containing 0.1% by weight of Tween 20), and this suspension was added to 2 mL of a new Protein LoBind tube (Eppendorf). Product, # 0030108132). After collecting antibody-bound magnetic particles using a magnetic flux collecting rack, 0.4 mL of the supernatant was extracted, and microRNA was recovered from the remaining 0.1 mL of the antibody-bound particle solution and quantified. The results are shown in FIGS. 7-1 and 7-2.
In addition, miRNeasy Serum / Plasma Kit (50) (QIAGEN, # 217184) was used for microRNA collection. In addition, for quantification of microRNA (miR-21), TaqMan MicroRNA Reverse Transcription Kit (manufactured by Life Technologies, # 4365597), TaqMan MicroRNA Assays (manufactured by Life Technologies, # 1867948384), TaqMan UniMinGunUrGianUNG Using Life Technologies, # 4440040), the recommended protocol was followed for operation.

For comparison, nucleic acids in vesicles were detected using an ultracentrifugation method, which is a standard method for recovering exosomes. Specifically, 11 mL of each of the specimens 10 and 12 shown in Test Example 3 was centrifuged at 100,000 × g and 4 ° C. for 70 minutes using an ultracentrifuge (Optima XE-90 manufactured by Beckman). The supernatant was extracted, and 11 mL of PBS was added to the precipitate and suspended. Again, the mixture was centrifuged at 100,000 rpm at 4 ° C. for 70 minutes using an ultracentrifuge (Optima XE-90 manufactured by Beckman). The supernatant was extracted, and 0.11 mL of PBS was added to the precipitate for suspension. 3 μL of the suspension was used for collecting and quantifying microRNA. The results are shown in FIGS. 7-1 and 7-2.
MicroRNA recovery and microRNA (miR-21) quantification were performed in the same manner as described above.

As shown in Fig. 7-1, it was confirmed that nucleic acids in vesicles derived from serum of healthy human subjects can be detected at the same level as in ultracentrifugation by reacting in the presence of sodium chloride with a final concentration of 0.25M. It was done.
As shown in FIG. 7-2, nucleic acids in vesicles derived from heparin plasma from healthy human subjects could not be detected using the ultracentrifugation method. It is considered that heparin plasma of healthy human subjects generally contains substances that cause PCR inhibition, and substances that inhibit PCR remain in samples collected by ultracentrifugation. In contrast, when the reaction was carried out in the presence of sodium chloride having a final concentration of 0.25 M, nucleic acids in vesicles derived from human healthy human heparin plasma could be detected.
From these results, it was found that by reacting vesicles and particles in the presence of sodium chloride having a final concentration of 0.25 M, nucleic acids in the vesicles can be detected regardless of the type of specimen such as serum and plasma.

(Test Example 8 Detection of protein on vesicle surface)
Ten 2 ml of Protein LoBind tubes (Eppendorf, # 0030108132) were prepared and numbered with tubes 21-30. Tubes 21 to 22 each contain 0.2 mg of anti-CD9 antibody-binding magnetic particles, tubes 23 to 24 each contain 0.2 mg of anti-CD63 antibody-binding magnetic particles, and tubes 25 to 26 contain anti-CD81 antibody-binding magnetic particles 0 mg. 0.2 mg each in tubes 27-28, 0.2 mg each in anti-EpCAM antibody-coupled magnetic particles, and tubes 29-30 in Composite antibody-coupled magnetic particles (anti-CD9 antibody-coupled magnetic particles, anti-CD63 antibody-coupled magnetic particles and anti-CD81 0.2 mg each of antibody-bound magnetic particles and anti-EpCAM antibody-bound magnetic particles mixed at 1: 1: 1: 1) was added.
Next, 1.0 mL of the reaction buffer of Example 3 was added to tubes 21 to 30 so that the final concentration of sodium chloride was 0.25M. The sample 10 shown in Test Example 3 was added to the odd-numbered tube, and 1.0 mL each of the sample 12 shown in Test Example 3 was added to the even-numbered tube, and reacted at 25 ° C. for 3 hours.
Next, each particle after the reaction was washed three times with 0.5 mL of a washing buffer (TBS containing 0.1% by weight of Tween 20), and this suspension was added to 2 mL of a new Protein LoBind tube (Eppendorf). Product, # 0030108132).
Next, after collecting the magnetic flux and discarding the washing solution, the antibody-bound magnetic particles were suspended in 1 × sample buffer 20 μL, and allowed to stand at 95 ° C. for 5 minutes. A total amount (20 μL) of each sample was applied, and SDS-PAGE was performed. The gel was transferred to a PVDF membrane, and then shaken in a blocking buffer (TBS containing 1% (w / v) BSA and 0.1% (w / v) Tween 20) at 37 ° C. for 2 hours. This was washed with a washing buffer (TBS containing 0.1% (w / v) Tween 20).
Anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody are used as primary antibodies, and HRP-labeled anti-mouse IgG antibody (Mouse TrueBlot ULTRA: Anti-Mouse IgG HRP, Rockland 18-8817-33) is used as a labeled antibody. This was allowed to react for a period of time, washed with a washing buffer (TBS containing 0.1% (w / v) Tween 20), then reacted with a luminescent substrate, and a Western blot image was obtained with a luminescence measuring device (LAS-3000, FUJIFILM). confirmed. The results are shown in FIGS. 8-1 and 8-2.

For comparison, proteins on the vesicle surface were detected using the ultracentrifugation method, which is a standard method for recovering exosomes. Specifically, 11 mL of each of the specimens 10 and 12 shown in Test Example 3 was centrifuged at 100,000 × g and 4 ° C. for 70 minutes using an ultracentrifuge (Optima XE-90 manufactured by Beckman). The supernatant was extracted, and 11 mL of PBS was added to the precipitate and suspended. Again, centrifugation was performed at 100,000 × g and 4 ° C. for 70 minutes using an ultracentrifuge (Optima XE-90 manufactured by Beckman). The supernatant was extracted, and 0.11 mL of PBS was added to the precipitate for suspension. To 10 μL of the suspension, 5 μL of 4 × sample buffer and 5 μL of PBS were added, and the mixture was allowed to stand at 95 ° C. for 5 minutes. A total amount (20 μL) of each sample was applied, and SDS-PAGE was performed. The gel was transferred to a PVDF membrane, and then shaken in a blocking buffer (TBS containing 1% (w / v) BSA and 0.1% (w / v) Tween 20) at 37 ° C. for 2 hours. This was washed with a washing buffer (TBS containing 0.1% (w / v) Tween 20).
Anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody are used as primary antibodies, and HRP-labeled anti-mouse IgG antibody (Mouse TrueBlot ULTRA: Anti-Mouse IgG HRP, Rockland 18-8817-33) is used as a labeled antibody. This was allowed to react for a period of time, washed with a washing buffer (TBS containing 0.1% (w / v) Tween 20), then reacted with a luminescent substrate, and a Western blot image was obtained with a luminescence measuring device (LAS-3000, FUJIFILM). confirmed. The results are shown in FIGS. 8-1 and 8-2.

As shown in FIGS. 8-1 and 8-2, by reacting vesicles with particles in the presence of sodium chloride having a final concentration of 0.25M, the surface of vesicles derived from human healthy human serum or human healthy heparin plasma It was confirmed that this protein can be detected at the same level as the ultracentrifugation method. In addition, when anti-EpCAM antibody-binding magnetic particles were used, no vesicle surface protein was detected. EpCAM protein is hardly present in the blood of healthy individuals, and its blood concentration is thought to increase due to diseases such as cancer. It was confirmed that nonspecific reaction could be suppressed by reacting vesicles and particles in the presence of sodium chloride having a final concentration of 0.25M.

(Test Example 9-1 Detection of Protein Maintaining Three-dimensional Structure of Native Foam on Vesicle Surface (1))
Eight 8 ml of Protein LoBind tubes (Eppendorf, # 0030108132) were prepared and numbered with tubes 31-38. Tubes 31 and 32 contain 0.1 mg of anti-CD9 antibody-coupled magnetic particles, tubes 33 and 34 contain 0.1 mg of anti-CD63 antibody-coupled magnetic particles, tubes 35 and 36 contain 0.1 mg of anti-CD81 antibody-coupled magnetic particles, and tube 37 38, 0.1 mg of anti-EpCAM antibody-coupled magnetic particles was added. 0.3 mL of the reaction buffer of Example 3 was added to tubes 31 to 38 so that the final concentration of sodium chloride was 0.25M.
Next, 0.3 mL of the specimen 10 shown in Test Example 3 was added to the tubes 31, 33, 35, and 37 and reacted at 25 ° C. overnight. In addition, 0.3 mL of the specimen 12 shown in Test Example 3 was added to the tubes 32, 34, 36, and 38, and the reaction was performed overnight at 25 ° C.
Next, each particle after the reaction is washed twice with 0.25 mL of a washing buffer (TBS containing 0.1% by weight of Tween 20), and after collecting the magnetic flux, the supernatant is removed and suspended in 1 mL of phosphate buffered saline. did.
40 new 2 ml Protein LoBind tubes (Eppendorf, # 0030108132) prepared separately from the above were prepared and numbered with tubes 39-78. From the tube 31, 0.1 mL of the suspension was dispensed into tubes 39 to 43. From the tube 32, 0.1 mL of the suspension was dispensed into tubes 44 to 48. From the tube 33, 0.1 mL of the suspension was dispensed into tubes 49 to 53. From the tube 34, 0.1 mL of the suspension was dispensed into tubes 54 to 58. From the tube 35, 0.1 mL of the suspension was dispensed into tubes 59 to 63. From the tube 36, 0.1 mL of the suspension was dispensed into tubes 64-68. From the tube 37, 0.1 mL of the suspension was dispensed into tubes 69 to 73. From the tube 38, 0.1 mL of the suspension was dispensed into tubes 74 to 78.
Next, as a control, phycoerythrin (hereinafter referred to as PE) labeled anti-mouse IgG antibody (manufactured by SONY) was added to the tubes 39, 44, 49, 54, 59, 64, 69, 74 as tubes 40, 45, 50. , 55, 60, 65, 70, 75 are PE-labeled anti-CD9 antibodies (manufactured by SONY), and tubes 41, 46, 51, 56, 61, 66, 71, 76 are PE-labeled anti-CD63 antibodies ( (Sony) and tubes 42, 47, 52, 57, 62, 67, 72, 77, PE labeled anti-CD81 antibody (manufactured by SONY), tubes 43, 48, 53, 58, 63, 68, In 73 and 78, 5 μL each of PE-labeled anti-EpCAM antibody (manufactured by SONY) was added. Thereafter, the tubes 39 to 78 were suspended for 1 hour under the conditions of 25 ° C. and 1000 rpm.
Next, each particle after the reaction was washed twice with 0.5 mL of phosphate buffered saline, and then the fluorescence signal of each sample was confirmed using a flow cytometer (BD Accuri C6, manufactured by BD). The results are shown in FIGS. 9-1 and 9-2.

As shown in FIGS. 9-1 and 9-2, by reacting vesicles and particles in the presence of sodium chloride having a final concentration of 0.25M, the surface of vesicles derived from human healthy human serum and human healthy human heparin plasma It was confirmed that CD9, CD63, and CD81 can be detected.
In addition, in the case of using anti-CD9 antibody-binding magnetic particles, anti-CD63 antibody-binding magnetic particles, and anti-CD81 antibody-binding magnetic particles, the peak detected by anti-EpCAM antibody that was thought to be expressed by carcinogenesis was used as a control. It is considered that the peak is not shifted compared with the anti-mouse IgG antibody, and it is not detected from the specimen of a healthy person.
Furthermore, when anti-EpCAM antibody-binding magnetic particles are used as the solid phase carrier, the peak detected with the anti-mouse IgG antibody and the peak detected with the anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and anti-EpCAM antibody are: It didn't change much. That is, when anti-EpCAM antibody-binding magnetic particles are used as a solid phase carrier, exosomes are not obtained from a healthy subject's sample, and it is considered that nonspecific adsorption is low.
In addition, it is considered that the samples used for analysis were not treated with heat, drugs, etc. that change the three-dimensional structure of the protein, and the native forms of CD9, CD63, and CD81 present on the vesicle surface could be detected. .

(Test Example 9-2 Detection of Protein Maintaining Three-dimensional Structure of Native Foam on Vesicle Surface (2))
Prior to the test, the following specimens 18 and 19 were prepared.
Specimen 18 (HT29 sup. Spike Serum): A sample obtained by adding HT29 cell culture supernatant 100-fold concentrated concentrate as a spike to pooled serum (manufactured by Kojin Bio Inc.) to a 10-fold dilution Specimen 19 (HT29 sup. Spike Plasma) (Heparin): Pooled heparin plasma (manufactured by Kojin Bio Inc.), spiked with HT29 cell culture supernatant 100-fold concentrated solution diluted 10-fold

Eight 8 ml of Protein LoBind tubes (Eppendorf, # 0030108132) were prepared and numbered with tubes 79-86. Tubes 79 and 80 contain 0.1 mg of anti-CD9 antibody-coupled magnetic particles, tubes 81 and 82 contain 0.1 mg of anti-CD63 antibody-coupled magnetic particles, tubes 83 and 84 contain 0.1 mg of anti-CD81 antibody-coupled magnetic particles, and tube 85. 86, 0.1 mg of anti-EpCAM antibody-coupled magnetic particles was added. 0.3 mL of the reaction buffer of Example 3 was added to tubes 79 to 86 so that the final concentration of sodium chloride was 0.25M.
Next, 0.3 mL of the specimen 18 was added to the tubes 79, 81, 83, and 85 and reacted at 25 ° C. overnight. In addition, 0.3 mL of the specimen 19 was added to the tubes 80, 82, 84, and 86, and reacted at 25 ° C. overnight.
Next, each particle after the reaction is washed twice with 0.25 mL of a washing buffer (TBS containing 0.1% by weight of Tween 20), and after collecting the magnetic flux, the supernatant is removed and suspended in 1 mL of phosphate buffered saline. did.
40 new 2 ml Protein LoBind tubes (Eppendorf, # 0030108132) other than the above were prepared and numbered with tubes 87-126. From the tube 79, 0.1 mL of the suspension was dispensed into tubes 87 to 91. From the tube 80, 0.1 mL of the suspension was dispensed into tubes 92 to 96. From the tube 81, 0.1 mL of the suspension was dispensed into tubes 97 to 101. From the tube 82, 0.1 mL of the suspension was dispensed into the tubes 102 to 106. From the tube 83, 0.1 mL of the suspension was dispensed into the tubes 107 to 111. From the tube 84, 0.1 mL of the suspension was dispensed into the tubes 112 to 116. From the tube 85, 0.1 mL of the suspension was dispensed into tubes 117 to 121. From the tube 86, 0.1 mL of the suspension was dispensed into tubes 122 to 126.
Next, in tubes 87, 92, 97, 102, 107, 112, 117, 122, PE-labeled anti-mouse IgG antibody (manufactured by SONY) was used as a control, and tubes 88, 93, 98, 103, 108, 113, 118 were used. , 123 is a PE-labeled anti-CD9 antibody (manufactured by SONY), and tubes 89, 94, 99, 104, 109, 114, 119, and 124 are PE-labeled anti-CD63 antibody (manufactured by SONY) and tube 90 , 95, 100, 105, 110, 115, 120, 125 are PE-labeled anti-CD81 antibody (manufactured by SONY), and tubes 91, 96, 101, 106, 111, 116, 121, 126 are labeled with PE. 5 μL of anti-EpCAM antibody (manufactured by SONY) was added. Thereafter, the tubes 87 to 126 were suspended for 1 hour at 25 ° C. and 1000 rpm.
Next, each particle after the reaction was washed twice with 0.5 mL of phosphate buffered saline, and then the fluorescence signal of each sample was confirmed using a flow cytometer (BD Accuri C6, manufactured by BD). The results are shown in FIGS. 9-3 and 9-4.

As shown in FIGS. 9-3 and 9-4, by reacting vesicles and particles in the presence of sodium chloride having a final concentration of 0.25 M, human healthy human serum, human healthy heparin plasma, human colon cancer HT29 It was confirmed that CD9, CD63, and CD81 present on the surface of vesicles derived from cells can be detected.
Moreover, the peak shift which confirmed that EpCAM considered to express by carcinogenesis with the anti- EpCAM antibody was able to confirm the peak shift compared with the anti-mouse IgG antibody used as control. This peak shift was not confirmed in FIGS. 9-1 and 9-2, in which only healthy subjects were used as samples, indicating that the disease can be determined using the present invention.
Furthermore, when anti-EpCAM antibody-binding magnetic particles are used as the solid phase carrier, the peak detected with the anti-mouse IgG antibody and the peak detected with the anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and anti-EpCAM antibody are: It didn't change much. That is, when anti-EpCAM antibody-binding magnetic particles are used as a solid phase carrier, exosomes are not obtained from a healthy subject's sample, and it is considered that nonspecific adsorption is low.
In addition, it is considered that the samples used for analysis were not treated with heat, drugs, etc. that change the three-dimensional structure of the protein, and the native forms of CD9, CD63, and CD81 present on the vesicle surface could be detected. .

(Test Example 10)
Twenty 20 mL of Protein LoBind tubes (Eppendorf, # 0030108132) were prepared and numbered with tubes 127-146. Tubes 127 to 130 each contain 0.2 mg of anti-CD9 antibody-coupled magnetic particles, tubes 131 to 134 each contain 0.2 mg of anti-CD63 antibody-coupled magnetic particles, and tubes 135 to 138 contain anti-CD81 antibody-coupled magnetic particles 0 mg. 0.2 mg each in tubes 139-142, 0.2 mg each in anti-EpCAM antibody-coupled magnetic particles and tubes 143-146 in Composite antibody-coupled magnetic particles (anti-CD9 antibody-coupled magnetic particles, anti-CD63 antibody-coupled magnetic particles and anti-CD81 0.2 mg each of antibody-bound magnetic particles and anti-EpCAM antibody-bound magnetic particles mixed at 1: 1: 1: 1) was added.
Next, 0.1 mL of the reaction buffer of Example 3 was added to tubes 127 to 146 so that the final concentration of sodium chloride was 0.25M. Samples 10 shown in Test Example 3 are placed in tubes 127, 131, 135, 139, and 143, and Samples 18 shown in Test Example 9-2 are placed in tubes 128, 132, 136, 140, and 144, and tubes 129, 133, and 137 are shown in FIG. , 141, 145, and 0.1 mL of the specimen 12 shown in Test Example 9-2 are added to the tubes 130, 134, 138, 142, 146, respectively, and the mixture is added at 25 ° C. for 24 hours. Reacted.
Next, each particle after the reaction was washed three times with 0.5 mL of a washing buffer (TBS containing 0.1% by weight of Tween 20), and this suspension was added to 2 mL of a new Protein LoBind tube (Eppendorf). Product, # 0030108132).
Next, after collecting the magnetic flux and discarding the washing solution, the antibody-bound magnetic particles were suspended in 1 × sample buffer 20 μL, and allowed to stand at 95 ° C. for 5 minutes. A total amount (20 μL) of each sample was applied, and SDS-PAGE was performed. The gel was transferred to a PVDF membrane, and then shaken in a blocking buffer (TBS containing 1% (w / v) BSA and 0.1% (w / v) Tween 20) at 37 ° C. for 2 hours. This was washed with a washing buffer (TBS containing 0.1% (w / v) Tween 20).
Anti-CD9 antibody, anti-EpCAM antibody, and anti-Alix antibody are used as the primary antibody, and HRP-labeled anti-mouse IgG antibody (Mouse TrueBlot ULTRA: Anti-Mouse IgG HRP, Rockland 18-8817-33) is used as the labeled antibody. This was allowed to react for a period of time, washed with a washing buffer (TBS containing 0.1% (w / v) Tween 20), then reacted with a luminescent substrate, and a Western blot image was obtained with a luminescence measuring device (LAS-3000, FUJIFILM). confirmed. The results are shown in FIGS. 10-1 and 10-2.

For comparison, proteins on the vesicle surface were detected using the ultracentrifugation method, which is a standard method for recovering exosomes. That is, 11 mL each of specimens 10, 12, 18, and 19 were centrifuged at 100,000 × g and 4 ° C. for 70 minutes using an ultracentrifuge (Optima XE-90 manufactured by Beckman). The supernatant was extracted, and 11 mL of PBS was added to the precipitate and suspended. Again, centrifugation was performed at 100,000 × g and 4 ° C. for 70 minutes using an ultracentrifuge (Optima XE-90 manufactured by Beckman). The supernatant was extracted, and 0.11 mL of PBS was added to the precipitate for suspension. 5 μL of 4 × sample buffer and 14 μL of PBS were added to 1 μL of the suspension, and the mixture was allowed to stand at 95 ° C. for 5 minutes. A total amount (20 μL) of each sample was applied, and SDS-PAGE was performed. The gel was transferred to a PVDF membrane, and then shaken in a blocking buffer (TBS containing 1% (w / v) BSA and 0.1% (w / v) Tween 20) at 37 ° C. for 2 hours. This was washed with a washing buffer (TBS containing 0.1% (w / v) Tween 20).
Anti-CD9 antibody, anti-EpCAM antibody, and anti-Alix antibody are used as the primary antibody, and HRP-labeled anti-mouse IgG antibody (Mouse TrueBlot ULTRA: Anti-Mouse IgG HRP, Rockland 18-8817-33) is used as the labeled antibody. This was allowed to react for a period of time, washed with a washing buffer (TBS containing 0.1% (w / v) Tween 20), then reacted with a luminescent substrate, and a Western blot image was obtained with a luminescence measuring device (LAS-3000, FUJIFILM). confirmed. The results are shown in FIGS. 10-1 and 10-2.

As shown in FIGS. 10-1 and 10-2, by reacting vesicles and particles in the presence of sodium chloride having a final concentration of 0.25 M, human healthy human serum, human healthy heparin plasma, human colon cancer HT29 It was confirmed that proteins on the surface of vesicles derived from cells could be detected at a level equivalent to or higher than that of ultracentrifugation. In addition, when detected using an anti-EpCAM antibody, no band is detected in the serum of human healthy subjects or heparin plasma, and when a culture supernatant of human colon cancer HT29 cells is added thereto, a band is detected. Therefore, it was found that the disease can be determined using the present invention.

Claims (23)

1. A biological sample containing a vesicle having a lipid bilayer membrane is contacted with a solid phase carrier to which a ligand that recognizes a surface antigen present on the surface of the vesicle is bound, and the vesicle and the solid phase carrier are combined. A complex forming step for forming a body;
   A washing step of washing the complex,
   At least one of the complex formation step and the washing step is performed in the presence of an inorganic salt or organic salt having a final concentration of 0.15 to 2M,
   A method for separating vesicles having a lipid bilayer membrane.
2. 2. The separation method according to claim 1, wherein the complex forming step is performed in the presence of an inorganic salt or organic salt having a final concentration of 0.15 to 2M.
3. The separation method according to claim 1 or 2, wherein the biological sample is a body fluid or a cell culture supernatant.
4. The separation method according to any one of claims 1 to 3, wherein the biological sample is a body fluid.
5. The separation method according to any one of claims 1 to 4, wherein the inorganic salt or organic salt is an inorganic salt.
6. The separation method according to claim 5, wherein the inorganic salt is an alkali metal halide.
7. The separation method according to any one of claims 1 to 6, wherein a reaction temperature of the complex formation reaction in the complex formation step is in a range of 2 to 42 ° C.
8. The separation method according to any one of claims 1 to 7, wherein the ligand is an antibody that recognizes a surface antigen present on the surface of a vesicle.
9. The separation method according to any one of claims 1 to 8, wherein the vesicle is an exosome.
10. The separation method according to any one of claims 1 to 9, wherein the solid phase carrier is a magnetic particle.
11. The cleaning step includes a magnetic flux collecting step of collecting the magnetic particles by a magnetic force to separate the magnetic particles from a liquid phase, and a dispersing step of dispersing the magnetic particles separated in the magnetic flux collecting step in the cleaning liquid. The separation method according to 10.
12. After the separation method according to any one of claims 1 to 11,
    The method further comprises a nucleic acid detection step of detecting nucleic acid in the vesicle,
    A method for detecting nucleic acids in vesicles.
13. After the separation method according to any one of claims 1 to 11,
    The method further comprises a protein detection step of detecting a protein present on at least one of the inside and the surface of the vesicle,
    A method for detecting a protein derived from a vesicle.
14. After the separation method according to any one of claims 1 to 11,
    The method further comprises a signal measurement step of measuring a signal intensity derived from a vesicle forming the complex.
    Signal measurement method derived from vesicles.
15. A method for determining whether a subject has developed a disease,
    Using a biological sample derived from a subject, and measuring a signal intensity derived from a vesicle formed with the complex by the signal measurement method according to claim 14,
    How to determine the disease.
16. A method for evaluating the efficacy of a drug for treating a disease,
    Using a biological sample derived from a subject before and after administration of a drug for treating a disease, by the signal measurement method according to claim 14, comprising measuring a signal intensity derived from a vesicle forming the complex. It is characterized by
    A method for evaluating the efficacy of a drug for treating a disease.
17. A solid phase carrier bound with a ligand that recognizes a surface antigen present on the surface of a vesicle having a lipid bilayer; and
    A liquid composition containing an inorganic salt or an organic salt, and the content of the inorganic salt or organic salt is such that the final concentration in the system can be adjusted to 0.15 to 2M,
    kit.
18. The kit according to claim 17, which is used for disease determination or evaluation of drug efficacy.
19. A biological sample containing a vesicle having a lipid bilayer membrane is contacted with a solid phase carrier to which a ligand that recognizes a surface antigen present on the surface of the vesicle is bound, and the vesicle and the solid phase carrier are combined. At least one of the complex forming step and the washing step used in a method for separating a vesicle having a lipid bilayer, comprising a complex forming step for forming a body and a washing step for washing the complex. A liquid composition for adding crab,
    Including an inorganic salt or an organic salt, and the content of the inorganic salt or organic salt is an amount capable of adjusting the final concentration in the system to 0.15 to 2M,
    Liquid composition.
20. In order to form a complex of the vesicle and the insoluble carrier used in a method of separating a vesicle having a lipid bilayer membrane from a biological sample containing the vesicle having a lipid bilayer membrane using an insoluble carrier A sample dilution solution of
    Including an inorganic salt or an organic salt, and the content of the inorganic salt or organic salt is an amount capable of adjusting the final concentration in the system to 0.15 to 2M,
    Sample diluent.
21. 21. The specimen diluent according to claim 20, wherein the inorganic salt or organic salt is an inorganic salt.
22. The specimen diluent according to claim 21, wherein the inorganic salt is an alkali metal halide.
23. A method for separating vesicles having a lipid bilayer from a biological sample containing vesicles having a lipid bilayer using an insoluble carrier,
    A specimen diluent containing an inorganic salt or an organic salt and having an inorganic salt or organic salt content that can adjust the final concentration in the system to 0.15 to 2M is added to the system. ,
    A method for separating vesicles having a lipid bilayer membrane.

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