Extracellular vesicle adsorbent and method for detecting and separating extracellular vesicles using the same

Abstract

To provide a method for detecting extracellular vesicles with high sensitivity and high accuracy, and a method that can separate high-purity extracellular vesicles in large quantities by simple operation.SOLUTION: An extracellular vesicle adsorbent comprises a sphingomyelin recognition molecule comprising sphingomyelin-binding protein such as equinatoxin and lysenin, and an insoluble carrier having the sphingomyelin recognition molecule immobilized thereto.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to an extracellular vesicle adsorbent capable of selectively adsorbing extracellular vesicles contained in a solution, and a method for detecting and separating extracellular vesicles using the extracellular vesicle adsorbent.

Background Art

[0002] Extracellular vesicles contain substances such as nucleic acids such as microRNA and messenger RNA and proteins inside, and are considered to be responsible for intercellular information transmission. Therefore, they are not only attracting attention as biomarkers for various diseases such as cancer, but also expected to be applied as new therapeutic agents and drug delivery systems (DDS) (Non-Patent Document 1).

[0003] As a technique for isolating and detecting extracellular vesicles, methods using heat shock proteins (HSP70, HSP90) and tetraspanins (CD9, CD63, CD81) as markers for extracellular vesicles are known. However, since the expression levels of these markers vary depending on the type of cells that release extracellular vesicles, it is a problem that they lack sensitivity and quantitativeness (Non-Patent Document 2).

[0004] Also, as techniques for isolating extracellular vesicles, ultracentrifugation, ultrafiltration, density gradient centrifugation, polymer precipitation, immunoprecipitation, etc. are known. However, among these methods, there is no method that satisfies all three of the following: obtaining high-purity and high-concentration extracellular vesicles, obtaining extracellular vesicles in large quantities and at low cost, and having a short and simple operation.

[0005] As another technique for isolating extracellular vesicles, methods using annexin V, which is a protein having binding affinity to phosphatidylserine of phospholipids, and Tim4 protein are known (Patent Document 1 and Non-Patent Document 2). However, the detection rate and recovery rate of extracellular vesicles in the above method are not sufficient.

Prior Art Documents

Patent Documents

[0006] [Patent Document 1] WO2016 / 088689 issue [Non-patent literature]

[0007] [Non-Patent Document 1] Proteomics, vol. 13, pp. 1637-1653, 2013 [Non-Patent Document 2] Japanese Journal of Pharmacology, Vol. 149, pp. 119-122, 2017. [Overview of the project] [Problems that the invention aims to solve]

[0008] The objective of this invention is to provide a method for detecting extracellular vesicles with high sensitivity and accuracy, and a method for separating large quantities of high-purity extracellular vesicles with simple operations, in order to advance the industrial use of extracellular vesicles. [Means for solving the problem]

[0009] The inventors have diligently studied to solve the above problems and have found an insoluble carrier and a sphingomyelin-recognizing component immobilized on the carrier. Child and Sphingomyelin recognition components that include Kogata We discovered that the chromatograph can detect and separate extracellular vesicles contained in solution with high sensitivity and accuracy, which led to the completion of this invention.

[0010] In other words, the present invention encompasses the embodiments described in [1] to [8] below.

[0011] [1] An insoluble carrier and a sphingomyelin-recognizing component containing a sphingomyelin-binding protein immobilized on the carrier. Child and An extracellular vesicle adsorbent containing [this ingredient].

[0012] [2] Sphingomyelin recognition component ChildThe adsorbent according to [1] further comprises an immobilization auxiliary protein having the ability to bind to an insoluble carrier.

[0013] [3] The adsorbent according to [1] or [2], wherein the sphingomyelin-binding protein is equinatoxin and / or lysenin.

[0014] [4] The adsorbent according to [3], wherein the equinatoxin is a protein selected from any of (a) to (c) below. (a) A protein comprising at least amino acid residues consisting of the amino acid sequence described in Sequence ID No. 1; (b) A protein having sphingomyelin-binding ability, comprising at least an amino acid residue consisting of the amino acid sequence described in Sequence ID No. 1, further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions within that amino acid residue; (c) A protein that contains at least one amino acid residue consisting of an amino acid sequence having 70% or more homology to the entire amino acid sequence described in Sequence ID No. 1, and that has sphingomyelin-binding ability.

[0015] [5] The adsorbent according to [3], wherein lysenin is a protein selected from any of (d) to (f) below. (d) A protein comprising at least amino acid residues consisting of the amino acid sequence described in Sequence ID No. 3; (e) A protein comprising at least an amino acid residue consisting of the amino acid sequence described in Sequence ID No. 3, wherein it further comprises one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions within that amino acid residue, and which has sphingomyelin-binding ability; (f) A protein that contains at least one amino acid residue consisting of an amino acid sequence having 70% or more homology to the entire amino acid sequence described in Sequence ID No. 3, and that has sphingomyelin-binding ability.

[0016] A method for detecting extracellular vesicles, comprising the steps of adding a solution containing extracellular vesicles to an adsorbent according to any one of [6][1] to [5] to adsorb the extracellular vesicles to the adsorbent, and detecting the extracellular vesicles adsorbed to the adsorbent.

[0017] An extracellular vesicle separation column filled with an adsorbent according to any one of [7][1] to [5].

[0018] A method for separating extracellular vesicles, comprising the steps of adding a solution containing extracellular vesicles to the column according to [8][7] to adsorb the extracellular vesicles to the adsorbent filled in the column, and eluting the extracellular vesicles adsorbed to the adsorbent using an eluent.

[0019] The present invention will be described in more detail below.

[0020] Sphingomyelin, one of the phospholipids, is known to be abundantly present on the surface of extracellular vesicles (Prog. Lipid Res., Vol. 66, pp. 30-41, 2017). The present invention is an invention achieved as a result of paying attention to and studying this finding.

[0021] In the present specification, extracellular vesicles refer to particles composed of a lipid bilayer membrane secreted from eukaryotic cells, and the particle size is 20 nm or more and several μm or less. Depending on their size and origin, they are classified into exosomes, microvesicles, and apoptotic vesicles. Among them, exosomes and microvesicles are mentioned in that they have been recognized as effective for disease diagnosis and treatment (for example, Biology (Basel), 2021, Vol. 10, p. 359). In terms of corresponding to these vesicles, the particle size is preferably 50 nm or more and 500 nm or less.

[0022] The insoluble carrier constituting the extracellular vesicle adsorbent of the present invention is not particularly limited as long as it is insoluble in aqueous solution. Examples include carriers made from polysaccharides such as agarose, alginate (alginate salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and carriers made from ceramics such as silica. Among these, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Specifically, examples include polymethacrylate gels with introduced hydroxyl groups such as Toyopal (manufactured by Tosoh), agarose gels such as Sepharose (manufactured by Cytiva), and cellulose gels such as Cellfine (manufactured by JNC). The shape of the insoluble carrier is not particularly limited and may be granular or non-granular, porous or non-porous. In particular, carriers with a large surface area and sphingomyelin recognition components are preferred. child Granular material is preferred because it allows for a larger immobilization amount. Furthermore, the particle size is preferably 1 μm to 10 μm in diameter, as this allows extracellular vesicles to pass through the gaps in the insoluble carrier without clogging and is readily available.

[0023] Sphingomyelin-recognizing component constituting the extracellular vesicle adsorbent of the present invention The childThe protein must contain at least a protein that has the ability to specifically bind sphingomyelin (hereinafter also referred to as a "sphingomyelin-binding protein"), and a polypeptide or molecule that does not have sphingomyelin-binding ability may be attached to the N-terminus or C-terminus of the protein. Examples of polypeptides or molecules that may be attached include protein tags such as SpyTag or SpyCatcher, the Z domain of the highly soluble protein Staphylococcus aureus-derived Protein A (SpA), linker sequences such as the GS linker (a linker consisting of repeating glycine residues and serine residues), protease recognition sequences such as the HRV 3C protease recognition sequence, separation and purification tags such as polyhistidine, c-myc tag, and FLAG tag, and proteins for immobilization to insoluble carriers such as glutathione S-transferase (GST), maltose-binding protein (MBP), and cellulose-binding domain (CBD). In particular, SpA's Z domain is preferable because it can improve the productivity of recombinant sphingomyelin-binding proteins by E. coli, and SpyCatcher is preferable because it can control the molecular density and orientation of sphingomyelin-binding proteins.

[0024] Examples of sphingomyelin-binding proteins used herein include equinatoxin II (hereinafter also referred to as "EqtII") from Actinia equina, lysenin (hereinafter also referred to as "Lysn") from Eisenia foetida, and pleurotolysin A2 and ostreolysin A from Pleurotus ostreatus. Among these, EqtII and Lysn are considered preferred embodiments of sphingomyelin-binding proteins because they are easily recombinant proteins produced by E. coli.

[0025] The amino acid sequence of the natural (unmutated) EqtII is shown as Sequence ID No. 1, and the amino acid sequence of the C-terminal domain of the natural (unmutated) Lysn is shown as Sequence ID No. 3. Sequence ID No. 1 corresponds to the amino acid sequence from position 36 to 214 of UniProt Accession No. P61914, and Sequence ID No. 3 corresponds to the amino acid sequence from position 160 to 297 of UniProt Accession No. O18423. Furthermore, the protein consisting of the sequence described in Sequence ID No. 3 is a protein in which the natural Lysn, a pore-forming toxin, has been detoxified and its safety enhanced (J. Biol. Chem., Vol. 280, pp. 24072-24084, 2005).

[0026] In the present invention, the term "sphingomyelin-binding protein" is not limited to naturally occurring sphingomyelin-binding proteins (including partial regions thereof; the same applies hereinafter), but may also include one or more amino acid residue substitutions, deletions, insertions, and additions, as long as it possesses sphingomyelin-binding ability (hereinafter also referred to as "sphingomyelin-binding protein mutant"). The substitutions, deletions, insertions, and additions of amino acid residues can be carried out using genetic engineering methods well known to those skilled in the art.

[0027] Specific examples of sphingomyelin-binding protein mutants include proteins that possess sphingomyelin-binding ability, as shown in (i) or (ii) below. (i) A protein comprising at least an amino acid residue consisting of the amino acid sequence described in Sequence ID No. 1 or 3, further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions within that amino acid residue; (ii) A protein comprising at least one amino acid residue consisting of an amino acid sequence having 70% or more homology to the entire amino acid sequence described in SEQ ID NO: 1 or 3.

[0028] In (i) above, "one or several" means one or more amino acid residues, although this can vary depending on the position and type of amino acid residues in the three-dimensional structure of the protein. For example, it can mean one to twenty, one to ten, one to five, or one to three. Furthermore, "one or more of substitutions, deletions, insertions, and additions" as described in (i) above also includes naturally occurring mutations (mutants or variants) based on individual differences in the organism from which the gene originates, differences in species, etc.

[0029] An example of the "substitution" described in (i) is the substitution of valine at position 8 and lysine at position 69 in SEQ ID NO: 1 with cysteine ​​(SEQ ID NO: 2). This substitution detoxifies the native EqtII (SEQ ID NO: 1), which is a pore-forming toxin, and enhances its safety (J. Biol. Chem., Vol. 279, pp. 46509-46517, 2004).

[0030] (ii) The homology of amino acid sequences in (ii) only needs to be 70% or more, and may have higher homology (for example, 80% or more, 85% or more, 90% or more, or 95% or more). In this invention, homology may mean similarity or identity, and may particularly mean identity. Identity between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, February 2013, Vol. 31 No. 3, Yodosha). Similarity between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, February 2013, Vol. 31 No. 3, Yodosha). The homology of amino acid sequences can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0031] Sphingomyelin-recognizing molecules complex 1 piecesThere is no limit to the number of sphingomyelin-binding proteins contained in each molecule, but it is preferable to have 3 to 9 proteins, as these have high affinity for sphingomyelin and are easy to produce using recombinant proteins in E. coli. Recognition molecule complex 1 pieces There are no restrictions on the types of sphingomyelin-binding proteins included in each sample. For example, two or more of the following can be selected as sphingomyelin-recognizing molecules: the native EqtII consisting of the amino acid sequence described in SEQ ID NO: 1, the detoxified EqtII consisting of the amino acid sequence described in SEQ ID NO: 2 (hereinafter also referred to as "NT-EqtII"), and the C-terminal domain of the native Lysn consisting of the amino acid sequence described in SEQ ID NO: 3 (detoxified Lysn, hereinafter also referred to as "NT-Lysn"). complex 1 pieces It may also be included in the surrounding area. Also, one sphingomyelin recognition molecule complex Sphingomyelin recognition molecules used in immobilization carriers complex There are no restrictions on the type, and two or more sphingomyelin-recognizing molecules. complex It's fine if it's fixed.

[0032] Sphingomyelin of the present invention recognition molecule A concrete example of this is the protein consisting of the amino acid sequences described in SEQ ID NOs. 4 and 5. Of SEQ ID NOs. 4, the amino acid sequence from position 5 to 183 is that of NT-EqtII (SEQ ID NOs. 2), positions 189 to 202 is that of the GS linker, and positions 207 to 322 is that of the protein tag SpyCatcher (Chain A and B [116 residues] of Protein Data Bank No. 4MLI). Furthermore, of SEQ ID NOs. 5, positions 5 to 141 is that of NT-Lysn (SEQ ID NOs. 3), positions 2 to 138 is that of NT-Lysn, positions 147 to 160 is that of the GS linker, and positions 165 to 280 is that of the protein tag SpyCatcher.

[0033] Furthermore, in the extracellular vesicle adsorbent of the present invention, a substance that does not recognize sphingomyelin (hereinafter also referred to as "sphingomyelin-non-binding substance") may be bound (immobilized) to the insoluble carrier, as long as the sphingomyelin-binding protein has a specific binding ability to extracellular vesicles. Examples of sphingomyelin-non-binding substances include immobilization auxiliary proteins, proteins that prevent nonspecific adsorption on the surface of the insoluble carrier such as bovine serum albumin, and non-protein components that improve the hydrophilicity of the carrier surface such as dextran. Among these, the immobilization auxiliary protein is sphingomyelin recognition molecule It has the ability to bind to and sphingomyelin recognition molecule This protein has the function of controlling the density and orientation of the protein when it is immobilized on an insoluble carrier. There are no restrictions on the number or type of sphingomyelin-unbound substances bound to a single sphingomyelin-recognizing molecular complex immobilization carrier, and two or more types of sphingomyelin-unbound substances may be immobilized (bound) to it.

[0034] As a sphingomyelin-non-binding substance to be immobilized on an insoluble carrier, an immobilization support protein is preferred in that it controls the density and orientation of sphingomyelin-binding proteins and optimizes their binding to extracellular vesicles. An immobilization support protein having a trimer or nonamer structure is even more preferred because it improves affinity due to the multivalent effect of sphingomyelin-binding proteins and is also easy to produce.

[0035] When using a sphingomyelin-binding protein with the aforementioned SpyCatcher attached to its C-terminus and an insoluble carrier on which biotin-binding proteins such as avidin and streptavidin are immobilized, specific examples of immobilization support proteins include proteins consisting of the amino acid sequences described in SEQ ID NOs: 6 to 8.

[0036] Of sequence number 6, the 5th is a cysteine ​​residue used for biotin labeling when immobilizing onto the insoluble carrier, positions 15 to 72 are the amino acid sequence of the SpA Z domain (positions 4 to 61 of GenBank Accession No. AL052730), positions 78 to 92 are the GS linker sequence, positions 95 to 108 are the α-helix linker sequence, and positions 116 to 128 are the amino acid sequence of the SpyTag protein tag (Chain C [13 residues] of Protein Data Bank No. 4MLI) that has binding affinity to the aforementioned SpyCatcher.

[0037] Furthermore, in sequence number 7, the 5th residue is the cysteine ​​residue used for biotin labeling as described above, positions 15 to 72 are the Z domain of SpA, positions 78 to 92, 112 to 126, and 162 to 176 are the GS linker sequence, positions 129 to 142, and 179 to 192 are the α-helix linker sequence, and positions 95 to 107, 145 to 157, and 200 to 212 are the amino acid sequence of the protein tag SpyTag.

[0038] Furthermore, in sequence number 8, the 5th to 10th positions are polyhistidine sequences, and the 15th to 169th positions are sequences of BC2LCN lectin amino acid substitutions (GenBank Accession). The sequence consists of the amino acid sequence from the 2nd to the 156th position of No.WP_006490828, wherein the 40th glutamine is replaced with leucine and the 82nd glutamic acid is replaced with cysteine ​​(corresponding to the biotin labeling site mentioned above; Japanese Patent Publication No. 2020-025535), the 176th to 190th, 210th to 224th, and 260th to 274th positions are the GS linker sequence, the 193rd to 205th, 243rd to 255th, and 298th to 310th positions are the amino acid sequence of the protein tag SpyTag, and the 227th to 240th and 277th to 290th positions are the α-helix linker sequence.

[0039] There are no particular restrictions on the method for producing the aforementioned sphingomyelin-binding protein and immobilization auxiliary protein; for example, it may be carried out by the method commonly used by those skilled in the art, as disclosed in Japanese Patent Publication No. 2018-000038. Specifically, the process involves transforming a host with an expression plasmid containing a polynucleotide encoding the amino acid sequence of the sphingomyelin-binding protein or immobilization auxiliary protein, culturing the resulting transformant, and obtaining a culture (including the cultured transformant itself, secretions, and the culture medium used for cultivation). The process involves centrifugation of the obtained culture to suspend the transformed cells in an appropriate buffer, disruption of the cells by a surfactant or other chemical agent or physical disruption by ultrasound, and removal of the disruption residue by centrifugation to obtain a soluble protein extract containing sphingomyelin-binding protein or immobilization auxiliary protein. The obtained soluble protein extract is purified by liquid chromatography such as affinity chromatography, gel filtration chromatography, ion exchange chromatography, and hydrophobic interaction chromatography. The desired sphingomyelin-binding protein or immobilization support protein can then be produced.

[0040] To obtain transformants capable of producing recombinant sphingomyelin-binding proteins and / or immobilization auxiliary proteins, a method commonly used by those skilled in the art may be used. This involves inserting the polynucleotide encoding the protein at an appropriate position in a vector based on bacteriophages, cosmids, or plasmids commonly used for the transformation of prokaryotic or eukaryotic cells to create an expression vector for the protein, and then transforming a host using this vector. While there are no particular restrictions on the host, Escherichia coli is preferred due to the ease of genetic engineering experiments. Examples of preferred Escherichia coli strains include JM109, BL21(DE3), NiCo21(DE3), and W3110. Furthermore, the transformation of the host using the expression vector may be carried out using a method commonly used by those skilled in the art.

[0041] Sphingomyelin recognition Child The process for immobilizing the substance on an insoluble support is not particularly limited as long as it is a general process that is normally used by those skilled in the art. For example, after introducing reactive functional groups such as maleimide groups or carboxyl groups to the insoluble support, the sphingomyelin recognition component can be attached to the functional group. Child Alternatively, the sphingomyelin may be immobilized directly, or a sphingomyelin-recognizing molecular complex may be prepared by linking a sphingomyelin-binding protein and an immobilization support protein via a multimer-forming protein containing the amino acid sequence of SpyTag / SpyCatcher, and then immobilized on an insoluble carrier to which a biotin-binding protein such as avidin or streptavidin has been introduced, via the biotin present in the recognition molecular complex, or a sphingomyelin-non-binding substance such as an immobilization support protein may be immobilized on an insoluble carrier via a reactive functional group, and then sphingomyelin may be immobilized. recognition moleculeThe sphingomyelin may be immobilized on the insoluble carrier by binding it to a sphingomyelin-unbound substance, or the sphingomyelin recognition molecule complex may be brought into contact with the insoluble carrier and the recognition molecule complex may be immobilized on the insoluble carrier by physical adsorption. recognition molecule By immobilizing it on an insoluble carrier via an immobilization auxiliary protein, the sphingomyelin-recognized component is immobilized. Child Immobilizing the sphingomyelin-binding protein on an insoluble support is preferable because it results in a milder reaction during the immobilization process, reducing denaturation of the sphingomyelin-binding protein, and allows for control over the orientation and density of the sphingomyelin-binding protein on the support.

[0042] The step of adsorbing extracellular vesicles onto the extracellular vesicle adsorbent of the present invention is not particularly limited as long as it is a general method commonly used by those skilled in the art. The solution containing extracellular vesicles is not particularly limited and may be derived from living organisms such as biological tissue or cultured cells, or it may be a solution in which extracellular vesicles are suspended in a culture medium or buffer solution. The solvent for preparing the solution containing extracellular vesicles is not particularly limited as long as it can stably maintain the extracellular vesicles and does not inhibit the adsorption of extracellular vesicles onto the adsorbent of the present invention, but the sphingomyelin recognition component Child In order to prevent the denaturation of extracellular vesicles contained in the solution, a buffer solution prepared to a pH of 6.0 to 8.0 is preferred, and specific examples include TBS (Tris Buffered Saline), PBS (Phosphate Buffered Saline), and HBS (HEPES[4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid] Buffered Saline). Furthermore, the solvent contains sphingomyelin recognition components. Child and To prevent nonspecific adsorption with components in the solution, a surfactant may be further included, specifically, 0.00001% (w / v) to 0.5% (w / v) of a nonionic surfactant. A preferred example of a nonionic surfactant is Tween 20 (trade name).

[0043] The temperature at which the extracellular vesicle adsorbent of the present invention is brought into contact with a solution containing extracellular vesicles is determined by the recognition of extracellular vesicles and sphingomyelin. child A temperature of 2°C to 40°C is preferred to prevent denaturation. Furthermore, the contact time between the extracellular vesicle adsorbent of the present invention and the solution containing extracellular vesicles is important for sphingomyelin recognition. Child and In order to ensure sufficient time for extracellular vesicles to be in contact and to prevent degeneration of extracellular vesicles, a contact period of 30 minutes to 24 hours is preferred, and 1 hour to 12 hours is more preferred.

[0044] The washing step to remove impurities nonspecifically adsorbed onto the extracellular vesicle adsorbent of the present invention can be appropriately selected from methods commonly used by those skilled in the art. The buffer solution and pH for washing can be the same as those used to prepare the solution containing the extracellular vesicles described above. The temperature can also be the same as that used for the step of adsorbing extracellular vesicles onto the extracellular vesicle adsorbent of the present invention.

[0045] The step of detecting extracellular vesicles adsorbed onto the extracellular vesicle adsorbent of the present invention is not particularly limited and can be carried out by methods commonly used by those skilled in the art. Specifically, examples include detecting extracellular vesicles using antibodies specific to extracellular vesicles by Western blotting or ELISA (Enzyme-Linked Immuno Sorbent Assay).

[0046] When detecting by Western blotting, for example, extracellular vesicles can be eluted from the adsorbent of the present invention using a suitable buffer, subjected to electrophoresis, and then detected using an antibody against the extracellular vesicle marker. To elute extracellular vesicles from the extracellular vesicle adsorbent of the present invention, one can use a solvent for preparing the above-mentioned solution containing extracellular vesicles, which contains several hundred mM arginine, or a denaturing agent such as SDS (Sodium Dodecyl Sulfate) to elute the sphingomyelin-recognizing component. ChildOne example of a method is to denature the cells and elute the extracellular vesicles. For example, extracellular vesicles can be detected by separating the eluate containing the extracellular vesicles using SDS-PAGE (SDS-polyacrylamide gel electrophoresis), transferring the proteins to a membrane, and then detecting them using an antibody against an extracellular vesicle marker.

[0047] When using the ELISA method, the extracellular vesicle adsorbent of the present invention, on which extracellular vesicles are adsorbed, is used as the solid phase, and an antibody, appropriately diluted with a buffer or the like for preparing a solution containing the extracellular vesicles, is added, and the extracellular vesicles on the complex are detected by the antibody. The antibody detection method can be a method commonly used by those skilled in the art, such as a colorimetric method or a fluorescence method. The reaction temperature and reaction time of the antibody can also be a method commonly used by those skilled in the art, but a reaction temperature of 4°C to 40°C and a reaction time of 30 minutes to 12 hours are preferred in terms of high stability of the antibody and the complex in the ELISA method. [Effects of the Invention]

[0048] The adsorbent of the present invention contains a sphingomyelin-binding protein that has the ability to bind to extracellular vesicles on which sphingomyelin is present, and a sphingomyelin-recognizing component. Child This material is immobilized on an insoluble carrier, and by using this carrier, extracellular vesicles can be selectively, sensitively, and accurately detected and separated from a solution containing extracellular vesicles. [Brief explanation of the drawing]

[0049] [Figure 1] Schematic diagrams of the sphingomyelin recognition molecule, immobilization auxiliary protein, sphingomyelin recognition molecule complex, and sphingomyelin recognition molecule complex immobilization support prepared in Examples 1 to 12 and Comparative Examples 1 to 3. [Figure 2] This figure shows the results of detecting extracellular vesicles contained in solution using the immobilized carriers of Example 10 and Comparative Example 3. [Figure 3] This figure shows the results of detecting extracellular vesicles contained in solution using the immobilized carriers of Examples 11 and 12 and Comparative Examples 1 and 2. [Figure 4] This figure shows the results of detecting extracellular vesicles isolated from the culture supernatant using the immobilized carriers of Example 9 and Comparative Example 2. [Examples]

[0050] The present invention will be described in more detail below with reference to examples of preparation, experiments, examples, and comparative examples, but the present invention is not limited to these examples.

[0051] Example 1: Sphingomyelin recognition molecule creation Sphingomyelin recognition molecule As such, the proteins shown in <Example 1> and <Example 2> below were designed, and these proteins were expressed using genetic engineering technology. <Example 1> Fusion protein of detoxified equinatoxin (NT-EqtII, SEQ ID NO: 2) and SpyCatcher (Chain A and B [116 residues] of Protein Data Bank No. 4MLI, SpyC) ((NT-EqtII)-SpyC, SEQ ID NO: 4) <Example 2> Fusion protein of detoxified lysenin (NT-Lysn, SEQ ID NO: 3) and SpyC ((NT-Lysn)-SpyC, SEQ ID NO: 5) Below, each molecule The method for producing it will be explained in detail.

[0052] <Example 1> Preparation of (NT-EqtII)-SpyC (Sequence ID 4) <1> A polynucleotide (SEQ ID NO: 10) encoding the fusion protein (SpA-Z)-(NT-EqtII)-SpyC (SEQ ID NO: 9), which is a fusion protein of the Z domain of Protein A from Staphylococcus aureus (SpA-Z, a polypeptide consisting of amino acid residues from position 4 to 61 of GenBank Accession No. AL052730) and (NT-EqtII)-SpyC (SEQ ID NO: 4), was inserted into the multi-cloning site of pET28a(+) (Merck) to create the vector pET_(SpA-Z)-(NT-EqtII)-SpyC (SEQ ID NO: 9), which is a fusion protein of (SpA-Z)-(NT-EqtII)-SpyC (SEQ ID NO: 9), into which (SpA-Z)-(NT-EqtII)-SpyC (SEQ ID NO: 9) was created. This allowed the expression of (SpA-Z)-(NT-EqtII)-SpyC (SEQ ID NO: 9) in E. coli.

[0053] <2> <1> The expression vector pET_(SpA-Z)-(NT-EqtII)-SpyC, which was prepared using [method / tool], was used to transform E. coli BL21(DE3) strain, and recombinant E. coli (transformed cells) were obtained.

[0054] <3> The resulting transformants were inoculated into LB (Luria-Bertani) medium (10 g / L Tryptone, 5 g / L Yeast extract, and 5 g / L NaCl) supplemented with 30 μg / mL kanamycin, and pre-cultured by shaking overnight at 37°C.

[0055] <4> <3> The pre-culture medium was inoculated into Terrific broth (TB) medium (24 g / L Yeast extract, 12 g / L Tryptone, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, and 4 mL / L Glycerol) supplemented with 30 μg / mL kanamycin, and cultured with shaking at 37°C. The turbidity (OD) of the culture medium was measured. 600nm When the ratio reached approximately 0.6, the culture temperature was switched to 20°C, 0.1 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) was added, and the cells were cultured overnight to express (SpA-Z)-(NT-EqtII)-SpyC.

[0056] <5> Using the BugBuster Protein Extraction Kit (Merck), soluble protein extracts were recovered from bacterial cells according to the manufacturer's protocol. (SpA-Z)-(NT-EqtII)-SpyC was purified from the soluble protein extracts by nickel chelate affinity chromatography, and the solution was replaced with a storage buffer (50 mM Tris-HCl (pH 7.5) containing 150 mM sodium chloride) using an ultrafiltration filter.

[0057] <6> To remove the histidine tag and SpA-Z from (SpA-Z)-(NT-EqtII)-SpyC, (SpA-Z)-(NT-EqtII)-SpyC was digested using HRV 3C protease (Merck) fused with a histidine-tagged peptide, according to the manufacturer's protocol.

[0058] <7> After adding 5 mM imidazole to the digested fluid, the solution was purified by nickel chelate affinity chromatography to obtain (NT-EqtII)-SpyC (SEQ ID NO: 4).

[0059] <Example 2> (NT-Lysn)-SpyC, SEQ ID NO. 5) <1> By inserting a polynucleotide (SEQ ID NO: 12) encoding the fusion protein (SpA-Z)-(NT-Lysn)-SpyC (SEQ ID NO: 11), a fusion protein of SpA-Z and (NT-Lysn)-SpyC (SEQ ID NO: 5), into the multi-cloning site of pET28a(+) (Merck), we created the vector pET_(SpA-Z)-(NT-Lysn)-SpyC (SEQ ID NO: 11) that can express (SpA-Z)-(NT-Lysn)-SpyC (SEQ ID NO: 11) in E. coli.

[0060] <2> <Example 1> <2> Recombinant Escherichia coli (transformed organism) capable of expressing (SpA-Z)-(NT-Lysn)-SpyC (SEQ ID NO: 11) was obtained by the same method as described in <Example 1> <3> from <7> (NT-Lysn)-SpyC (SEQ ID NO: 5) solution was obtained using the same method as described above.

[0061] Example 2: Preparation of immobilization support proteins The following are examples of sphingomyelin-unbound substances: from <c>We designed the immobilization support protein shown in [figure] and expressed it using genetic engineering technology. < / c> A fusion protein (Cys-(SpA-Z)-1SpyT, SEQ ID NO: 6) of SpA-Z, which has a biotin-labeled cysteine ​​residue (Cys) added to its N-terminus, and SpyTag (Chain B [13 residues] of Protein Data Bank No. 4MLI, SpyT). A fusion protein (Cys-(SpA-Z)-3SpyT, SEQ ID NO: 7) of SpA-Z with a biotin-labeled cysteine ​​residue (Cys) added to its N-terminus. <c>A fusion protein ((BC2LCN-m2)-3SpyT, SEQ ID NO: 8) of a BC2LCN lectin amino acid substitution polypeptide (a polypeptide consisting of amino acid residues 2 through 156 of GenBank Accession No. WP_006490828, in which glutamine at position 40 is substituted with leucine and glutamic acid at position 82 is substituted with cysteine ​​(for biotin labeling)) and three copies of SpyT. The following describes in detail the methods for preparing each immobilization support protein.

[0062] < / c> Cys-(SpA-Z)-1SpyT(Sequence ID 6) <a-1>The polynucleotide encoding Cys-(SpA-Z)-1SpyT (SEQ ID NO: 6) was inserted into the multi-cloning site of the pBR322 plasmid pGEX (manufactured by Cytiva). pGEX has a polynucleotide encoding GST (Glutathione S-transferase, a polypeptide consisting of amino acid residues 1 through 218 of GenBank Accession No. QLV95778) and a protease recognition site upstream of the multi-cloning site. This insertion yields the vector pGEX_GST-Cys-(SpA-Z)-1SpyT, which can express the fusion protein GST-Cys-(SpA-Z)-1SpyT (SEQ ID NO: 13) in E. coli. The sequence of the polynucleotide encoding GST-Cys-(SpA-Z)-1SpyT is shown in SEQ ID NO: 14.

[0063] <a-2> <a-1>The E. coli BL21 strain was transformed with the expression vector pGEX_GST-Cys-(SpA-Z)-1SpyT, which was prepared using [method / technology], to obtain recombinant E. coli (transformed cells).

[0064] <a-3>The obtained transformants were inoculated into LB medium supplemented with 60 μg / mL of carbenicillin and pre-cultured overnight at 37°C by shaking.

[0065] <a-4> <a-3>The pre-culture solution was inoculated into LB medium supplemented with 60 μg / mL of carbenicillin and cultured with shaking at 37°C. The turbidity of the culture medium (OD) was measured. 600nm When the ratio reached approximately 0.6, the culture temperature was switched to 30°C, 0.5 mM IPTG was added, and the cells were cultured for 5 hours to express GST-Cys-(SpA-Z)-1SpyT.

[0066] <a-5>Using the BugBuster Protein Extraction Kit (Merck), soluble protein extracts were recovered from bacterial cells according to the manufacturer's protocol. Purification of GST-Cys-(SpA-Z)-1SpyT from the soluble protein extracts was performed by affinity chromatography using a glutathione-immobilized support.

[0067] <a-6>GST-Cys-(SpA-Z)-1SpyT bound to the aforementioned immobilized carrier was digested using GST-fused HRV 3C protease (Merck) according to the manufacturer's protocol, and the supernatant was collected to obtain a Cys-(SpA-Z)-1SpyT (SEQ ID NO: 6) solution.

[0068] Cys-(SpA-Z)-3SpyT (Sequence ID 7) <b-1>By inserting the polynucleotide encoding Cys-(SpA-Z)-3SpyT (SEQ ID NO: 7) into the multi-cloning site of pGEX (Cytiva), a vector pGEX_GST-Cys-(SpA-Z)-1SpyT (SEQ ID NO: 15), a fusion protein of GST and Cys-(SpA-Z)-3SpyT (SEQ ID NO: 7), was created that can express the protein in E. coli. The sequence of the polynucleotide encoding GST-Cys-(SpA-Z)-1SpyT is shown in SEQ ID NO: 16.

[0069] <b-2> <a-2>Recombinant E. coli (transformed organisms) capable of expressing GST-Cys-(SpA-Z)-3SpyT were obtained using the same method as described above. <a-3>from <a-6>Cys-(SpA-Z)-3SpyT (SEQ ID NO: 7) solution was obtained using the same method as described above.

[0070] <c>(BC2LCN-m2)-3SpyT(Sequence ID 8) <c-1>By inserting the polynucleotide encoding (SEQ ID NO: 17) of (BC2LCN-m2)-3SpyT (SEQ ID NO: 8) into the multi-cloning site of pET28a(+) (Merck), a vector pET_(BC2LCN-m2)-3SpyT capable of expressing (BC2LCN-m2)-3SpyT (SEQ ID NO: 8) in E. coli was constructed. (BC2LCN-m2)-3SpyT has a structure in which three copies of SpyTag are added to the C-terminus of BC2LCN-m2, and since BC2LCN-m2 forms a trimer structure, the recombinant protein expressed is 1 pieces Each contains 9 SpyTag sequences.

[0071] <c-2><Example 1> <2> Recombinant Escherichia coli (transformed organism) capable of expressing (BC2LCN-m2)-3SpyT (SEQ ID NO: 8) was obtained by the same method as described in <Example 1> <3> and <4> (BC2LCN-m2)-3SpyT was expressed using the same method as described.

[0072] <c-3>Using the BugBuster Protein Extraction Kit (Merck), soluble protein extracts were collected from bacterial cells according to the manufacturer's protocol. (BC2LCN-m2)-3SpyT was purified from the soluble protein extract by nickel chelate affinity chromatography. The (BC2LCN-m2)-3SpyT (SEQ ID NO: 8) solution was obtained by replacing the solution with the preservation buffer D-PBS(+) (137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, pH 7.4) using an ultrafiltration filter.

[0073] Fabrication Example 3: Fabrication of a sphingomyelin recognition molecule complex immobilization support. Sphingomyelin prepared in example 1 recognition molecule A sphingomyelin-recognizing molecule complex immobilization carrier was created by immobilizing it onto an insoluble carrier via a non-sphingomyelin-non-binding substance (immobilization auxiliary protein) prepared in Production Example 2.

[0074] <1> Fabrication of a sphingomyelin recognition molecular complex Sphingomyelin prepared in example 1 recognition molecule A sphingomyelin-recognizing molecular complex was created by binding the non-sphingomyelin-binding protein (immobilization support protein) prepared in Preparation Example 2 to the sphingomyelin-recognizing molecular complex using the method shown below, thereby immobilizing it on an insoluble carrier. recognition molecule The combinations of these proteins with immobilization support proteins are summarized in Table 1.

[0075] [Table 1]

[0076] <1-1> The immobilization support protein prepared in Preparation Example 2 was replaced with D-PBS(+) buffer using an ultrafiltration filter, and then biotin was labeled to the sulfhydryl groups of cysteine ​​residues present in the immobilization support protein using EZ-Link Maleimide-PEG2-Biotin (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0077] <1-2> Based on the combinations listed in Table 1, the biotin-labeled immobilization auxiliary protein prepared in <1-1> and the sphingomyelin prepared in Preparation Example 1 were used. recognition molecule They were combined. Specifically, biotin-labeled immobilization support protein and sphingomyelin. recognition molecule Each of the two proteins was suspended in D-PBS(+) or TBS-T (25 mM Tris-HCl (pH 7.2) containing 0.15 M NaCl and 0.05% (w / v) Tween 20 (trade name)) with a pH of 7.0 to 8.0, at a concentration of 0.3 mg / mL or higher. After mixing the two suspensions, they were left at 4°C for at least 24 hours to produce a sphingomyelin-recognizing molecular complex via the binding reaction between the protein tags SpyT and SpyC. The molar ratio during mixing was 1 part biotin-labeled immobilization auxiliary protein to 1 part sphingomyelin recognition molecule This was designated as 3 (Examples 3 and 6) or 9 (Examples 4, 5, and 7).

[0078] <2> Sphingomyelin-recognizing molecules complex Immobilization to an insoluble carrier <2-1> As an insoluble carrier, Magnoshere MS300 / Streptavidin (manufactured by JSR Life Sciences), which is streptavidin-immobilized magnetic nanoparticle, was used. 100 μL of a slurry solution of the magnetic nanoparticles (1% (w / v) microparticle content) was collected, and the supernatant was removed.

[0079] <2-2> The magnetic particles from which the supernatant was removed were washed with a fixation buffer (10 mM Tris-HCl (pH 7.4) containing 0.5 mM EDTA, 1 M NaCl, and 0.05% (w / v) Tween 20 (trade name)).

[0080] <2-3> Replaced with fixation buffer using an ultrafiltration membrane or the like in advance. <1> Sphingomyelin recognition molecule created complex The solution was adjusted to a concentration of 0.1 mg / mL or higher, calculated as the concentration of the immobilization auxiliary protein. Then, 4.5 μg (calculated as the amount of immobilization auxiliary protein) was added to the magnetic particles washed in <2-2>, and the mixture was mixed at room temperature for 10 minutes.

[0081] <2-4> After mixing, remove the supernatant, wash three times with the fixation buffer, then wash once with the exosome binding buffer (10 mM HEPES [4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid], pH 7.3) to identify the sphingomyelin recognition molecule. complex An immobilized carrier was fabricated.

[0082] <2-5> As a control, sphingomyelin recognition molecule A biotin-labeled immobilization support protein that is not bound to <2-2> was immobilized on magnetic particles washed with <2-2> in the same manner as described in <2-3> and <2-4> to prepare the immobilization support protein immobilization carrier.

[0083] The sphingomyelin-recognizing molecule used in the preparation of the immobilized support was complex Examples 8 to 12 or biotin-labeled immobilization auxiliary proteins (control, Comparative Examples 1 to 3) are shown in Table 2.

[0084] [Table 2]

[0085] Experimental Example 1: Separation of extracellular vesicles from a solution containing extracellular vesicles <1> Preparation of extracellular vesicle solution <1-1> Prostate cancer cells (PC3 cells) were cultured at 37°C in a 5% CO2 environment using Ham's F-12K medium (manufactured by Fujifilm Wako Pure Chemical Industries) containing 15% (v / v) FBS (Fetal Bovine Serum).

[0086] <1-2> Cultured PC3 cells 2.5 × 10 5 After suspending the cells in Ham's F-12K medium to a concentration of cells / mL, the cells were seeded at 2 mL / well in a 6-well plate, suspended, and cultured for a further 3 days.

[0087] <1-3> After culturing, the entire culture supernatant (approximately 2 mL) was collected and centrifuged at 300 × G for 10 minutes at room temperature to remove suspended cells, and 1.5 mL of the supernatant was collected (culture supernatant, hereafter also referred to as "CM").

[0088] <1-4>CM was further centrifuged at 3000×G for 10 minutes at 4°C to remove cell debris, and 1.2 mL of the supernatant was collected. The collected supernatant was further centrifuged at 16000×G for 60 minutes at 4°C, and 1 mL of the supernatant was transferred to another tube.

[0089] <1-5> 1 mL of the supernatant transferred to another tube was mixed with 1 mL of PBS (Phosphate buffered saline), and the mixture was ultracentrifuged at 259,000 × G for 70 minutes at 4°C. 1.8 mL of the supernatant was removed. The remaining precipitate was suspended in 1.8 mL of PBS, ultracentrifuged at 259,000 × G for 70 minutes at 4°C, washed, and 1.8 mL of the supernatant was removed. The remaining 0.2 mL containing the precipitate was used as the extracellular vesicle solution.

[0090] <2> Separation of extracellular vesicles <2-1> Example of production 3 <2> Of the immobilized carriers prepared (Table 2), a container was placed containing one of the immobilized carriers from Examples 10 to 12 and Comparative Examples 1 to 3. <1> After adding 20 μL of the extracellular vesicle solution prepared by [method omitted], the container was shaken at 10°C for 3 hours. After shaking, 7.5 μL of the supernatant was collected and designated as the "supernatant."

[0091] <2-2> After removing the remaining supernatant, the sample was washed twice with TBS-T buffer. 7.5 μL of 2% (w / v) SDS (Sodium Dodecyl Sulfate) aqueous solution was added, and the mixture was vigorously stirred with a vortex mixer and then allowed to stand for 15 minutes. The mixture was stirred again with a vortex mixer, and 7.5 μL of 2× sample buffer (DTT-free) (manufactured by Atto) was added. The entire volume was collected and used as the "eluate".

[0092] <2-3> After heat treatment of the supernatant and eluate, the samples were subjected to SDS-PAGE (SDS-polyacrylamide gel electrophoresis), transferred to a PVDF membrane, and blocked with 3% skim milk-containing TBS-T buffer. When performing SDS-PAGE, extracellular vesicle specimens were used. <1> The extracellular vesicle solution prepared using [method / method] was provided simultaneously.

[0093] <2-4> Extracellular vesicles were detected by Western blotting using an anti-CD9 mouse antibody as the primary antibody and an HRP (horseradish peroxidase)-labeled anti-mouse antibody as the secondary antibody.

[0094] Figure 2 shows the results of extracellular vesicle separation using immobilized carriers for Example 10 and Comparative Example 3, and Figure 3 shows the results of extracellular vesicle separation using immobilized carriers for Examples 11 and 12 and Comparative Examples 1 and 2. Sphingomyelin-recognizing molecules, including sphingomyelin-binding proteins. complex When extracellular vesicle separation was performed using the immobilized carriers of Examples 10 to 12, which were immobilized with sphingomyelin-binding proteins, a clear band corresponding to the extracellular vesicle specimen (lane 1 in Figures 2 and 3) was observed in the eluate (Example 10: lane 5 in Figure 2, Example 11: lane 5 in Figure 3, Example 12: lane 9 in Figure 3). On the other hand, when extracellular vesicle separation was performed using the immobilized carriers of Comparative Examples 1 to 3, which were immobilized with only immobilization auxiliary proteins and did not contain sphingomyelin-binding proteins, a band corresponding to the extracellular vesicle specimen (lane 1 in Figures 2 and 3) was hardly observed or not observed at all in the eluate (Comparative Example 1: lane 3 in Figure 3, Comparative Example 2: lane 7 in Figure 3, Comparative Example 3: lane 3 in Figure 1).

[0095] Based on the above results, the insoluble carrier and the sphingomyelin-recognizing component, which includes equinatoxin (EqtII) or lysenin (Lysn), a sphingomyelin-binding protein immobilized on the carrier, are considered to be the most effective components. Child and It was found that the sphingomyelin-recognizing molecule complex immobilization carrier, which includes [specific component], can selectively capture extracellular vesicles. Furthermore, since the sphingomyelin-recognizing molecule complex immobilization carrier can selectively capture extracellular vesicles, it is suggested that this immobilization carrier can selectively separate and detect extracellular vesicles contained in solution.

[0096] Experimental Example 2: Isolation of extracellular vesicles from cell culture supernatant <1> Preparation of cell culture supernatant <1-1> Prostate cancer cells (PC3 cells) were cultured at 37°C in a 5% CO2 environment using Ham's F-12K medium (manufactured by Fujifilm Wako Pure Chemical Industries) containing 15% (v / v) FBS.

[0097] <1-2> Cultured PC3 cells were suspended in Ham's F-12K medium containing 15% (v / v) FBS (exosomes had been removed beforehand by ultrafiltration membrane treatment), cultured for 2 days, and the culture supernatant was collected.

[0098] <1-3> The collected culture supernatant was centrifuged at 300×G for 5 minutes at 4°C to remove suspended cells, and the supernatant was collected. Further centrifuged at 1200×G for 20 minutes at 4°C to remove cell debris, and the supernatant was collected.

[0099] <1-4> The collected supernatant was further centrifuged at 10,000 × G for 30 minutes at 4°C to remove large extracellular vesicles, and the supernatant was collected and designated as the "cell culture supernatant".

[0100] <2> Separation of extracellular vesicles <2-1> Example of production 3 <2> Of the immobilized carriers prepared (Table 2), 100 μL of the immobilized carrier from Example 9 or Comparative Example 2 was placed in a container as a slurry. <1> After adding 750 μL of the cell culture supernatant prepared using the method described above, the container was shaken at 10°C for 3 hours.

[0101] <2-2> After removing the supernatant that did not adsorb to the immobilized carrier, the eluate was obtained in the same manner as in Experimental Example 1 <2-2>.

[0102] <2-3> After heat treatment of the cell culture supernatant and eluate, the cells were subjected to SDS-PAGE, transferred to a PVDF membrane, and blocked with 3% skim milk-containing TBS-T buffer. When performing SDS-PAGE, extracellular vesicle specimens were used. <1> The extracellular vesicle solution prepared using [method / method] was provided simultaneously.

[0103] <2-4>Extracellular vesicles were detected by Western blotting using the antibody described in Experiment 1<2-4>.

[0104] Figure 4 shows the results of extracellular vesicle separation. Sphingomyelin-recognizing molecules, including sphingomyelin-binding proteins, are shown. complex When extracellular vesicle separation was performed using the immobilized carrier of Example 9, which had the sphingomyelin-binding protein immobilized on it, a clear band corresponding to the extracellular vesicle sample (lane 1 in Figure 4) was observed in the eluate (lane 4). On the other hand, when extracellular vesicle separation was performed using the immobilized carrier of Comparative Example 2, which had only the immobilization auxiliary protein immobilized and did not contain the sphingomyelin-binding protein, no band corresponding to the eluate (lane 3) was observed. From these results, the sphingomyelin-recognizing molecule complex This suggests that the immobilized carrier can selectively separate extracellular vesicles contained in the cell culture supernatant.

[0105] Furthermore, the band of extracellular vesicles separated by the immobilized carrier in Example 9 (lane 4) shows a significantly stronger signal intensity compared to the band of the culture supernatant before contact with the carrier (lane 2), suggesting that extracellular vesicles contained in the cell culture supernatant can be selectively concentrated on the immobilized carrier. < / c> < / b-2> < / a-4> < / a-2>

Claims

1. An extracellular vesicle adsorbent comprising an insoluble carrier and a sphingomyelin-recognizing molecule immobilized on the carrier, The adsorbent comprising the sphingomyelin-recognizing molecule equinatoxin.

2. The adsorbent according to claim 1, further comprising an immobilization auxiliary protein having the ability to bind to a sphingomyelin-recognizing molecule and an insoluble carrier.

3. The adsorbent according to claim 1 or 2, wherein the equina toxin is a protein selected from any of (a) to (c) below. (a) A protein comprising at least amino acid residues consisting of the amino acid sequence described in Sequence ID No. 1; (b) A protein comprising at least an amino acid residue consisting of the amino acid sequence described in Sequence ID No. 1, further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions within that amino acid residue, and having sphingomyelin-binding ability; (c) A protein that contains at least one amino acid residue consisting of an amino acid sequence having 90% or more identity with the entire amino acid sequence described in Sequence ID No. 1, and that has sphingomyelin-binding ability.

4. A method for detecting extracellular vesicles, comprising the steps of: adding a solution containing extracellular vesicles to an adsorbent according to any one of claims 1 to 3 to adsorb the extracellular vesicles onto the adsorbent; and detecting the extracellular vesicles adsorbed onto the adsorbent.

5. An extracellular vesicle separation column packed with an adsorbent according to any one of claims 1 to 3.

6. A method for separating extracellular vesicles, comprising the steps of: adding a solution containing extracellular vesicles to a column according to claim 5 to adsorb the extracellular vesicles onto an adsorbent packed in the column; and eluting the extracellular vesicles adsorbed onto the adsorbent using an eluent.

Images