JP2018186739A - Method of recovering vesicle aggregate, method of recovering vesicle, and complex and kit for recovering vesicle aggregate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new means that can efficiently concentrate vesicles from a biological sample.SOLUTION: A method of recovering a vesicle aggregate includes: an aggregation step in which a biological sample containing a vesicle is brought into contact with a material A having one or more sites to adsorb or bind to the vesicle surface for forming the vesicle aggregate, and a recovery step in which the vesicle aggregate is recovered from the biological sample.SELECTED DRAWING: None

Description

  The present invention relates to a method for collecting vesicle aggregates, a method for collecting vesicles, and a complex and kit for collecting vesicle aggregates.

  Vesicles have a structure encased in a lipid bilayer. Among such vesicles, exosomes are known as vesicle granules present in body fluids in vivo. 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.

  In order to use vesicles for analysis and diagnosis, it is first necessary to concentrate and collect vesicles from a biological sample. Conventional methods for concentrating and recovering vesicles such as exosomes include ultracentrifugation, ultrafiltration, precipitation with volume-excluding polymers such as PEG and PVA, and separation using magnetic particles The method is known (patent documents 1 to 3).

International Publication No. 2013/158203 Pamphlet JP 2013-188212 A Special table 2012-533308 gazette Special table 2013-521258 gazette

However, in order to recover vesicles from a large amount of biological sample by using ultracentrifugation or magnetic particles, it is necessary to divide the biological sample several times and perform many operations. There was a problem of needing. In particular, the ultracentrifugation method requires an expensive apparatus and requires skilled skills to satisfy the reproducibility of the concentration / recovery efficiency.
In addition, the ultrafiltration method and the precipitation method using PEG and the like have a problem that it is unavoidable that impurities other than the target vesicle (for example, high molecular weight protein) are mixed.
On the other hand, Patent Document 4 describes a method using a dimer of an Fc binding domain of protein A or protein G as a method for selectively concentrating a target protein containing an immunoglobulin or Fc domain. However, this method does not concentrate and collect vesicle aggregates and vesicles, and the concentration and collection of vesicle aggregates and vesicles using the affinity sinking method have not been studied so far.
The problem to be solved by the present invention is to provide a new means capable of efficiently concentrating vesicles from a biological sample.

  The problem to be solved by the present invention has been solved by the following means.

<1> A vesicle aggregate is formed by bringing a biological sample containing vesicles into contact with a substance A (hereinafter also simply referred to as substance A) having one or more sites that adsorb or bind to the surface of the vesicles. A method for recovering vesicle aggregates (hereinafter also referred to as a vesicle aggregate recovery method of the present invention), comprising an aggregation step and a recovery step for recovering the vesicle aggregates from the biological sample.
<2> In the aggregation step, in addition to the biological sample and the substance A, a plurality of substances B (hereinafter simply referred to as substances) in which a plurality of the substances A are bound at a part different from the part adsorbed or bound to the surface of the vesicle The method according to <1>, which is an agglomeration step in which a vesicle aggregate is formed by contacting with B).
<3> The method according to <2>, wherein the aggregation step is an aggregation step in which the biological sample is brought into contact with a complex composed of the substance A and the substance B to form a vesicle aggregate.
<4> The aggregation step is an aggregation step in which the biological sample and the substance A are brought into contact with each other and the substance B is brought into contact with the complex formed of the obtained vesicle and the substance A to form a vesicle aggregate. The method according to <2>.

<5> The substance A is at least one selected from an antibody specifically recognizing a molecule present on the surface of a vesicle and a biotinylated antibody specifically recognizing a molecule present on the surface of the vesicle. > Method.
<6> The method according to <1>, wherein the substance A is one or more selected from annexin V and biotinylated annexin V.

<7> The substance A is a biotinylated antibody, biotinylated annexin V, or biotinylated Tim protein that specifically recognizes a molecule present on the surface of a vesicle, and the substance B is an avidin or avidin The method according to any one of <2> to <4>, which is a similar binding carrier.
<8><2> to <4>, wherein the substance A is an antibody that specifically recognizes a molecule present on the surface of a vesicle, and the substance B is protein A, protein G, or protein L The method according to any one.
<9><2> to <4>, wherein the substance A is an antibody, annexin V, or Tim protein that specifically recognizes a molecule present on the surface of a vesicle, and the substance B is a latex carrier. The method in any one of.

<10> The method according to any one of <2> to <4>, wherein the substance B is a substance having 2 to 4 sites that bind to the substance A.
<11> The vesicle is an exosome, microvesicle, nanovesicle, apoptotic body, dexosome, bleb, vesicle, prostasome, microparticle, intraluminal vesicle, endosomal vesicle or extracellular vesicle, The method according to any one of 1> to <10>.

  <12> An aggregation step of forming a vesicle aggregate by bringing a biological sample containing vesicles into contact with a substance A having one or more sites that adsorb or bind to the surface of the vesicles; A method for recovering vesicles (hereinafter referred to as vesicles of the present invention), comprising a recovery step of recovering vesicle aggregates, and a dissociation step of dissociating vesicles from the vesicle aggregates recovered in the recovery step. Also called recovery method).

<13> A substance A having one or more sites adsorbed or bound to the surface of the vesicle, and a substance B to which a plurality of the substances A are bound at a site different from the site adsorbed or bound to the surface of the vesicle A complex for collecting vesicle aggregates (hereinafter also referred to as a complex for collecting vesicle aggregates of the present invention).
<14> A substance A having one or more sites that are adsorbed or bonded to the surface of the vesicle, and a substance B to which a plurality of the substances A are bonded at a site different from the site that is adsorbed or bonded to the surface of the vesicle A kit for collecting vesicle aggregates (hereinafter also referred to as a kit for collecting vesicle aggregates of the present invention).

According to the vesicle aggregate recovery method of the present invention, a vesicle concentrate can be efficiently obtained as an aggregate.
Moreover, according to the vesicle collection method of the present invention, vesicles can be collected efficiently.

It is a schematic diagram of an aggregation process when an antibody that specifically recognizes a molecule present on the surface of a vesicle is used as the substance A. It is a schematic diagram of an aggregation process when a biotinylated antibody that specifically recognizes a molecule present on the surface of a vesicle is used as substance A and avidins are used as substance B. It is a schematic diagram of the aggregation step (2) when biotinylated annexin V is used as substance A and avidins are used as substance B. It is a schematic diagram of the aggregation step (3) when biotinylated annexin V is used as substance A and avidins are used as substance B. It is a figure which shows the collection | recovery result of the exosome aggregate at the time of using an anti-CD9 antibody as the substance A. It is a figure which shows the collection | recovery result of the exosome aggregate when a biotinylated antibody is used as the substance A, and when a biotinylated antibody is used as the substance A and streptavidin is used as the substance B, respectively. It is a figure which shows the collection | recovery result of the exosome aggregate at the time of using a biotinylated antibody as the substance A and streptavidin as the substance B, respectively. It is a figure which shows the collection | recovery result of the exosome aggregate when a biotinylated annexin V is used as the substance A, and when a biotinylated annexin V is used as the substance A and streptavidin is used as the substance B, respectively.

  Hereinafter, the present invention will be described in detail. In addition, description of "a-b" etc. showing a numerical range is synonymous with "a or more and b or less", and A and B are included in the numerical range.

<Vesicle aggregate collection method>
The method for collecting vesicle aggregates according to the present invention is a method for forming a vesicle aggregate by contacting a biological sample containing vesicles with a substance A having one or more sites that adsorb or bind to the surface of the vesicles. And a recovery step of recovering the vesicle aggregate from the biological sample.

(Vesicle)
As vesicles in the present invention, exosomes, microvesicles (microvesicles), nanovesicles, apoptotic bodies, dexosomes, blebs, blebs, prostasomes, microparticles, intraluminal vesicles (intraluminal) vesicle), endosomal vesicles, extracellular vesicles and the like. Among these, the vesicle aggregate recovery method of the present invention is particularly suitable for recovery of exosome aggregates.
The diameter of the vesicle is preferably 1 to 10,000 nm, more preferably 5 to 1000 nm, still more preferably 10 to 800 nm, still more preferably 15 to 500 nm, still more preferably 20 to 200 nm, and particularly preferably 30 to 150 nm.

(Biological sample)
The biological sample is not particularly limited as long as it contains vesicles, and examples thereof include various liquids such as body fluid, fungal fluid, cell culture medium, cell culture supernatant, and tissue cell disruption fluid. Cells contained in the culture medium, culture supernatant, and disrupted solution may be derived from eukaryotes or bacteria. Among these, body fluids and cell culture supernatants are preferred as biological samples.
Body fluids include whole blood, serum, plasma, blood components, various blood cells, blood clots, platelets, peripheral blood, cord blood, and other blood and blood composition components, as well as sweat, gastric fluid, interstitial fluid, joint fluid, synovial fluid , Aqueous humor, sputum, pleural effusion, oral mucosa group, bone marrow fluid, semen, prostate fluid, cowper gland fluid, pre-ejaculation fluid, female ejaculate, menstrual fluid, cyst fluid, pericardial fluid, digestion Fluid, tissue fluid, body cavity fluid, saliva, bile, vaginal fluid, tears, milk, urine, cerebrospinal fluid, pancreatic fluid, nasal fluid, feces, stool, chym, chyl, ascites, amniotic fluid, pus , Sebum, blastocyst fluid, vomit, mucous secretion, lymph, nasal wash, bronchoalveolar lavage, etc., preferably a blood composition component. The blood composition component may be treated with an anticoagulant such as citric acid or EDTA.
The collection source of the biological sample includes all animals having the vesicles. Examples include mammals, human or non-human primates, dogs, cats, horses, cows, other farm animals or rodents (eg, mice, rats, guinea pigs, etc.). Biological samples may be pre-treated by concentration or purification by ultrafiltration, ultracentrifugation, magnetic separation, etc., but those taken from living organisms or collected culture supernatants are used as they are. You can also

(Aggregation process)
The aggregation step is a step of forming a vesicle aggregate by bringing a biological sample containing vesicles into contact with a substance A having one or more sites that adsorb or bind to the surface of the vesicles.

  The concentration of vesicles is not particularly limited, but is preferably 1 to 5000 μg / mL, more preferably 5 to 1000 μg / mL with respect to the total amount of liquid phase in the system (total volume including vesicles and buffers). 10 to 500 μg / mL is particularly preferable.

In the aggregation step, the temperature at which the biological sample containing vesicles is brought into contact is preferably in the range of 2 to 42 ° C, more preferably in the range of 2 to 32 ° C. Further, the total time for contacting the biological sample containing vesicles is preferably 1 to 120 hours, more preferably 3 to 60 hours. The contact of the biological sample containing vesicles is preferably performed in an aqueous medium. Examples of the aqueous medium include various buffers such as phosphate buffer, HEPES buffer, Tris buffer, carbonate buffer, MES buffer and the like in addition to water.
Moreover, the pH in the system when contacting a biological sample containing vesicles is not particularly limited, but is preferably in the range of pH 3 to 11, more preferably in the range of pH 5 to 9. In order to maintain the target pH, the above buffer is used.

(Substance A)
The substance A is a substance having one or more sites that adsorb or bind to the surface of the vesicle.
The substance A preferably has a plurality of sites that adsorb or bind to the surface of the vesicle, and more preferably has 2 to 4 sites that adsorb or bind to the surface of the vesicle, from the viewpoint of recovery efficiency.

Examples of the substance A include an antibody that specifically recognizes a molecule present on the vesicle surface, a biotinylated antibody that specifically recognizes a molecule present on the vesicle surface, annexin V, biotinylated annexin V, and Tim protein , Biotinylated Tim protein, DNA, RNA, aptamer (DNA / RNA), peptide, peptoid, zDNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), lectin, dendrimer, other drugs and labeling reagents It is done. Of these, one kind may be used alone, or two or more kinds may be used in combination.
Among these, as substance A, in terms of recovery efficiency, an antibody that specifically recognizes a molecule present on the vesicle surface, a biotinylated antibody that specifically recognizes a molecule present on the vesicle surface, annexin V, Biotinylated Annexin V, Tim protein, and biotinylated Tim protein are preferred. Here, FIG. 1 shows a schematic diagram of the aggregation process when an antibody that specifically recognizes a molecule present on the surface of the vesicle is used as the substance A. An antibody that specifically recognizes a molecule present on the surface of a vesicle has two sites that adsorb or bind to the surface of the vesicle.
For biotinylation, not only biotin but also derivatives thereof can be used. Examples of biotin and biotin derivatives include those described in International Publication No. 2011/034115, International Publication No. 2014/129446, International Publication No. 2015/125820.

Here, an antibody that specifically recognizes a molecule present on the vesicle surface and an “antibody” in a biotinylated antibody that specifically recognizes a molecule present on the vesicle surface will be described.
As antibodies, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, synthetic antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab fragments, Fab ′ fragments, F (ab ′) 2 fragments, Fv fragments, Fv 'Fragments, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, and any of the epitope binding fragments described above. Examples of antibody classes include IgG, IgE, IgM, IgD, and IgA. The subclass is not particularly limited.

  As the above-mentioned antibodies, anti-B7-H3 antibody, anti-CD63 antibody, anti-CD9 antibody, anti-CD81 antibody, anti-EpCAM antibody, anti-MFG-E8 antibody, anti-PCSA antibody, anti-PSMA antibody are suitable for collecting exosome aggregates. Anti-PSCA antibody, anti-Rab antibody, anti-STEAP antibody, anti-TNFR antibody and anti-5T4 antibody are preferred. Among them, antibodies that recognize tetraspanins such as exosome surface antigens CD9, CD63, CD81 are particularly preferable.

  The “Tim protein” in the Tim protein and biotinylated Tim protein means a T cell immunoglobulin / mucin domain protein, specifically, a T cell immunoglobulin / mucin domain-containing molecule 1 (Tim1). Protein, T cell immunoglobulin / mucin domain-containing molecule 3 (Tim3) protein, T cell immunoglobulin / mucin domain-containing molecule 4 (Tim4) protein, and the like.

  Further, when the above annexin V, biotinylated annexin V, Tim protein, or biotinylated Tim protein is used as the substance A, the aggregation step is performed in the presence of alkaline earth metal ions such as calcium ions from the viewpoint of promoting aggregation. Is preferably performed. When such agglomeration is performed, a calcium halide such as calcium chloride may be added to the system of the agglomeration step.

  Although the usage-amount of the substance A is not specifically limited, 0.0001-10% (w / v) is preferable with respect to the total amount of the liquid phase in a system, 0.0005-5% (w / v) is more preferable .

(Substance B)
In the aggregation step, the biological sample, the substance A, and the substance B to which a plurality of substances A bind at a part different from the part that is adsorbed or bound to the surface of the vesicle are brought into contact in terms of recovery efficiency. A step of forming vesicle aggregates is preferred. Substance B binds to substance A at a site that does not inhibit adsorption or binding between substance A and vesicles.

As a technique of the aggregation process in the case of using the substance B, a biological sample containing vesicles, a substance A and a substance B coexist at the same time, and these are brought into contact with each other to form a vesicle aggregate (the aggregation process by this technique) Agglomeration step (also referred to as (1)), a method of forming a vesicle aggregate by bringing a biological sample containing vesicles into contact with a complex composed of substance A and substance B (the agglomeration step by this technique is agglomeration step (2)) A method of bringing a biological sample containing vesicles into contact with the substance A and bringing the substance B into contact with the resulting vesicle and the complex composed of the substance A to form vesicle aggregates (aggregation by this technique). The step is also referred to as an aggregating step (3)). Among these, the aggregation steps (2) and (3) are preferable in terms of recovery efficiency and handling properties.
In the aggregation step (2), the complex composed of the substance A and the substance B can be formed by bringing the substance A and the substance B into contact with each other. The contact time of the substance A and the substance B at this time is 5 Minutes to about 24 hours may be sufficient.

The substance B preferably has two or more sites that bind to the substance A in terms of recovery efficiency, more preferably has 2 to 20 sites that bind to the substance A, and a site that binds to the substance A. Those having 2 to 4 are particularly preferred. By using such a substance B, a plurality of substances A are bridged and vesicles are easily aggregated, and the recovery efficiency is improved.
Although the usage-amount of the substance B is not specifically limited, 0.00001-10% (w / v) is preferable with respect to the total amount of the liquid phase in a system, and 0.00001-5% (w / v) is more preferable. .
The molar ratio [(A) :( B)] of the amounts used of substance A and substance B is preferably 100: 0 to 30:70, more preferably 95: 5 to 40:60, in terms of recovery efficiency. 90:10 to 50:50 is particularly preferred.

  Examples of the substance B include water-soluble or water-insoluble compounds and solid phase carriers. As the solid phase carrier, a carrier in which a compound that binds to a plurality of substances A is carried on the carrier surface can be used. Specific examples of the solid phase carrier include a latex carrier and magnetic particles. Among such substances B, a water-soluble compound and a latex carrier are preferable, and a water-soluble compound is more preferable in terms of low cost. Moreover, when aggregating by the technique of the aggregating step (2), it is preferable that the complex composed of the substance A and the substance B is a water-soluble compound. In addition, the solubility with respect to water may change with temperature and pH conditions. As the substance B, a low molecular compound having a plurality of binding sites such as reactive functional groups can also be used. Examples of the low molecular weight compound include polyfunctional epoxy compounds (preferably 2 to 4 functional epoxy compounds).

The substance B preferably has a high binding property with the substance A to be used and is easily aggregated.
Specifically, when an antibody that specifically recognizes a molecule present on the surface of the vesicle is used as the substance A, the substance B is preferably a carrier carrying an antibody-binding substance or an antibody-binding protein.
In the case where a biotinylated antibody that specifically recognizes a molecule present on the surface of a vesicle is used as the substance A, the substance B is preferably an avidin or an avidin-binding carrier in terms of recovery efficiency and handling properties. . Here, FIG. 2 shows a schematic diagram of the aggregation step when a biotinylated antibody that specifically recognizes a molecule present on the vesicle surface is used as substance A and avidins are used as substance B. A biotinylated antibody that specifically recognizes a molecule present on the surface of a vesicle has two sites that adsorb or bind to the surface of the vesicle, and avidins are sites that bind to the biotinylated antibody. Have two.
When Annexin V or Tim protein is used as the substance A, the substance B is preferably a solid phase carrier from the viewpoint of recovery efficiency.
In the case of using biotinylated annexin V or biotinylated Tim protein as the substance A, the substance B is preferably an avidin or an avidin-binding carrier in terms of recovery efficiency and handling properties. Here, FIG. 3 shows a schematic diagram of the aggregation step (2) when biotinylated annexin V is used as substance A and avidins are used as substance B. FIG. 4 shows a schematic diagram of the aggregation step (3) when biotinylated annexin V is used as substance A and avidins are used as substance B. Biotinylated annexin V and biotinylated Tim protein have one site that adsorbs or binds to the surface of the vesicle, and avidins include the biotinylated annexin V and biotinylated Tim protein. It has four binding sites. That is, when biotinylated annexin V, biotinylated Tim protein is used as substance A, and avidin is used as substance B, substances A and B are complex like annexin V tetramer as shown in FIG. Therefore, vesicles are efficiently aggregated and excellent recovery efficiency can be obtained.

Examples of the antibody binding protein include protein A, protein G, protein L, a multimer of the protein, and a complex of two or more antibody binding proteins and a water-soluble compound. Examples of the multimer and complex include a dimer of an Fc binding domain of protein A or protein G as described in JP 2013-521258, a compound formed by linking two proteins of the dimer with a water-soluble compound, Examples include proteins in which a plurality of protein A domains are linked. Among antibody binding proteins, Fc binding protein is preferable, and protein A is particularly preferable.
The antibody-binding protein is preferably used without being bound to a solid phase carrier.

  Examples of the avidins include avidin, streptavidin, tamavidin, neutravidin, and modifications thereof. Examples of the modified products include those obtained by modifying avidin with poly (meth) acrylic acid, JP-A-2006-41075, JP-A-2011-055827, WO2012 / 091101 and WO2009 / 028625. Modified biotin-binding proteins described in No. pamphlets and the like. As avidins, avidin and streptavidin are preferable.

In the present invention, the substance A and the substance B are preferably used in the following modes (α) to (ε) in terms of recovery efficiency, handling properties, and cost, and the modes (α) to (γ): It is more preferable to use in (γ).
(Α) An embodiment wherein the substance A is at least one selected from an antibody that specifically recognizes a molecule present on the vesicle surface and a biotinylated antibody that specifically recognizes a molecule present on the vesicle surface (substance A And B only substance A is used).
(Β) An embodiment in which substance A is one or more selected from annexin V and biotinylated annexin V (only substance A is used among substances A and B).
(Γ) Substance A is a biotinylated antibody, biotinylated annexin V, or biotinylated Tim protein that specifically recognizes molecules present on the surface of vesicles, and substance B binds to avidins or avidins An embodiment that is a carrier. Among these embodiments, an aspect in which the substance A is a biotinylated antibody that specifically recognizes a molecule present on the surface of the vesicle and the substance B is an avidin or an avidin-binding carrier in terms of recovery efficiency. Particularly preferred.
(Δ) A mode in which substance A is an antibody that specifically recognizes a molecule present on the surface of a vesicle, and substance B is protein A, protein G, or protein L.
(Ε) An embodiment in which substance A is an antibody, annexin V, or Tim protein that specifically recognizes a molecule present on the surface of a vesicle, and substance B is a latex carrier. In the embodiment (ε), when the substance A is an antibody that specifically recognizes a molecule present on the vesicle surface, a latex carrier on which an antibody-binding substance is supported is more preferable.

  Depending on the type of substance A, a water-soluble polymer can be used as substance B, and depending on the type of substance B, a water-soluble polymer can be used as substance A. As a combination of the substance A and the substance B in the case of using the water-soluble polymer, the substance A is an antibody, annexin V or Tim protein that specifically recognizes a molecule present on the surface of the vesicle, and the substance B is water-soluble. And a combination in which the substance A is a biotinylated water-soluble polymer and the substance B is an avidin.

As said water-soluble polymer, what has an acidic group is preferable. Examples of the acidic group include a carboxy group, a phosphate group, a sulfo group, a phenolic hydroxyl group, a dihydroxyboryl group, and salts and ions thereof.
The polymer having an acidic group may be a natural polymer or a synthetic polymer, and may be used alone or in combination of two or more. When the polymer has a carboxy group as an acidic group, part of the carboxy group may be esterified with ethylene glycol, propylene glycol, or the like, and the polymer having an acidic group has a phenolic hydroxyl group. In the case of having an alcoholic hydroxyl group in addition to the acidic group, a part of the phenolic hydroxyl group or a part or all of the alcoholic hydroxyl group may be reacted with acetic acid, nitric acid, sulfuric acid, phosphoric acid, etc. .

  Natural polymers having an acidic group include milk proteins having an acidic group; heparin and derivatives thereof; polypeptides such as polyglutamic acid and polyaspartic acid, xanthan gum, carrageenan, alginic acid, pectin, farcelan, gum arabic, and gucci gum , Karaya gum, tragacanth gum, agar, hyaluronic acid, chondroitin sulfate, dextran sulfate, pentosan polysulfate, carboxymethylated or succinylated polysaccharide (cellulose, dextran, starch, chitosan etc. carboxymethylated or succinylated), These salts may be mentioned, and one of these may be used alone, or two or more may be used in combination. Examples of the milk protein having an acidic group include casein, whey protein concentrate, non-fat dry milk, and skim milk. In addition, you may use derivatives of milk proteins, such as a partial hydrolyzate and crosslinked protein, as milk protein which has an acidic group. Examples of heparin derivatives include untreated heparin, low molecular weight heparin, de-N-sulfated heparin, de-2-O-sulfated heparin, and de-6-O-sulfated heparin.

Examples of the synthetic polymer having an acidic group include a polymer or copolymer having a repeating unit derived from a polymerizable unsaturated monomer having an acidic group or a salt thereof, and inorganic polyphosphoric acid.
Here, the polymerizable unsaturated monomer having an acidic group means a compound having a polymerizable unsaturated group and an acidic group in one molecule. Examples of the polymerizable unsaturated monomer having an acidic group include a polymerizable unsaturated monomer having a phosphoric acid group, a polymerizable unsaturated monomer having a carboxy group, and a polymerizable unsaturated monomer having a sulfo group. The seeds may be used alone or in combination of two or more. Among these, a polymerizable unsaturated monomer having a phosphate group and a polymerizable unsaturated monomer having a carboxy group are preferable.

The polymerizable unsaturated monomer having a phosphoric acid group is preferably a phosphoric acid group-containing (meth) acrylic acid ester, such as 2- (meth) acryloyloxyethyl acid phosphate, 2- (meth) acryloyloxypropyl acid phosphate. Is mentioned.
Examples of the polymerizable unsaturated monomer having a carboxy group include unsaturated groups such as (meth) acrylic acid, crotonic acid, vinylbenzoic acid, cinnamic acid, and hexahydrophthalic acid mono-2- (meth) acryloyloxyethyl. Monocarboxylic acids; unsaturated dicarboxylic acids such as fumaric acid, maleic acid, and itaconic acid; and partially esterified products of unsaturated dicarboxylic acids such as monomethyl maleate, monoethyl maleate, monomethyl itaconate, monoethyl itaconate, etc. Acid anhydrides of unsaturated dicarboxylic acids such as maleic acid and itaconic anhydride can also be used.
Examples of the polymerizable unsaturated monomer having a sulfo group include styrene sulfonic acid, vinyl sulfonic acid, allyl sulfonic acid, methallyl sulfonic acid, 2-sulfoethyl (meth) acrylate, and 2- (meth) acrylamide-2-methylpropane sulfonic acid. Etc.

The synthetic polymer having an acidic group may have a repeating unit other than the repeating unit derived from a polymerizable unsaturated monomer having an acidic group or a salt thereof (hereinafter also referred to as other repeating unit).
Other repeating units include aromatic vinyl monomers, (meth) acrylate monomers, (meth) acrylamide monomers, esters of saturated carboxylic acids and unsaturated alcohols, alkyl vinyl ethers, vinyl group-containing alcohols, etc. The repeating unit which originates is mentioned. Of these, one kind may be used alone, or two or more kinds may be used in combination.

Examples of the aromatic vinyl monomer include styrene, α-methylstyrene, vinyltoluene, 2-methylstyrene, 3-methylstyrene, 4-methylstyrene, 4-ethylstyrene, 4-tert-butylstyrene, 3, 4-dimethylstyrene, 4-methoxystyrene, 4-ethoxystyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 2,4-dichlorostyrene, 2,6-dichlorostyrene, 4-chloro-3- In addition to styrene monomers such as methylstyrene, 1-vinylnaphthalene, 2-vinylpyridine, 4-vinylpyridine and the like can be mentioned.
Examples of the (meth) acrylate monomer include methyl (meth) acrylate, ethyl (meth) acrylate, n-butyl (meth) acrylate, isobutyl (meth) acrylate, tert-butyl (meth) acrylate, 2-ethylhexyl ( Alkyl (meth) acrylates such as meth) acrylate and lauryl (meth) acrylate; (meth) acrylates having an aromatic ring structure such as benzyl (meth) acrylate; isobornyl (meth) acrylate, adamantyl (meth) acrylate, dicyclopentanyl (Meth) acrylate etc. which have alicyclic structures, such as (meth) acrylate, are mentioned.
Examples of the (meth) acrylamide monomer include Nt-butyl (meth) acrylamide, (meth) octylacrylamide, diacetone (meth) acrylamide, and the like.
Examples of the esters of saturated carboxylic acid and unsaturated alcohol include vinyl acetate, vinyl propionate, and vinyl neodecanoate.
Examples of alkyl vinyl ethers include methyl vinyl ether and ethyl vinyl ether.
Examples of vinyl group-containing alcohols include vinyl alcohol.

  As a combination of a polymerizable unsaturated monomer having an acidic group or a salt thereof and a monomer for deriving another repeating unit, a combination of a polymerizable unsaturated monomer having a phosphate group or a salt thereof and a (meth) acrylate monomer , Preferably a combination of a polymerizable unsaturated monomer having a carboxy group or a salt thereof and an aromatic vinyl monomer, a combination of a polymerizable unsaturated monomer having a phosphate group or a salt thereof and butyl (meth) acrylate, a carboxy group A combination of a polymerizable unsaturated monomer having a salt or a salt thereof and one or two selected from styrene and α-methylstyrene is more preferable.

(Recovery process)
The recovery step is a step of recovering the vesicle aggregate formed in the aggregation step from the biological sample.
Examples of the recovery method include a method of removing the supernatant from the system in the aggregation step. Specific examples include centrifugation and filtration. Centrifugation is preferable from the viewpoint of recovery efficiency. When collecting by centrifugation, vesicle aggregates are collected as a residue.
Centrifugation includes non-ultracentrifugation and ultracentrifugation, but non-ultracentrifugation is preferred. According to the vesicle aggregate recovery method of the present invention, vesicle aggregates can be efficiently recovered by non-ultracentrifugation even when ultracentrifugation is not performed.

As a centrifugal force in centrifugation, 100-100000 * g is preferable, 500-50000 * g is more preferable, 1000-30000 * g is still more preferable, 3000-22000 * g is especially preferable.
According to the vesicle aggregate recovery method of the present invention, vesicle aggregates can be efficiently recovered even by the centrifugal force as described above.

The centrifugation time is preferably 20 minutes to 24 hours, more preferably 30 minutes to 12 hours, still more preferably 45 minutes to 6 hours, and particularly preferably 60 minutes to 3 hours per centrifugation.
Moreover, the frequency | count of centrifugation is 1 time or more, 1-10 times are more preferable, 1-5 times are especially preferable.
In addition, when collect | recovering by centrifugation, you may wash | clean the obtained residue with water and various buffer solutions.

  And according to the vesicle aggregate recovery method of the present invention, the vesicle concentrate can be efficiently obtained as an aggregate. In addition, it is easy to scale up.

<Vesicle collection method>
The method for collecting vesicles of the present invention comprises an aggregation step in which a biological sample containing vesicles is contacted with a substance A having one or more sites that adsorb or bind to the surface of the vesicles to form vesicle aggregates; A recovery step of recovering the vesicle aggregate from the biological sample; and a dissociation step of dissociating the vesicle from the vesicle aggregate recovered in the recovery step.
The aggregation process and the recovery process are the same as the aggregation process and the recovery process in the vesicle aggregate recovery method of the present invention.

(Dissociation process)
Dissociation of vesicles can be performed with reference to known methods described in JP 2013-521258 A, WO 2016/088869 pamphlet and the like.
Specifically, when annexin V, biotinylated annexin V, Tim protein, or biotinylated Tim protein is used as substance A, a chelating agent such as EDTA is brought into contact with vesicle aggregates to Can be dissociated.
In addition, biotinylated antibody that specifically recognizes molecules present on the surface of vesicle, biotinylated annexin V, biotinylated Tim protein was used as substance A, and avidin or avidin binding carrier was used as substance B. In this case, the avidin-biotin bond between the substance A and the substance B is dissociated by adding another biotin having higher affinity with the avidin or avidin-binding carrier than the substance A, and the avidins And vesicles can be dissociated from the avidin-binding carrier.
When an antibody that specifically recognizes a molecule present on the vesicle surface is used as the substance A and an antibody-binding protein is used as the substance B, the acidic condition (preferably in the range of pH 2 to 4.5) is used. By adjusting, vesicles can be dissociated.

  The vesicles can also be efficiently recovered by dissociating the vesicles from the vesicle aggregates in this way. The collected vesicles can be used for analysis and diagnosis.

<Complex and kit for collecting vesicle aggregates>
The complex for collecting vesicle aggregates of the present invention comprises a substance A having one or more sites that adsorb or bind to the surface of the vesicle, and a plurality of sites other than the site that adsorbs or binds to the surface of the vesicle. And a substance B to which the substance A is bonded. Specific examples include a complex containing biotinylated annexin V and / or biotinylated Tim protein and an avidin or avidin-binding carrier, and biotinylated annexin V and avidin or an avidin-binding carrier. A complex comprising 4 biotinylated annexins V and 1 avidin is particularly preferred.
The kit for collecting vesicle aggregates of the present invention comprises a substance A having one or more sites that are adsorbed or bound to the surface of the vesicle, and a plurality of the above-mentioned sites at sites other than the site that is adsorbed or bound to the surface of the vesicle And a substance B to which the substance A binds.
In the kit, the substance A and the substance B may or may not form a complex. Specifically, as the kit, a kit including a liquid agent containing substance A or substance A, a liquid agent containing substance B or substance B, and a buffer solution, a complex composed of substance A and substance B, or the complex A kit comprising a solution containing the body and a buffer solution can be mentioned. Moreover, the kit may be provided with instructions describing a method for collecting vesicle aggregates.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

[Test Example 1]
(Example 1-1)
(1) D-MEM / Ham'sF-12 medium (manufactured by Wako Pure Chemical Industries) containing 10% (w / v) fetal bovine serum (manufactured by EQUITECH) of HT29 cells (human colorectal cancer-derived cell line) Was used until the cell density reached about 80% (w / v). Next, the cells were washed twice with PBS (manufactured by Wako Pure Chemical Industries, Ltd.), cultured for about 3 days in serum-free D-MEM / Ham'sF-12 medium (manufactured by Wako Pure Chemical Industries, Ltd.), and the culture supernatant was collected. . The culture supernatant is centrifuged at 4 ° C. and 300 × g for 10 minutes to recover the supernatant, and the recovered supernatant is centrifuged at 4 ° C. and 2000 × g for 10 minutes to recover the supernatant. Then, it was filtered with a filter having a pore size of 0.22 μm. The culture supernatant was concentrated 100 times using an ultrafiltration membrane (Amicon Ultra-15 (MWCO: 100K) manufactured by Millipore) having a fractional molecular weight of 100K. The concentrate obtained here is referred to as “concentrate X”.
Next, 1 μg of an anti-CD9 antibody (D252-3 manufactured by MBL) was added to 50 μL of the concentrated solution X, and PBS was added to make 100 μL. In the sample obtained here, the concentration of the anti-CD9 antibody is about 0.001% (w / v). The sample was allowed to stand overnight at 4 ° C., and then centrifuged at 15000 rpm and 4 ° C. for 90 minutes (apparatus: CF16RN, rotor: T15A39 (Hitachi Kogyo Co., Ltd.), centrifugal force: about 21500 × g), and the supernatant And divided into residues. The supernatant collected by this centrifugation is referred to as “Sample A”.
Next, the residue is washed with 0.5 mL of PBS, centrifuged at 15000 rpm and 4 ° C. for 90 minutes (apparatus: CF16RN, rotor: T15A39 (Hitachi Kogyo Co., Ltd.), centrifugal force: about 21500 × g), and the supernatant is discarded. The residue was collected with 100 μL of PBS. The sample obtained here is referred to as “Sample B”.
(2) Next, exosomes contained in Sample A and Sample B were detected by Western blotting.
That is, SDS-PAGE was performed on sample A and sample B. Next, using a trans blot Turbo blotting system (BIO-RAD), the separation gel was transferred to a PVDF membrane (Trans blot Turbo transfer pack PVDF (Midi) (BIO-RAD)), and the membrane after transfer Was immersed in 50% Blocking One (manufactured by Nacalai Tesque) / PBS (manufactured by Sigma) at 37 ° C. for about 2 hours. The primary antibody Anti-CD9 mAb (manufactured by MBL, MEX001-3) was added thereto at 1 μg / mL, and immersed at 37 ° C. for about 1 hour. This was washed 3 times with a washing buffer (TBS containing 0.1% by mass of Tween 20), and then 1 μg / mL of HRP-labeled anti-mouse IgG antibody (Mouse TrueBlot ULTRA: Anti-Mouse IgG HRP, Rockland 18- 8817-33) was reacted at 25 ° C. for 1 hour. This was washed 5 times with a washing buffer (TBS containing 0.1% by weight of Tween 20), then reacted with a luminescent substrate (SuperSignal West Femto Maximum Sensitive substrate, Thermo scientific Cat # 34095), and a luminescence measuring machine (LAS-500). The Western blot image was confirmed by GE Healthcare.
(Example 1-2)
Sample A and sample B were obtained in the same manner as in Example 1-1 (1) except that the amount of anti-CD9 antibody used was changed to 10 μg. Exosomes were detected by Western blotting in the same manner as in (2) of 1.
(Comparative Example 1)
Sample A and sample B were obtained in the same manner as in Example 1-1 (1) except that the treatment was carried out without adding the anti-CD9 antibody. Exosomes were detected by Western blotting in the same manner as in (2).

The results of Test Example 1 are shown in FIG. 5 (lane 1: Sample A of Comparative Example 1, Lane 2: Sample B of Comparative Example 1, Lane 3: Sample A of Example 1-1, Lane 4: Example 1) Sample B of 1; Lane 5: Sample A of Example 1-2; Lane 6: Sample B of Example 1-2).
As shown in FIG. 5, many exosome aggregates were contained in the specimen B of Examples 1-1 and 1-2.
From this result, it was found that exosome aggregates can be efficiently recovered by forming exosome aggregates using anti-CD9 antibody as substance A.

[Test Example 2]
(Example 2-1)
(1) An anti-CD81 antibody (MEX003-3 manufactured by MBL) was biotinylated using an antibody biotin labeling kit (Thermo # 21335). The obtained biotinylated antibody is referred to as “biotinylated antibody Y”. Biotinylation was performed according to the attached protocol.
(2) 10 μg of biotinylated antibody Y was added to 50 μL of the concentrated solution X prepared in the same manner as in Example 1-1, and allowed to stand overnight at 4 ° C., then 0.25 μg of streptavidin (Roche Corporation 353545103) was added. PBS was added to make 100 μL. In the sample obtained here, the concentration of the biotinylated antibody is about 0.01% (w / v), and the concentration of streptavidin is about 0.00025% (w / v). The sample was allowed to stand at 25 ° C. for 60 minutes, and then centrifuged at 15000 rpm and 4 ° C. for 90 minutes (apparatus: CF16RN, rotor: T15A39 (Hitachi Kogyo Co., Ltd.), centrifugal force: about 21500 × g), and the supernatant And divided into residues. The supernatant collected by this centrifugation is referred to as “Sample C”.
Next, the residue is washed with 0.5 mL of PBS, centrifuged at 15000 rpm and 4 ° C. for 90 minutes (apparatus: CF16RN, rotor: T15A39 (Hitachi Kogyo Co., Ltd.), centrifugal force: about 21500 × g), and the supernatant is discarded. The residue was collected with 100 μL of PBS. The sample obtained here is referred to as “Sample D”.
(3) Next, exosomes contained in the samples C and D were detected by Western blotting. Western blotting was performed in the same manner as (2) of Example 1-1 except that Anti-CD81 mAb (manufactured by MBL, MEX003-3) was used as the primary antibody.

(Example 2-2)
Sample C and sample D were obtained in the same manner as (2) of Example 2-1 except that the treatment was performed without adding streptavidin, and the obtained sample C and sample D were obtained in Example 2-1. Exosomes were detected by Western blotting as in (3).

The results of Test Example 2 are shown in FIG. 6 (Lane 1: Sample C of Example 2-2, Lane 2: Sample D of Example 2-2, Lane 3: Sample C of Example 2-1, Lane 4: Sample D) of Example 2-1.
As shown in FIG. 6, the specimen D of Examples 2-1 and 2-2 contained many exosome aggregates. In particular, the specimen D of Example 2-1 contained more exosome aggregates.
From this result, an exosome aggregate is formed using a biotinylated antibody as substance A (Example 2-2), and an exosome aggregate is formed using biotinylated antibody as substance A and streptavidin as substance B, respectively. (Example 2-1) allows efficient recovery of exosome aggregates. In particular, by forming exosome aggregates using biotinylated antibody as substance A and streptavidin as substance B, exosome aggregation It was found that things can be recovered more efficiently.

[Test Example 3]
(Example 3-1)
Sample C and sample D were obtained in the same manner as (2) of Example 2-1, except that the amount of biotinylated antibody Y was changed to 1 μg and the amount of streptavidin was changed to 0.025 μg. About the obtained sample C and sample D, the exosome was detected by the western blotting method like (2-1) of Example 2-1.
(Example 3-2)
HT29 cells (a cell line derived from human colon cancer) were used with D-MEM / Ham'sF-12 medium (manufactured by Wako Pure Chemical Industries) containing 10% (w / v) fetal bovine serum (manufactured by EQUITECH). The cells were cultured until the cell density reached about 80% (w / v). Next, the cells were washed twice with PBS (manufactured by Wako Pure Chemical Industries, Ltd.), cultured for about 3 days in serum-free D-MEM / Ham'sF-12 medium (manufactured by Wako Pure Chemical Industries, Ltd.), and the culture supernatant was collected. . The culture supernatant was centrifuged at 4 ° C. and 300 × g for 10 minutes to recover the culture supernatant.
Except for using the obtained culture supernatant in place of the concentrate X, a specimen D was obtained in the same manner as in Example 3-1, and the obtained specimen D was obtained in the same manner as in Example 3-1. The exosome was detected by Western blotting.

The results of Test Example 3 are shown in FIG. 7 (lane 1: sample C of Example 3-1, lane 2: sample D of Example 3-1, lane 3: sample D of Example 3-2).
As shown in FIG. 7, the specimen D of Example 3-2 was also rich in exosome aggregates, like the specimen D of Example 3-1.
From this result, it is possible to efficiently collect exosome aggregates even when the concentration step by ultrafiltration is not performed by using biotinylated antibody as substance A and streptavidin as substance B to form exosome aggregates. I understood.

[Test Example 4]
(Example 4-1)
(1) To 50 μL of a 0.1 mg / mL biotinylated annexin V solution (solvent: PBS), 6.25 μg of streptavidin (Roche 3535452103) was added and allowed to stand at 25 ° C. for 60 minutes. Concentration solution X 50 μL prepared in the same manner as in -1, 5 × Binding Buffer (50 mM HEPES (pH 7.4), 750 mM NaCl, 25 mM KCl, 5 mM MgCl ₂ , 9 mM CaCl ₂ ) 25 μL was added. In the sample obtained here, the concentration of biotinylated annexin V is about 0.004% (w / v), and the concentration of streptavidin is about 0.005% (w / v). The sample was allowed to stand overnight at 4 ° C., and then centrifuged at 15000 rpm and 4 ° C. for 90 minutes (apparatus: CF16RN, rotor: T15A39 (Hitachi Kogyo Co., Ltd.), centrifugal force: about 21500 × g), and the supernatant And divided into residues.
Next, the residue is washed with 0.5 mL of PBS, centrifuged at 15000 rpm and 4 ° C. for 90 minutes (apparatus: CF16RN, rotor: T15A39 (Hitachi Kogyo Co., Ltd.), centrifugal force: about 21500 × g), and the supernatant is discarded. The residue was collected with 100 μL of PBS. The sample obtained here is referred to as “Sample E”.
(2) Next, exosomes contained in the specimen E were detected by Western blotting. Western blotting was performed in the same manner as in Example 1-1 (2).
(Example 4-2)
Except for the treatment without adding streptavidin, specimen E was obtained in the same manner as (1) of Example 4-1, and the obtained specimen E was obtained in the same manner as (2) of Example 4-1. The exosome was detected by Western blotting.

The results of Test Example 4 are shown in FIG. 8 (lane 1: sample E of Example 4-2, lane 2: sample E of Example 4-1).
As shown in FIG. 8, the specimen E of Examples 4-1 and 4-2 contained many exosome aggregates. In particular, the specimen E of Example 4-1 contained more exosome aggregates.
From this result, exosome aggregates were formed using biotinylated annexin V as substance A (Example 4-2), biotinylated annexin V as substance A, and streptavidin as substance B. (Example 4-1), exosome aggregates can be efficiently recovered. In particular, by using biotinylated annexin V as substance A and streptavidin as substance B, exosome aggregates can be formed. It was found that exosome aggregates can be recovered more efficiently.

Considering the results of Test Examples 1 to 4 comprehensively and considering that dissociation of annexin V and exosome is extremely easy, among the combinations of substances A and B of each Example, substance A is biotinylated. It is presumed that a combination that is an antibody, substance B is streptavidin, substance A is biotinylated annexin V, and substance B is streptavidin is suitable for recovery of exosome aggregates. In particular, a combination in which the substance A is a biotinylated antibody and the substance B is streptavidin has a particularly good recovery efficiency of exosome aggregates, and is particularly suitable for recovery of exosome aggregates.
In addition, exosomes could be easily dissociated from the exosome aggregates obtained in each Example.

Claims (14)

An aggregation step of bringing a biological sample containing vesicles into contact with a substance A having one or more sites that adsorb or bind to the surface of the vesicles to form vesicle aggregates; and the vesicle aggregation from the biological sample Including a recovery step of recovering the object,
A method for collecting vesicle aggregates.   In the aggregation step, in addition to the biological sample and the substance A, a plurality of substances B to which the substance A binds are brought into contact with a part other than the part to be adsorbed or bound to the surface of the vesicle to thereby aggregate the vesicle The method according to claim 1, wherein the method is an agglomeration step of forming a slag.   The method according to claim 2, wherein the aggregating step is an aggregating step in which the biological sample is brought into contact with a complex composed of substance A and substance B to form a vesicle aggregate.   The aggregation step is an aggregation step in which the biological sample and the substance A are brought into contact with each other, and a substance B is brought into contact with the complex composed of the obtained vesicle and the substance A to form a vesicle aggregate. The method described in 1.   The substance A is at least one selected from an antibody specifically recognizing a molecule present on the vesicle surface and a biotinylated antibody specifically recognizing a molecule present on the vesicle surface. the method of.   The method according to claim 1, wherein the substance A is one or more selected from annexin V and biotinylated annexin V.   The substance A is a biotinylated antibody that specifically recognizes a molecule present on the surface of a vesicle, biotinylated annexin V, or biotinylated Tim protein, and the substance B is an avidin or avidin binding carrier The method according to any one of claims 2 to 4, wherein   The substance A is an antibody that specifically recognizes a molecule present on the surface of a vesicle, and the substance B is protein A, protein G, or protein L according to any one of claims 2 to 4. The method described.   5. The substance according to claim 2, wherein the substance A is an antibody, annexin V, or Tim protein that specifically recognizes a molecule present on the surface of a vesicle, and the substance B is a latex carrier. The method described in 1.   The method according to any one of claims 2 to 4, wherein the substance B is a substance having 2 to 4 sites for binding to the substance A.   The vesicle is an exosome, microvesicle, nanovesicle, apoptotic body, dexosome, bleb, vesicle, prostasome, microparticle, intraluminal vesicle, endosomal vesicle or extracellular vesicle. 11. The method according to any one of items 10. An aggregation step of forming a vesicle aggregate by contacting a biological sample containing the vesicle with a substance A having one or more sites that adsorb or bind to the surface of the vesicle;
A recovery step of recovering the vesicle aggregate from the biological sample, and a dissociation step of dissociating the vesicle from the vesicle aggregate recovered in the recovery step.
How to collect vesicles. A substance A having one or a plurality of sites that adsorb or bind to the surface of the vesicle, and a substance B that binds a plurality of the substances A at a site different from the site that adsorbs or binds to the surface of the vesicle Features
Complex for collecting vesicle aggregates. A substance A having one or a plurality of sites that adsorb or bind to the surface of the vesicle, and a substance B that binds a plurality of the substances A at a site different from the site that adsorbs or binds to the surface of the vesicle Features
Kit for collecting vesicle aggregates.