A method for detecting extracellular membrane vesicles in a sample and use of a detection kit

Abstract

The present invention relates to a method for detecting extracellular membrane vesicles in a sample and the use of a detection kit. The method comprises the following steps: (1) a complex formation step, which is a step of forming a complex of a T cell immunoglobulin and mucin domain molecule 4 (Tim4) protein bound to a carrier and extracellular membrane vesicles in a sample in the presence of calcium ions; (2) a detection step, which is a step of detecting the complex using an antibody that binds to the extracellular membrane vesicles. The present invention can easily and effectively obtain or remove extracellular membrane vesicles present in a sample in a good purity and in an intact state, and detect extracellular membrane vesicles with high sensitivity.

Description

[0001] The present application is a divisional application. The international application number of the original application is PCT / JP2015 / 083505, the Chinese national application number is 201580066077.1, the application date is November 27, 2015, and the name of the invention is "Tim protein-binding carrier, method for obtaining, removing, detecting and kit comprising the extracellular membrane vesicles and viruses using the carrier". Technical Field

[0002] The present invention relates to a Tim protein-binding carrier, a method for obtaining, removing, and detecting extracellular membrane vesicles and viruses using the carrier, and a kit comprising the carrier. Background Art

[0003] As is known to all, extracellular membrane vesicles contain proteins, small RNA (microRNA) and other nucleic acids inside their particles, which are responsible for the transfer of substances between cells. Extracellular membrane vesicles are also secreted into body fluids such as blood, and proteins and small RNA (microRNA) in extracellular membrane vesicles have attracted attention as diagnostic markers for diseases. In addition, their application as a delivery tool for nucleic acid drugs has also attracted attention.

[0004] In addition, there are a large number of enveloped viruses whose surfaces are covered with a membrane-like envelope. Most of the enveloped viruses are viruses that cause diseases such as influenza virus and human immunodeficiency virus, and therefore have attracted attention as research objects. In addition, enveloped viruses are inactivated and used as vaccines, carriers, etc., and are applied to the prevention and treatment of diseases.

[0005] Thus, when applying extracellular membrane vesicles to diagnosis or pharmaceuticals, using enveloped viruses as vaccines or carriers, and conducting basic research such as functional analysis of extracellular membrane vesicles or enveloped viruses, a process for obtaining extracellular membrane vesicles and enveloped viruses with high purity is essential, and people are eagerly looking forward to a method that can easily obtain extracellular membrane vesicles and enveloped viruses with high purity. In addition, in order to prevent contamination of extracellular membrane vesicles and enveloped viruses from organisms, people are looking forward to developing a method for effectively removing extracellular membrane vesicles and enveloped viruses from materials from body fluids used. Furthermore, a method for highly sensitively detecting the obtained extracellular membrane vesicles, enveloped viruses, and extracellular membrane vesicles and enveloped viruses in the subject is also eagerly expected.

[0006] As a method for obtaining extracellular membrane vesicles, the most conventional method is to perform ultracentrifugation separation on the sample to obtain extracellular membrane vesicles as a precipitate component (Non-patent Document 1). However, for this method, in addition to extracellular membrane vesicles, protein complexes or aggregates, lipoproteins such as HDL, etc. contained in the sample also produce co-precipitations, making it difficult to obtain extracellular membrane vesicles with high purity. The precipitate component obtained by the ultracentrifugation separation treatment is density graded by the sucrose density gradient method, so that the protein complex and aggregate can be separated, but it is difficult to separate from HDL of the same density. In addition, for this method, since ultracentrifugation separation treatment is required, it is difficult to process multiple specimens at the same time. Moreover, ultracentrifugation separation treatment requires expensive instruments.

[0007] In addition, there is a method in which a commercially available reagent such as ExoQuick (manufactured by System Biosciences) or Total Exosome Isolation Reagent (manufactured by Thermo Fisher Scientific) is added and centrifuged to obtain extracellular membrane vesicles as a precipitate component (Non-Patent Document 1). However, this method has a problem in that the purity of the extracellular membrane vesicles obtained is lower than that of the method using the ultracentrifugation separation treatment.

[0008] In addition to the above-mentioned conventional methods, there is also a method for obtaining extracellular membrane vesicles by using an antibody against the surface antigen protein of extracellular membrane vesicles and the affinity between the surface antigen protein and the antibody (anti-CD63 antibody immobilization method, Exosome-Human CD63 Isolation / Detection (manufactured by Thermo Fisher Scientific) etc.) (Non-patent document 1). Although the above-mentioned method can obtain highly pure extracellular membrane vesicles, there are the following problems: only extracellular membrane vesicles with surface antigen proteins against antibodies can be obtained, the yield of extracellular membrane vesicles is small, surfactants, acidic buffers, etc. are required to dissolve the extracellular membrane vesicles from the antibodies, and it is difficult to obtain intact extracellular membrane vesicles (i.e., in a state where the original function of the extracellular membrane vesicles is maintained).

[0009] As a method for obtaining viruses, the most common method is to perform ultracentrifugation on the sample to obtain the virus as a precipitate component (Non-Patent Document 1). However, for this method, in addition to the virus, the protein complex or aggregate, lipoproteins such as HDL, etc. contained in the sample also produce co-precipitation, making it difficult to obtain a virus with high purity. The precipitate component obtained by the ultracentrifugation separation treatment is density graded by the sucrose density gradient method, so that the protein complex and aggregate can be separated, but it is difficult to separate from the aggregate with equal density. In addition, there is also the problem of decreased activity of the virus due to the ultracentrifugation separation treatment (Non-Patent Document 2). In addition, since the method requires ultracentrifugation separation treatment, it is difficult to process multiple specimens at the same time. Moreover, ultracentrifugation separation treatment requires expensive instruments.

[0010] In addition to the method, it is known that a method for purifying viruses by ion exchange chromatography (non-patent literature 3) is used. However, for this method, it is necessary to set optimum conditions for various viruses. In addition, it is sometimes difficult to set conditions, so that it is difficult to be applied to the purification of all viruses.

[0011] As a method for removing extracellular membrane vesicles, the most common method known is to perform ultracentrifugation separation on a sample, precipitate and fractionate the extracellular membrane vesicles, and obtain the supernatant fraction as the removed sample. However, it is difficult to completely remove extracellular membrane vesicles with this method, and extracellular membrane vesicles that have not been completely removed remain in the sample.

[0012] Furthermore, there is also a method in which a commercially available reagent represented by ExoQuick (System Biosciences) and Total Exosome Isolation Reagent (Thermo Fisher Scientific) is added, and centrifugation is performed to precipitate and classify the extracellular membrane vesicles, and the supernatant fraction is obtained as a removed sample. However, even in this method, the extracellular membrane vesicles cannot be completely removed, and remain in the sample, making it difficult to completely remove the extracellular membrane vesicles. In addition, since the added reagent is mixed into the sample, the reagent sometimes causes problems such as the biological activity of the extracellular membrane vesicles.

[0013] Similar methods are known as methods for removing viruses, but they have the same problems as those of methods for removing extracellular membrane vesicles.

[0014] As a method for detecting extracellular membrane vesicles, a sandwich ELISA using antibodies against surface antigens of extracellular membrane vesicles is known as a conventional method. However, it has been reported that the minimum detection sensitivity of the sandwich ELISA system using antibodies for detecting colorimetric signals is about 3 μg for purified exosomes, and sufficient sensitivity cannot be obtained when measuring body fluid samples such as serum (Non-patent Document 4). As a method for detecting viruses, the same method is also known, and sufficient sensitivity cannot be obtained for the detection of viruses.

[0015] In addition, flow cytometry is known as another method for detecting extracellular membrane vesicles. However, the conventional method of capturing extracellular membrane vesicles using a carrier on which antibodies against surface antigens of extracellular membrane vesicles have been immobilized cannot obtain sufficient sensitivity, and it is difficult to directly detect from samples such as culture supernatants or body fluids, so it is necessary to concentrate or purify extracellular membrane vesicles from the sample before detection.

[0016] Prior art literature

[0017] Non-patent literature

[0018] Non-patent literature 1: Kenneth W. Witwer et al. Journal of Extracellular Vesicles 2013 May 27; 2. doi:10.3402;

[0019] Non-patent document 2: GY Chen et al. Biotechnol. Prog. 25 (2009) 1669;

[0020] Non-patent document 3: Petra Gerster et al. Journal of Chromatography A, 1290 (2013) 36-45;

[0021] Non-patent document 4: Mariantonia Logozzi et al. PLoS ONE 4 (2009) 5219. Summary of the invention

[0022] Problems to be solved by the invention

[0023] As described above, it is difficult to obtain high-purity extracellular membrane vesicles or viruses in an intact state simply and effectively using existing methods for obtaining extracellular membrane vesicles or viruses. In addition, existing methods for removing extracellular membrane vesicles or viruses are difficult to effectively remove extracellular membrane vesicles or viruses from a sample. Furthermore, existing methods for detecting extracellular membrane vesicles or viruses cannot achieve sufficient sensitivity.

[0024] Therefore, an object of the present invention is to simply and effectively obtain or remove extracellular membrane vesicles or viruses present in a sample in a high purity and intact state, and to detect extracellular membrane vesicles or viruses with high sensitivity.

[0025] Technical solutions to problems

[0026] The present invention has been made to solve the above-mentioned problems and is composed of the following technical solutions.

[0027] 1. A vector (Tim vector) to which a protein (Tim protein) selected from the group consisting of a T cell immunoglobulin and mucin domain-containing molecule 4 (Tim4) protein, a T cell immunoglobulin and mucin domain-containing molecule 3 (Tim3) protein, and a T cell immunoglobulin and mucin domain-containing molecule 1 (Tim1) protein is bound.

[0028] 2. A method for obtaining extracellular membrane vesicles or viruses in a sample, characterized in that it comprises the following steps:

[0029] (1) a step of forming a complex between the Tim protein bound to the carrier and the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions (complex formation step);

[0030] (2) a step of separating the complex from the sample (complex separation step);

[0031] (3) A step of separating the extracellular membrane vesicles or the virus from the complex and obtaining the extracellular membrane vesicles or the virus (obtaining step).

[0032] 3. A method for removing extracellular membrane vesicles or viruses in a sample, characterized in that it comprises the following steps:

[0033] (1) a step of forming a complex between the Tim protein bound to the carrier and the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions (complex formation step);

[0034] (2) A step of separating the complex from the sample (complex separation step).

[0035] 4. A method for detecting extracellular membrane vesicles or viruses in a sample, comprising the following steps:

[0036] (1) A step of forming a complex between the Tim protein bound to the carrier and the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions (complex formation step)

[0037] (2) Step of detecting the complex (detection step)

[0038] 5. A cell extracellular membrane vesicle or virus capture kit, wherein it contains a Tim vector.

[0039] 6. A kit for capturing extracellular membrane vesicles or viruses, comprising a reagent containing a Tim protein and a reagent containing a carrier.

[0040] In view of the above situation, the present inventors conducted careful research and found that by using at least one protein selected from Tim1 protein, Tim3 protein and Tim4 protein, extracellular membrane vesicles or viruses having phosphatidylserine on the surface can be obtained with high purity, the extracellular membrane vesicles or viruses can be obtained in a complete state, the extracellular membrane vesicles or viruses can be effectively removed, and the extracellular membrane vesicles or viruses can be detected with high sensitivity, thereby completing the present invention.

[0041] Phosphatidylserine of phospholipids is present (exposed) on the surface of extracellular membrane vesicles. As proteins capable of binding to phosphatidylserine (hereinafter, sometimes referred to as "phosphatidylserine binding proteins" or "PS proteins"), for example, Annexin V (membrane adhesion protein-5), MFG-E8, Tim1 (T-cell immunoglobulin-mucin domain 1), Tim1 (T-cell immunoglobulin-mucin domain 1) protein, Tim3 (T-cell immunoglobulin-mucin domain 3) protein, Tim4 (T-cell immunoglobulin-mucin domain 4) protein, etc. are known (The Journal of Biochemistry 265, 4923-4928 (25 March 1990), Nature 417, 182-187 (9 May 2002), Nature 450,435-439(15 November 2007).

[0042] However, it has been found that, among the PS proteins, when PS proteins other than Tim1 protein, Tim3 protein, and Tim4 protein are used, it is difficult to obtain, remove, or detect extracellular membrane vesicles or viruses.

[0043] Effects of the Invention

[0044] According to the present invention, extracellular membrane vesicles or viruses present in a sample can be easily and efficiently obtained, removed, and detected with high sensitivity in a highly pure and intact state. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is an electrophoretic diagram of whether or not extracellular membrane vesicles were obtained by Western blotting in Example 1-8.

[0046] Figure 2 This is an electrophoretic diagram for confirming whether extracellular membrane vesicles were obtained by Western blotting in Example 9-16.

[0047] Figure 3 This is an electrophoretic diagram for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 17-18.

[0048] Figure 4 -A is an electrophoresis diagram for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 19-20 and Comparative Examples 1-3. Figure 4 -B is an electrophoresis diagram for confirming whether extracellular membrane vesicles were obtained by silver staining in Examples 19-20 and Comparative Examples 1-3.

[0049] Figure 5 These are the extracellular membrane vesicles obtained by the method of the present invention and observed by electron microscopy in Example 21.

[0050] Figure 6 This is an electrophoretic diagram for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 22-25.

[0051] Figure 7 This is an electrophoresis diagram for confirming whether extracellular membrane vesicles were obtained by silver staining in Examples 26-27 and Comparative Examples 4-9.

[0052] Figure 8 This is an electrophoretic diagram for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 28-33.

[0053] Fig. 9 This is an electrophoresis diagram for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 34-35 and Comparative Examples 10-13.

[0054] Fig.10 This is an electrophoresis diagram for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 36-38.

[0055] Fig.11This is an electrophoretic diagram for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 39-40.

[0056] Fig.12 This is an electrophoretic diagram for confirming whether or not extracellular membrane vesicles were obtained (removed) by Western blotting in Examples 41-47.

[0057] Fig.13 This is an electrophoretic diagram for confirming whether or not extracellular membrane vesicles were obtained (removed) by Western blotting in Examples 48-55.

[0058] Fig.14 These are electrophoretic diagrams for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 56 to 67 and Comparative Examples 14 to 15.

[0059] Fig.15 This is an electrophoresis diagram for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 68-79.

[0060] Fig.16 These are electrophoretic diagrams for confirming whether extracellular membrane vesicles were obtained by Western blotting in Examples 80 to 83 and Comparative Examples 16 to 19.

[0061] Fig.17 This is an electrophoresis diagram to confirm whether or not the virus was obtained by Western blotting in Examples 84 to 95 and Comparative Examples 20 to 21.

[0062] Fig.18 This is an electrophoresis diagram to confirm whether or not the virus was obtained by Western blotting in Examples 96 to 99 and Comparative Examples 22 to 25.

[0063] Fig.19 These are the results of detecting extracellular membrane vesicles by ELISA in Examples 100-105 and Comparative Examples 26-33.

[0064] Fig. 20 These are the results of detecting extracellular membrane vesicles by ELISA in Examples 106-109.

[0065] Fig.21 These are the results of virus detection by ELISA in Examples 110-115 and Comparative Examples 34-39.

[0066] Fig. 22 This is an electrophoresis diagram to confirm whether the virus was obtained by Western blotting in Examples 116-119.

[0067] Fig.23These are the results of detecting extracellular membrane vesicles by flow cytometry in Examples 120-121 and Comparative Examples 40-41.

[0068] Fig.24 This is an electrophoresis diagram to confirm whether or not the virus was obtained by Western blotting in Examples 122-123 and Comparative Examples 42-43.

[0069] Fig.25 This is an electrophoresis diagram to confirm whether or not the virus was obtained by Western blotting in Examples 124-125 and Comparative Examples 44-45. DETAILED DESCRIPTION

[0070] <1. Extracellular membrane vesicles of the present invention>

[0071] The extracellular membrane vesicles of the present invention are small membrane vesicles secreted by cells in vivo or cultured cells, composed of a lipid bilayer membrane and having phosphatidylserine on the membrane surface. The diameter of the vesicle is usually 20nm to 1000nm, preferably 50nm to 500nm, and more preferably 50nm to 200nm.

[0072] As for the extracellular membrane vesicles of the present invention, various vesicles classified according to the origin of formation and the size of small membrane vesicles, as described by Naoto Aoki et al. in the subject of Nature Reviews Immunology (9, 581-593 (August 2009)) and Fat Research (Vol. 13, No. 2 2007), can be cited. Specifically, exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, apoptotic vesicles, adiposomes, etc. can be cited.

[0073] Exosomes are small membrane vesicles derived from late endosomes, composed of a lipid bilayer membrane and having phosphatidylserine on the membrane surface. The diameter of the vesicle is usually 50nm to 200nm, preferably 50nm to 150nm, and more preferably 50nm to 100nm. It is well known that exosomes contain proteins such as tetraspanins such as CD63 and CD9, Alix, TSG101, Lamp-1, and Flotillin.

[0074] Microvesicles are small membrane vesicles derived from cell membranes (plasma membranes), composed of lipid bilayer membranes, and having phosphatidylserine on the membrane surface. Microvesicles are usually 100nm to 1000nm, preferably 100nm to 800nm, and more preferably 100nm to 500nm. It is well known that microvesicles contain proteins such as integrins, selectins, and CD40 ligands.

[0075] Ectoplasmic granules are small membrane vesicles derived from cell membranes (plasma membranes), composed of lipid bilayer membranes, and having phosphatidylserine on the membrane surface. Ectoplasmic granules are usually 50 nm to 200 nm, preferably 50 nm to 150 nm, and more preferably 50 nm to 100 nm. Ectoplasmic granules are known to contain CR1 and proteolytic enzymes, but do not contain CD63.

[0076] Membrane particles are small membrane vesicles derived from cell membranes (plasma membranes), composed of lipid bilayer membranes, and having phosphatidylserine on the membrane surface. Membrane particles are usually 50 nm to 80 nm. Membrane particles are known to contain CD133 but not CD63.

[0077] Exosome-like vesicles are small membrane vesicles derived from early endosomes, composed of a lipid bilayer membrane and having phosphatidylserine on the membrane surface. Exosome-like vesicles are usually 20nm to 50nm. Exosome-like vesicles are known to contain TNFRI (Tumor Necrosis Factor Receptor I).

[0078] Apoptotic vesicles are small membrane vesicles derived from apoptotic cells, composed of a lipid bilayer membrane and having phosphatidylserine on the membrane surface. Apoptotic vesicles are generally 50 nm to 500 nm, preferably 50 nm to 300 nm, and more preferably 50 nm to 200 nm. Apoptotic vesicles are known to contain histones.

[0079] Fat bodies are small membrane vesicles derived from adipocytes, composed of a lipid bilayer membrane and having phosphatidylserine on the membrane surface. Fat bodies are generally 100 nm to 1000 nm, preferably 100 nm to 800 nm, and more preferably 100 nm to 500 nm. Fat bodies are known to contain MFG-E8 (milk fat globule-EGF factor 8).

[0080] <2. Virus of the present invention>

[0081] The virus of the present invention is a virus having an envelope (envelop) composed of a lipid bilayer membrane derived from a cell membrane, nuclear membrane, Golgi apparatus, endoplasmic reticulum, etc. of a host cell in the capsid (shell) of the virus, and having phosphatidylserine on the surface of the envelope (hereinafter referred to as "enveloped virus"). The virus of the present invention is usually 20nm to 320nm in diameter. As the enveloped virus of the present invention, there can be cited a virus with an envelope belonging to the family recorded in the 2nd edition of the Biochemistry Dictionary (Tokyo Chemical Dojin, 1990, 1503p-1505p). Specific examples include Poxviridae, Baculoviridae, Rhabdoviridae, Bunyaviridae, Togaviridae, Herpesviridae, Paramyxoviridae, Orthomyxoviridae, Retrovirus, Arenaviridae, and Coronaviridae.

[0082] <3. Tim protein of the present invention>

[0083] The Tim protein of the present invention is at least one Tim protein selected from the group consisting of the T cell immunoglobulin and mucin domain-containing molecule 1 (Tim1) protein of the present invention (hereinafter, sometimes referred to as “the Tim1 protein of the present invention”), the T cell immunoglobulin and mucin domain-containing molecule 3 (Tim3) protein of the present invention (hereinafter, sometimes referred to as “the Tim3 protein of the present invention”), and the T cell immunoglobulin and mucin domain-containing molecule 4 (Tim4) protein of the present invention (hereinafter, sometimes referred to as “the Tim4 protein of the present invention”).

[0084] As the Tim4 (T cell immunoglobulin and mucin domain-containing molecule 4) protein of the present invention, any protein that can bind to the extracellular membrane vesicle or virus of the present invention can be used, and preferably the Tim4 protein is derived from an animal. Among them, the Tim4 protein possessed by humans or mice is preferred (hereinafter, the Tim4 protein possessed by humans is sometimes referred to as "human-derived Tim4 protein", and the Tim4 protein possessed by mice is sometimes referred to as "mouse-derived Tim4 protein").

[0085] More specifically, any protein having at least the amino acid sequence of the binding domain (IgV domain) for phosphatidylserine may be used, and may be a protein having the full-length amino acid sequence of the Tim4 protein or a portion of the Tim4 protein.

[0086] As the amino acid sequence of the binding domain (IgV domain) relative to the phosphatidylserine, for example, sequence number 1 (the 22nd to 135th amino acid domain of the N-terminal of the mouse Tim4 protein (RefSeq NP_848874.3), sequence number 2 (the 25th to 137th amino acid domain of the N-terminal of the human Tim4 protein (RefSeq NP_612388.2), etc. can be cited.

[0087] Examples of the full-length amino acid sequence of the Tim4 protein include sequence number 3 (amino acid domain 1 to 343 of the full-length sequence of mouse Tim4 protein (RefSeq NP_848874.3)), sequence number 4 (amino acid domain 1 to 378 of the full-length sequence of human Tim4 protein (RefSeq NP_612388.2)), and the like.

[0088] As a part of the Tim4 protein, there are exemplified sequences having an amino acid sequence of a binding domain (IgV domain) for phosphatidylserine and a mucin domain, such as SEQ ID NO: 5 (amino acid domain at positions 22 to 273 of the N-terminal of mouse Tim4 protein (RefSeq NP_848874.3)), SEQ ID NO: 6 (amino acid domain at positions 22 to 279 of the N-terminal of mouse Tim4 protein (RefSeq NP_848874.3), and SEQ ID NO: 7 (amino acid domain at positions 25 to 315 of the N-terminal of human Tim4 protein (RefSeq NP_612388.2). If necessary, the sequence may have a signal sequence.

[0089] The Tim1 (T cell immunoglobulin and mucin domain-containing molecule 1) protein of the present invention may be any protein that can bind to the extracellular membrane vesicle or virus of the present invention, and preferably a Tim1 protein derived from an animal. Among them, a Tim1 protein possessed by humans or mice is preferred (hereinafter, Tim1 protein possessed by humans may be referred to as “human-derived Tim1 protein”, and Tim1 protein possessed by mice may be referred to as “mouse-derived Tim1 protein”).

[0090] More specifically, any protein having at least the amino acid sequence of the binding domain (IgV domain) for phosphatidylserine may be used, and may be a protein having the full-length amino acid sequence of Tim1 protein or a portion of Tim1 protein.

[0091] As the amino acid sequence of the binding domain (IgV domain) for phosphatidylserine, for example, sequence number 8 (the 22nd to 131st amino acid domain of the N-terminal of mouse Tim1 protein (RefSeq NP_001160104.1), sequence number 9 (the 21st to 130th amino acid domain of the N-terminal of human Tim1 protein (RefSeq NP_036338.2), etc. can be cited.

[0092] Examples of the Tim1 protein full-length amino acid sequence include SEQ ID NO: 10 (amino acid domain 1 to 282 of the mouse Tim1 protein full-length sequence (RefSeq NP_001160104.1)) and SEQ ID NO: 11 (amino acid domain 1 to 364 of the human Tim1 protein full-length sequence (RefSeq NP_036338.2).

[0093] As a part of the Tim1 protein, there can be mentioned sequences having an amino acid sequence of a binding domain (IgV domain) for phosphatidylserine and a mucin domain, such as SEQ ID NO: 12 (amino acid domain at positions 22 to 212 of the N-terminal of mouse Tim1 protein (RefSeq NP_001160104.1)) and SEQ ID NO: 13 (amino acid domain at positions 21 to 295 of the N-terminal of human Tim1 protein (RefSeq NP_612388.2). If necessary, the sequence may have a signal sequence.

[0094] As the Tim3 (T cell immunoglobulin and mucin domain-containing molecule 3) protein of the present invention, any protein that can bind to the extracellular membrane vesicle or virus of the present invention can be used, and Tim3 protein derived from animals is preferred. Among them, Tim3 protein possessed by humans or mice is preferred (hereinafter, Tim3 protein possessed by humans is sometimes referred to as "human-derived Tim3 protein", and Tim3 protein possessed by mice is sometimes referred to as "mouse-derived Tim3 protein").

[0095] More specifically, any protein having at least the amino acid sequence of the binding domain (IgV domain) for phosphatidylserine may be used, and may be a protein having the full-length amino acid sequence of the Tim3 protein or a portion of the Tim3 protein.

[0096] As the amino acid sequence of the binding domain (IgV domain) for phosphatidylserine, for example, sequence number 14 (the 22nd to 134th amino acid domain of the N-terminal of the mouse Tim3 protein (RefSeq NP_599011.2), sequence number 15 (the 22nd to 135th amino acid domain of the N-terminal of the human Tim3 protein (RefSeq NP_116171.3), etc. can be cited.

[0097] Examples of the full-length amino acid sequence of the Tim3 protein include sequence number 16 (amino acid domain 1 to 281 of the full-length sequence of mouse Tim3 protein (RefSeq NP_599011.2)), sequence number 17 (amino acid domain 1 to 301 of the full-length sequence of human Tim3 protein (RefSeq NP_116171.3), and the like.

[0098] As a part of the Tim3 protein, there are sequences having an amino acid sequence of a binding domain (IgV domain) for phosphatidylserine and a mucin domain, such as SEQ ID NO: 18 (amino acid domain at positions 22 to 189 of the N-terminal of mouse Tim3 protein (RefSeq NP_599011.2)) and SEQ ID NO: 19 (amino acid domain at positions 22 to 200 of the N-terminal of human Tim3 protein (RefSeq NP_116171.3). If necessary, the sequence may have a signal sequence.

[0099] In addition, as long as the Tim protein of the present invention can bind to the extracellular membrane vesicles or viruses of the present invention, it can also be a variant formed by the deletion, substitution, insertion and / or addition of one or more amino acids in the above amino acid sequence.

[0100] The Tim protein of the present invention may be any protein as long as it has the above-mentioned properties, and any protein may be used, such as proteins extracted from cells (e.g., immune cells such as macrophages) and tissues of organisms having Tim proteins, such as animals such as mice and humans, and plants, or proteins prepared by genetic recombination technology based thereon.

[0101] When a protein produced by gene recombination technology is used as the Tim protein of the present invention, a protein having one or more affinity tags is preferred from the viewpoint of ease of purification.

[0102] As the affinity tag, any tag can be used as long as it is an affinity tag used when preparing proteins by genetic recombination technology, and examples thereof include affinity tags such as Fc tag, FLAG tag, His tag, GST tag, MBP tag, HA tag, Myc tag, Strep (II) tag, and PA tag.

[0103] The affinity tag is fused to the C-terminal side of the Tim protein of the present invention.

[0104] Therefore, the Tim protein of the present invention includes not only proteins consisting of the amino acid sequence of the Tim protein (full length or partial sequence) but also proteins having the amino acid sequence of the Tim protein (full length or partial sequence) and the amino acid sequence of the affinity tag as described above.

[0105] In addition, the affinity tag and the Tim protein may be directly bound, or may be bound via a spacer described in "Protein Expression Using a Cell-Free Protein Synthesis Kit Derived from Insect Cultured Cells (Transdirect Insect Cells)" (Eren Tetsu, Suzuki Takashi, Ito Masaaki, Shiho Masamitsu (Shimadzu Corporation / Analytical Measurement Division), published on June 9, 2008, Archives of the Protein Science Society, 1, e005 (2008)). Therefore, the Tim protein of the present invention also includes a protein having the amino acid sequence of the Tim protein (full length or a portion of the sequence), the amino acid sequence of the affinity tag described above, and the amino acid sequence of the spacer.

[0106] <4. Method for producing Tim protein of the present invention>

[0107] The Tim protein of the present invention can be produced by conventional chemical production methods based on its amino acid sequence. For example, the Tim protein of the present invention can be obtained by conventional chemical production methods (chemical synthesis methods) such as the fluorenylmethyloxycarbonyl method (Fmoc method) and the tert-butyloxycarbonyl method (tBoc method). In addition, chemical synthesis can be performed using a commercially available peptide synthesizer.

[0108] Furthermore, the Tim protein of the present invention can also be obtained by the following well-known method using gene recombination technology, that is, combining the nucleic acid molecule encoding the Tim protein of the present invention into an appropriate expression vector such as a plasmid or phage, transforming (or transducing) the host cell with the recombinant expression vector, and amplifying the obtained host cell and secreting it into or outside the cell.

[0109] The method for producing the Tim protein of the present invention will be described below with respect to the case where the Tim protein is produced by genetic recombination technology.

[0110] <Expression vector of the present invention>

[0111] As for the expression vector for expressing the Tim protein of the present invention (hereinafter referred to as the expression vector of the present invention), any vector can be used as long as it contains a nucleic acid sequence encoding the Tim protein of the present invention (hereinafter sometimes referred to as the "Tim coding sequence of the present invention").

[0112] In the Tim coding sequence of the present invention, as the nucleic acid sequence encoding Tim4 protein, there can be cited: for example, sequence number 20 (the base sequence of the cDNA encoding the amino acid domain of positions 1 to 343 of the full-length sequence of mouse Tim4 protein (RefSeq No.NM_178759.4). The terminal 3 bases include a stop codon (tga)), sequence number 21 (the base sequence of the cDNA encoding the amino acid domain of positions 1 to 378 of the full-length sequence of human Tim4 protein (RefSeq No.NM_138379.2). The terminal 3 bases include a stop codon (taa)), etc.

[0113] Examples of nucleic acid sequences encoding the Tim1 protein of the present invention include, for example, sequence number 22 (the base sequence of cDNA encoding the amino acid region from positions 1 to 282 of the full-length sequence of mouse Tim1 protein (RefSeq No. NM_001166632.1). The terminal 3 bases include a stop codon (tga)), sequence number 23 (the base sequence of cDNA encoding the amino acid region from positions 1 to 364 of the full-length sequence of human Tim1 protein (RefSeq No. NM_012206.3). The terminal 3 bases include a stop codon (taa)), etc.

[0114] Examples of nucleic acid sequences encoding the Tim3 protein of the present invention include, for example, sequence number 24 (the base sequence of cDNA encoding the amino acid domain from positions 1 to 281 of the full-length sequence of mouse Tim3 protein (RefSeq No. NM_134250.2). The terminal 3 bases include a stop codon (tga)), sequence number 25 (the base sequence of cDNA encoding the amino acid domain from positions 1 to 301 of the full-length sequence of human Tim3 protein (RefSeq No. NM_032782.4). The terminal 3 bases include a stop codon (tag)), etc.

[0115] As the expression vector of the present invention, the Tim coding sequence of the present invention can be genetically introduced into a commercially available vector according to conventional cloning methods. As the expression vector of the present invention, for example, sequence number 26 (cDNA encoding the N-terminal amino acid domain of mouse Tim4 protein at positions 1 to 273, the terminal 3 bases contain a stop codon (tga)), sequence number 27 (cDNA encoding the N-terminal amino acid domain of mouse Tim4 protein at positions 1 to 279, the terminal 3 bases contain a stop codon (tga)), sequence number 28 (cDNA encoding the N-terminal amino acid domain of human Tim4 protein at positions 1 to 315, the terminal 3 bases contain a stop codon (tga)), sequence number 29 (cDNA encoding the N-terminal amino acid domain of mouse Tim1 protein at positions 1 to 212, the terminal 3 bases contain a stop codon (tga)), sequence number 30 (cDNA encoding the human Tim1 A cDNA encoding the N-terminal amino acid domain of the 1st to 295th amino acid of the protein, the terminal 3 bases contain a stop codon (tga)), sequence number 31 (cDNA encoding the N-terminal amino acid domain of the mouse Tim3 protein, the terminal 3 bases contain a stop codon (tga)), or sequence number 32 (cDNA encoding the N-terminal amino acid domain of the human Tim3 protein, the terminal 3 bases contain a stop codon (tga)) is combined with an expression vector such as the commercially available pCAG-Neo vector (manufactured by Wako Pure Chemical Industries, Ltd.). As a vector for gene introduction into the Tim coding sequence, any vector that has the function of expressing and producing the Tim protein of the present invention in the host cell can be used, and it is convenient to use a commercially available vector. As commercially available vectors for the above purpose, when the host is an animal cell, pCAG-Neo vectors, pcDNA vectors, etc. can be mentioned.

[0116] <Host>

[0117] As a host, any host capable of expressing the Tim protein of the present invention can be used, for example, Escherichia coli, insect cells, mammalian cells, plant cells, yeast cells, etc., preferably mammalian cells. As mammalian cells, for example, HEK293T cells, COS-7 cells, CHO-K1 cells, CHO-S cells, etc. can be mentioned.

[0118] <Gene introduction into host>

[0119] The gene is introduced into the host by the expression vector of the present invention according to the conventional method of introducing a gene into a host described in "Protein Expression Plan Selected by Purpose" (Chapter 3 Protein Expression Plan, ISBN 978-4-7581-0175-2, Yodosha) and the like.

[0120] <Host cultivation>

[0121] The host into which the gene has been introduced is cultured according to a conventional method for culturing the host. The culture conditions vary depending on the host into which the gene has been introduced, but may be cultured according to a conventional method for each host. For example, if the host is an animal cell, the culture may be carried out under a temperature of generally 5 to 10%, preferably 5 to 8% CO. 2 , and is usually cultured at 36°C to 38°C, preferably 36.5°C to 37.5°C for 1 to 10 days, preferably 3 to 4 days. It should be noted that since the Tim protein of the present invention does not contain a transmembrane region and an intracellular region, it is expressed and secreted in the culture supernatant.

[0122] <Purification of Tim protein of the present invention>

[0123] Then, the culture fluid of the gene-introduced host is centrifuged (usually at 200 to 400 × g for 3 to 10 minutes, preferably at 300 × g for 3 to 6 minutes), and the culture supernatant is recovered. If necessary, (i) the recovered culture supernatant is centrifuged usually at 1000 to 2000 × g for 20 to 60 minutes, preferably at 1200 × g for 20 to 40 minutes, and / or (ii) filtered through a filter to separate impurities to obtain a culture supernatant filtrate.

[0124] Furthermore, if necessary, the obtained culture supernatant filtrate can be concentrated usually 5 to 20 times, preferably 8 to 12 times by a conventional method such as ultrafiltration to obtain a concentrated culture supernatant filtrate.

[0125] Then, when the Tim protein of the present invention has an affinity tag, the Tim protein of the present invention (a fusion protein of the Tim protein and an affinity tag) can be purified from the obtained culture supernatant, the obtained culture supernatant filtrate, or the obtained concentrate of the culture supernatant filtrate according to conventional methods for purifying proteins using affinity tags corresponding to each affinity tag described in "Protocol for Protein Expression Selected by Purpose" (Chapter 3 Protein Expression Protocol, Section 6 Protein Purification, Purification Using Tags, ISBN 978-4-7581-0175-2, Yodosha) and the like (for example, a method using a carrier on which a substance having an affinity for the affinity tag has been immobilized).

[0126] When the Tim protein of the present invention does not have an affinity tag, the Tim protein of the present invention can be purified from the obtained culture supernatant, the obtained culture supernatant filtrate, or the obtained culture supernatant filtrate concentrate by various chromatography according to conventional methods for protein purification described in "Protein Expression Protocol Selected by Purpose" (Chapter 3 Protein Expression Protocol, Section 6 Protein Purification, Purification by Chromatography, ISBN 978-4-7581-0175-2, Yodosha).

[0127] The purification can be carried out by appropriately combining the above purification methods.

[0128] <Specific method for producing Tim protein of the present invention>

[0129] As a specific method for preparing the Tim protein of the present invention, for example, when an Fc tag is used as an affinity tag, the following method can be cited. First, according to a conventional method, the Tim coding sequence of the present invention is combined with a pEF-Fc vector or a commercially available vector to construct an expression vector of the present invention. Then, according to a conventional method, the gene of the present invention is introduced into the expression vector of the present invention in a host cell at a concentration of usually 5 to 10%, preferably 5 to 8% CO. 2The culture medium is then centrifuged (usually at 200 to 400 × g for 3 minutes to 10 minutes, preferably at 300 × g for 3 minutes to 6 minutes) to recover the culture supernatant. If necessary, the recovered culture supernatant may be (i) centrifuged at 1000 to 2000 × g for 20 minutes to 60 minutes, preferably at 1200 × g for 20 minutes to 40 minutes, and / or (ii) filtered using a filter to separate impurities to obtain a culture supernatant filtrate. Further, if necessary, the obtained culture supernatant filtrate may be concentrated by conventional methods such as ultrafiltration to a concentration of usually 5 to 20 times, preferably 8 to 12 times, to obtain a concentrated culture supernatant filtrate. Then, when the Tim protein of the present invention has an affinity tag, the Tim protein of the present invention is purified from the obtained culture supernatant, the obtained culture supernatant filtrate, or the obtained culture supernatant filtrate concentrate according to a conventional purification method using an affinity tag corresponding to each affinity tag, thereby obtaining the Tim protein of the present invention.

[0130] <5. Tim vector of the present invention>

[0131] The Tim protein-bound carrier of the present invention (hereinafter, sometimes simply referred to as "the Tim carrier of the present invention") is a carrier in which the Tim protein of the present invention as described above is bound to the carrier of the present invention.

[0132] Specifically, examples of the Tim vector of the present invention include a vector to which the Tim1 protein of the present invention is bound (hereinafter, sometimes referred to as “the Tim1 vector of the present invention”), a vector to which the Tim3 protein of the present invention is bound (hereinafter, sometimes referred to as “the Tim3 vector of the present invention”), a vector to which the Tim4 protein of the present invention is bound (hereinafter, sometimes referred to as “the Tim4 vector of the present invention”), etc. In addition, a vector to which two or more proteins selected from the group consisting of the Tim1 protein of the present invention, the Tim3 protein of the present invention, and the Tim4 protein of the present invention are bound is also included in the Tim vector of the present invention.

[0133] As the Tim vector of the present invention, the Tim4 vector of the present invention is particularly preferred.

[0134] <Carrier of the present invention>

[0135] As the carrier of the present invention, any carrier can be used as long as it is an insoluble carrier used in conventional immunoassays, for example, organic substances such as polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, ethylene-maleic anhydride copolymer; inorganic substances such as glass, silicon oxide, diatomaceous earth, porous glass, frosted glass, aluminum oxide, silica gel, metal oxide; magnetic substances such as iron, cobalt, nickel, magnetite, chromite; and substances prepared from alloys of the magnetic substances. In addition, the carrier can be used in various forms such as microplates, tubes, discs, particles (beads), etc.

[0136] It should be noted that when used in the acquisition method of the present invention and the removal method of the present invention described later, it is preferably used in the form of particles (beads). There is no particular limitation on the size of the particles, but depending on the purpose and use, particles of usually 10 nm to 100 μm, preferably 100 nm to 10 μm can be cited.

[0137] In addition, when used for the detection method of the present invention described later, particles (beads) or microplates are preferred. There is no particular limitation on the size of the particles, but depending on the purpose and use, particles generally having a size of 10 nm to 100 μm, preferably 100 nm to 10 μm, can be cited. In addition, there is no particular limitation on the number and size of the holes of the microplate, but depending on the purpose and use, microplates generally having 12 to 1536 holes, preferably 96 to 384 holes can be cited.

[0138] <Method for binding the Tim protein of the present invention to the carrier of the present invention>

[0139] The method for binding the Tim protein of the present invention to the carrier of the present invention can be carried out according to a method known per se for binding a protein to a carrier, and examples thereof include: for example, a method for binding by affinity binding; a method for binding by chemical binding (for example, the method described in Japanese Patent No. 3269554 and WO2012 / 039395); a method for binding by physical adsorption (for example, the method described in Japanese Patent Publication No. 5-41946), etc., but the method for binding by affinity binding and the method for binding by physical adsorption are preferred.

[0140] It should be noted that when used in the acquisition method and removal method of the present invention described below, a method of binding by affinity binding is preferred. In addition, when used in the detection method of the present invention described below, a method of binding by affinity binding or physical adsorption is preferred.

[0141] <Methods for binding the Tim protein of the present invention to the carrier of the present invention>

[0142] As for the binding mode of the Tim protein of the present invention and the carrier of the present invention, there is no limitation as long as the carrier of the present invention binds to the Tim protein of the present invention, but the binding mode of the carrier of the present invention and the thiol group (SH group) of the Tim protein of the present invention is preferred. In addition, the Tim protein of the present invention may be directly bound to the carrier of the present invention, or indirectly bound via a chemical linker, an affinity substance [for example, a substance having an affinity for an affinity tag (described later), an antibody against the Tim protein of the present invention, biotin (described later), avidin (described later), an antibody, etc.], etc.

[0143] <Binding method by affinity binding>

[0144] As the method for binding by affinity binding, any method can be used as long as it is a method for binding by utilizing affinity binding (affinity) between substances, and examples thereof include the following (a) to (c).

[0145] (a) Binding method using affinity binding of biotin and avidin

[0146] For example, by using two or more substances (affinity substances) having affinity (affinity) for each other, such as a combination of biotin (biotin, iminobiotin, desthiobiotin, biocytin, biotin sulfoxide, etc.) and avidin (avidin, neutravidin (Tamavidin), neutravidin 2 (Tamavidin 2), streptavidin, etc.), the Tim protein of the present invention can be bound to the carrier of the present invention via the affinity substance.

[0147] It should be noted that any one of the affinity substances can be bound to the Tim protein of the present invention in advance, and the remaining one can be bound to the carrier of the present invention. However, for example, when biotin and avidin are used, usually, avidin is bound to the carrier of the present invention in advance, and biotin is bound to the Tim protein of the present invention.

[0148] (b) Binding method by affinity binding between an affinity tag and a substance having affinity for the affinity tag

[0149] For example, by using a substance (affinity substance) having affinity for the Tim protein of the present invention, such as a substance having affinity for an affinity tag (Protein A, Protein G, etc.), the Tim protein of the present invention can be bound to the carrier of the present invention via the affinity substance.

[0150] It should be noted that the affinity substance is usually bound to the carrier of the present invention in advance.

[0151] (c) Method of binding by affinity binding of an antibody against the Tim protein of the present invention to the Tim protein of the present invention

[0152] For example, by using antibodies against the Tim protein of the present invention (anti-FLAG tag antibody, anti-His tag antibody, anti-HA tag antibody, anti-Myc tag antibody, anti-MBP tag antibody, anti-GST tag antibody, anti-Strep (II) tag antibody, etc., and anti-TIM4 antibody (Anti-TIM4 Antibody) (clone RMT4-54) (manufactured by LifeSpan Biosciences)), etc.) that have affinity (affinity) for the Tim protein of the present invention (affinity substance), the Tim protein of the present invention can be bound to the carrier of the present invention through the affinity substance.

[0153] It should be noted that the affinity substance is usually bound to the carrier of the present invention in advance.

[0154] It should be noted that in the above method, a method of binding the Tim protein of the present invention or / and the carrier of the present invention to an affinity substance, a method of binding them by a physical adsorption method known per se, or a method of binding them by a chemical binding method can be used. The binding can be direct or indirect via a linker or the like.

[0155] <Amount of the Tim protein of the present invention bound to the carrier of the present invention>

[0156] The amount of the Tim protein of the present invention bound to the carrier of the present invention, for example, when the carrier of the present invention is beads, is generally 0.1 to 50 μg, preferably 0.5 to 30 μg, and more preferably 1.0 to 20 μg per 1 mg of the carrier.

[0157] When the carrier of the present invention is a microplate, the amount of the protein per well is usually 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg.

[0158] <Specific method for preparing the Tim carrier of the present invention>

[0159] Hereinafter, the specific method for preparing the Tim vector of the present invention will be described by taking the case where the methods (a) to (c) are used to prepare the Tim vector of the present invention as an example.

[0160] <(a) Binding method using affinity binding of biotin and avidin>

[0161] First, in method (a), the Tim protein of the present invention is prepared according to the method for preparing the Tim protein of the present invention. Then, the Tim protein of the present invention is bound to biotin (hereinafter, sometimes referred to as "biotin labeling" or "biotinylation") to form a Tim protein-biotin complex of the present invention. On the other hand, avidins are bound to the carrier of the present invention to form a carrier-avidin complex of the present invention (hereinafter, sometimes referred to as "the carrier of the present invention bound with avidins"). The obtained Tim protein-biotin complex of the present invention is brought into contact with the carrier-avidin complex of the present invention, so that the biotin in the Tim protein-biotin complex of the present invention is bound to the avidin in the carrier-avidin complex, thereby obtaining the Tim carrier of the present invention.

[0162] - Binding of the Tim protein of the present invention to biotin (biotin labeling) -

[0163] In the method (a), the binding of the Tim protein of the present invention to biotin can be carried out using a commercially available protein biotin labeling kit, or by appropriately adjusting the required reagents and performing the binding according to a conventional method for protein biotin labeling. As a method using a commercially available protein biotin labeling kit (biotinylation kit), the method can be carried out according to the method provided in the Biotin Labeling Kit-Sulphydryl (Biotin Labeling Kit-SH) (Co., Ltd., Dojin Scientific Laboratory) or the Biotin Labeling Kit-Amino (Biotin Labeling Kit-NH 2 The method was carried out according to the protocol of ) (Dojin Scientific Laboratory Co., Ltd.).

[0164] The amount of biotin to be bound to 1 μg of the Tim protein of the present invention is usually 10 ng to 1.0 μg, preferably 20 ng to 200 ng, and more preferably 30 ng to 150 ng.

[0165] As a site for binding biotin to the Tim protein of the present invention, a thiol group of the Tim protein of the present invention is preferred.

[0166] -The carrier-avidin complex of the present invention (the carrier of the present invention bound with avidins)-

[0167] In the method (a) described above, the carrier-avidin complex of the present invention (the carrier of the present invention bound to avidins) can use commercially available complexes, or can be prepared by appropriately adjusting the required reagents according to conventional methods. As the carrier-avidin complex of the present invention, for example, beads or microplates bound to avidin, beads or microplates bound to neutravidin (Tamavidin), beads or microplates bound to neutravidin 2 (Tamavidin 2), beads or microplates bound to streptavidin (streptavidin), etc. can be cited. As commercially available complexes, Dynabeads M-270 streptavidin C1 beads (Dynabeads M-270streptavidin C1) (trade name, manufactured by Thermo Fisher Scientific), streptavidin FG beads (FGビーズ)ストレプトアビジン) (manufactured by Tamagawa Seiki Co., Ltd.), affinity plain board (アビジンプレート) (manufactured by Sumitomo Bakelite Co., Ltd. (Sumitomo Bakelite Co., Ltd.)), etc.

[0168] For the binding of the carrier of the present invention and avidins, for example, when the carrier of the present invention is a bead, the amount of avidins in contact with 1 mg of the carrier of the present invention is usually 5.0 to 150 μg, preferably 10 to 100 μg, and more preferably 20 to 50 μg. For example, when the carrier of the present invention is a microplate, the amount of avidins in contact with one well is usually 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg.

[0169] - Binding of the carrier-avidin complex of the present invention to the Tim protein-biotin complex of the present invention

[0170] In the method (a), in order to obtain the Tim carrier of the present invention (to bind the carrier-avidin complex of the present invention to the Tim protein-biotin complex of the present invention), for example, when the carrier of the present invention is a bead, the carrier-avidin complex of the present invention, which is usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, and more preferably 0.5 to 3.0 mg, can be brought into contact with the Tim protein-biotin complex of the present invention, which is usually 1.0 to 50 μg, preferably 1.0 to 30 μg, and more preferably 1.0 to 20 μg, relative to 1 mg of the carrier-avidin complex of the present invention; or when the carrier of the present invention is a microplate, the carrier-avidin complex of the present invention in each well can be brought into contact with the Tim protein-biotin complex of the present invention, which is usually 1.0 to 20 μg, relative to 1 mg of the carrier-avidin complex of the present invention. The carrier-avidin complex of the present invention is contacted with 10 μg, preferably 1.0 to 5.0 μg, and more preferably 1.0 to 2.0 μg of the Tim protein-biotin complex of the present invention, and the reaction is carried out under the conditions of usually 4.0°C to 37°C, preferably 11°C to 30°C, and more preferably 20°C to 25°C for usually 0.5 to 24 hours, preferably 0.5 to 8.0 hours, and more preferably 0.5 to 2.0 hours to bind avidin and biotin. Thus, the carrier-avidin complex of the present invention is bound to the Tim protein-biotin complex of the present invention to obtain the Tim carrier of the present invention.

[0171] In general, the carrier-avidin complex of the present invention can be brought into contact with the Tim protein-biotin complex of the present invention by bringing a solution containing the Tim protein-biotin complex of the present invention into contact with the carrier-avidin complex of the present invention.

[0172] As a solution containing the Tim protein-biotin complex of the present invention, any solution can be used as long as the Tim protein-biotin complex of the present invention is dissolved in a stable state, and examples thereof include purified water, for example, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (for example, PBS, TBS, HBS, etc.). In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5.0 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. In addition, in the solution, as long as it is an amount that does not hinder the binding of the carrier-avidin complex of the present invention to the Tim protein-biotin complex of the present invention after the carrier-avidin complex of the present invention is brought into contact with the solution containing the Tim protein-biotin complex of the present invention, salts such as sugars, NaCl, surfactants, preservatives, proteins, etc. can be contained. As a surfactant, for example, Tween 20 can be mentioned, and the concentration of the surfactant in the solution containing the Tim protein-biotin complex of the present invention is usually 0.00001 to 0.2%, preferably 0.0005 to 0.1%. It should be noted that the solution obtained by dissolving the Tim protein-biotin complex of the present invention in the solvent is sometimes referred to as "a solution containing the Tim protein-biotin complex of the present invention".

[0173] -Specific preparation method of the Tim carrier of the present invention based on the method (a)-

[0174] The specific method for preparing the Tim carrier of the present invention in the method (a) can be carried out, for example, by the following method.

[0175] First, the Tim protein of the present invention is prepared according to the method for preparing the Tim protein of the present invention. Then, the Tim protein of the present invention is prepared according to the method for preparing the Tim protein of the present invention. 2 The Tim protein of the present invention is bound to biotin according to the protocol of ) (Dojin Scientific Laboratory Co., Ltd.) or a conventional method for biotin labeling of proteins, thereby forming a Tim protein-biotin complex of the present invention.

[0176] Then, when the carrier of the present invention is a bead, usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 to 3.0 mg of the carrier-avidin complex of the present invention is mixed with usually 50 μL to 1500 μL, preferably 100 μL to 1000 μL, more preferably 200 μL to 500 μL of a liquid containing usually 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg of the carrier-avidin complex of the present invention relative to 1 mg. The carrier of the present invention is brought into contact with a solution of the Tim protein-biotin complex of the present invention (for example, a solution containing the Tim protein-biotin complex of the present invention in purified water or a buffer solution having a buffering effect under the condition of pH 7.0 to 8.0, preferably 7.2 to 7.6), and the reaction is carried out at a temperature of usually 4.0° C. to 37° C., preferably 11° C. to 30° C., and more preferably 20° C. to 25° C., for usually 0.5 hour to 24 hours, preferably 0.5 hour to 8.0 hours, and more preferably 0.5 hour to 2.0 hours, so that the avidin in the carrier-avidin complex of the present invention is bound to the biotin in the Tim protein-biotin complex of the present invention, thereby obtaining the Tim carrier of the present invention.

[0177] When the carrier of the present invention is a microtiter plate, the carrier-avidin complex of the present invention in each well is mixed with usually 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 100 μL to 200 μL of a solution containing usually 1.0 to 10 μg, preferably 1.0 to 5.0 μg, more preferably 1.0 to 2.0 μg of the Tim protein-biotin complex of the present invention (for example, a buffer having a buffering effect in pure water or at pH 7.0 to 8.0, preferably 7.2 to 7.6). The Tim carrier of the present invention is brought into contact with a solution containing the Tim protein-biotin complex of the present invention, and the reaction is carried out at a temperature of usually 4.0°C to 37°C, preferably 11°C to 30°C, and more preferably 20°C to 25°C for usually 0.5 hour to 24 hours, preferably 0.5 hour to 8.0 hours, and more preferably 0.5 hour to 2.0 hours, so that the avidin in the carrier-avidin complex of the present invention is bound to the biotin in the Tim protein-biotin complex of the present invention, thereby obtaining the Tim carrier of the present invention.

[0178] <(b) Method of binding by affinity binding between an affinity tag and a substance having affinity for the affinity tag>

[0179] In the method (b), first, the Tim protein of the present invention having an affinity tag is prepared according to the method for preparing the Tim protein of the present invention. In addition, a substance having an affinity for the affinity tag (affinity substance) is bound to the carrier of the present invention to form the carrier-affinity substance complex of the present invention (hereinafter, sometimes simply referred to as "the carrier of the present invention bound with an affinity substance"). The obtained carrier-affinity substance complex of the present invention is brought into contact with the Tim protein of the present invention having an affinity tag, the affinity substance in the carrier-affinity substance complex of the present invention is bound to the affinity tag in the Tim protein of the present invention having an affinity tag, and the carrier-affinity substance complex of the present invention is bound to the Tim protein of the present invention having an affinity tag, thereby obtaining the Tim carrier of the present invention.

[0180] -Carrier-affinity substance complex of the present invention-

[0181] In the method (b) above, as the carrier-affinity substance complex of the present invention, commercially available products may be used, or the complex may be prepared according to a conventional method by appropriately adjusting the necessary reagents. Examples of the carrier-affinity substance complex of the present invention include beads or microplates bound to protein G, beads or microplates bound to protein A, and the like. Examples of commercially available products include Dynabeads Protein G (manufactured by Thermo Fisher Scientific), Dynabeads Protein A (manufactured by Thermo Fisher Scientific), FG beads Protein G (manufactured by Tamagawa Seiki Co., Ltd.), and FG beads Protein A (manufactured by Tamagawa Seiki Co., Ltd.).

[0182] Regarding the binding of the carrier of the present invention and the affinity substance, the amount of the affinity substance in contact with 1 mg of the carrier of the present invention is usually 5.0 to 50 μg, preferably 10 to 50 μg, and more preferably 20 to 50 μg when the carrier of the present invention is a bead. When the carrier of the present invention is a microplate, the amount of the affinity substance in contact with one well is usually 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg.

[0183] - Binding of the carrier-affinity substance complex of the present invention to the Tim protein of the present invention having an affinity tag-

[0184] In the method (b) described above, in order to obtain the Tim carrier of the present invention (through the binding of the carrier-affinity substance complex of the present invention to the Tim protein of the present invention having an affinity tag), the following method can be used, for example. When the carrier of the present invention is a bead, usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 to 3.0 mg of the carrier-affinity substance complex of the present invention is contacted with usually 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg of the Tim protein of the present invention having an affinity tag relative to 1 mg of the carrier-affinity substance complex of the present invention. When the carrier of the present invention is a microplate, the carrier-affinity substance complex of the present invention in each well is contacted with usually 1.0 to 10 μg, preferably 1.0 to 5.0 μg, more preferably 1.0 to 2.0 μg of the Tim protein of the present invention having an affinity tag, at a temperature of usually 4.0° C. to 37° C., preferably 11° C. to 30° C., more preferably 20° C. to 25° C., for usually 0.5 hour to 24 hours, preferably 0.5 hour to 8.0 hours, more preferably 0.5 hour to After 2.0 hours of reaction, the carrier-affinity substance complex of the present invention is bound to the Tim protein of the present invention having an affinity tag, thereby obtaining the Tim carrier of the present invention.

[0185] It should be noted that, usually, in the method (b), the contact between the carrier-affinity substance complex of the present invention and the Tim protein of the present invention having an affinity tag is carried out by contacting the carrier-affinity substance complex of the present invention with a solution containing the Tim protein of the present invention having an affinity tag.

[0186] As a solution containing the Tim protein of the present invention having an affinity tag, any solution can be used as long as it dissolves the Tim protein of the present invention having an affinity tag in a stable state, and examples thereof include purified water, for example, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (e.g., PBS, TBS, HBS, etc.). In addition, the concentration of the buffer in the buffer is usually appropriately selected from the range of 5.0 to 50 mM, preferably from the range of 10 to 30 mM, and the concentration of NaCl is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM.

[0187] In addition, the solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc., as long as the amount does not hinder the binding of the Tim protein of the present invention with the affinity tag to the carrier-affinity substance complex of the present invention after the solution containing the Tim protein of the present invention with the affinity tag is brought into contact with the carrier-affinity substance complex of the present invention. As the surfactant, for example, Tween 20 can be cited, and the concentration of the surfactant in the solution containing the Tim protein of the present invention with the affinity tag is usually 0.00001 to 0.2%, preferably 0.0005 to 0.1%. It should be noted that the solution obtained by dissolving the Tim protein of the present invention with the affinity tag in the solution is sometimes referred to as "a solution containing the Tim protein of the present invention with the affinity tag".

[0188] -Specific preparation method of the Tim carrier of the present invention based on the method (b)-

[0189] In the method (b), the specific preparation method of the Tim carrier of the present invention can be carried out by, for example, the following method.

[0190] First, according to the method for preparing the Tim protein of the present invention, the Tim protein of the present invention having an affinity tag is prepared.

[0191] Then, when the carrier of the present invention is a bead, usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 to 3.0 mg of the carrier-affinity substance complex of the present invention is contacted with usually 50 μL to 1500 μL, preferably 100 μL to 1000 μL, more preferably 200 μL to 500 μL of a solution containing usually 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg of the Tim protein of the present invention having an affinity tag per mg of the carrier-affinity substance complex of the present invention (for example, a solution containing the Tim protein of the present invention having an affinity tag in purified water, a buffer solution of pH 7.0 to 8.0, etc.), and the reaction is carried out at a temperature of usually 4.0° C. to 37° C., preferably 11° C. to 30° C., more preferably 20° C. to 25° C., for usually 0.5 hour to 24 hours, preferably 0.5 hour to 8.0 hours. The Tim carrier of the present invention is obtained by reacting the affinity substance in the carrier-affinity substance complex of the present invention and the affinity tag in the Tim protein of the present invention having an affinity tag for 0.5 to 2.0 hours, more preferably 0.5 to 2.0 hours.

[0192] When the carrier of the present invention is a microtiter plate, the carrier-affinity substance complex of the present invention is contacted with usually 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 100 μL to 200 μL of a solution containing 1.0 to 10 μg, preferably 1.0 to 5.0 μg, more preferably 1.0 to 2.0 μg of the Tim protein of the present invention having an affinity tag (for example, a solution containing the Tim protein-biotin complex of the present invention in purified water or a buffer having a buffering effect at pH 7.0 to 8.0, preferably 7.2 to 7.6) per well at a temperature of usually 4.0° C. to 37° C., preferably 11° C. to 30° C., more preferably 20° C. to 25° C., for usually 0.5 hour to 24 hours. The reaction is preferably carried out for 0.5 to 8.0 hours, more preferably for 0.5 to 2.0 hours, to allow the affinity substance in the carrier-affinity substance complex of the present invention to bind to the affinity tag in the Tim protein of the present invention having an affinity tag, thereby obtaining the Tim carrier of the present invention.

[0193] <(c) Method for binding by affinity binding of an antibody against the Tim protein of the present invention to the Tim protein of the present invention>

[0194] First, the Tim protein of the present invention is prepared according to the method for preparing the Tim protein of the present invention. On the other hand, an antibody against the Tim protein of the present invention (hereinafter, sometimes referred to as "anti-Tim antibody") is bound to the carrier of the present invention to form the carrier-anti-Tim antibody complex of the present invention. The obtained carrier-anti-Tim antibody complex of the present invention is brought into contact with the Tim protein of the present invention, and the antibody against the Tim protein in the carrier-anti-Tim antibody complex of the present invention is bound to the Tim protein of the present invention, thereby obtaining the Tim carrier of the present invention.

[0195] It should be noted that, in the above, when a Tim protein with an affinity tag (a protein having the amino acid sequence of a Tim protein and the amino acid sequence of an affinity tag) is used as the Tim protein of the present invention, an antibody that recognizes the Tim protein (an antibody that recognizes a protein portion based on the amino acid sequence of the Tim protein) or an antibody that recognizes an affinity tag (an antibody that recognizes a protein portion based on the amino acid sequence of the affinity tag) may be used as the anti-Tim antibody. In addition, when a Tim protein without an affinity tag (a protein consisting only of the amino acid sequence of the Tim protein) is used as the Tim protein of the present invention, an antibody that recognizes the Tim protein (an antibody that recognizes a protein portion based on the amino acid sequence of the Tim protein) may be used as the anti-Tim antibody.

[0196] -The vector-anti-Tim antibody complex of the present invention (the vector of the present invention bound with an antibody)-

[0197] In the method (c), as the carrier-anti-Tim antibody complex of the present invention, commercially available products can be used, or the required reagents can be appropriately adjusted and prepared according to conventional methods. As the carrier-anti-Tim antibody complex of the present invention, for example, beads or microplates bound to anti-Fc tag antibodies, beads or microplates bound to anti-FLAG tag antibodies, beads or microplates bound to anti-His tag antibodies, beads or microplates bound to anti-GST tags, beads or microplates bound to anti-MBP tags, beads or microplates bound to anti-HA tags, beads or microplates bound to anti-Myc tags, beads or microplates bound to anti-Strep (II) tags, beads or microplates bound to antibodies against anti-Tim proteins, etc., as commercially available products, anti-DYKDDDDK tag antibody magnetic beads (manufactured by Wako Pure Chemical Industries, Ltd.) and the like can be cited.

[0198] The source of the anti-Tim antibody is not particularly limited. Moreover, it can be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred. In addition, the anti-Tim antibody can use commercially available antibodies, or can be prepared according to conventional methods by appropriately adjusting the required reagents. Specific examples of anti-Tim antibodies are as described above.

[0199] For the binding of the carrier of the present invention and the anti-Tim antibody, when the carrier of the present invention is a bead, the amount of the antibody against the Tim protein of the present invention in contact with 1 mg of the carrier of the present invention is usually 5.0 to 50 μg, preferably 10 to 50 μg, and more preferably 20 to 50 μg when used in the acquisition method of the present invention and the removal method of the present invention. When the carrier of the present invention is a microplate, the amount per well is usually 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg.

[0200] - Binding of the carrier-anti-Tim antibody complex of the present invention to the Tim protein of the present invention -

[0201] In the method (c) described above, examples of methods for obtaining the Tim vector of the present invention by binding the vector-anti-Tim antibody complex of the present invention to the Tim protein of the present invention include the following methods. That is, when the carrier of the present invention is a bead, usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 to 3.0 mg of the carrier-anti-Tim antibody complex of the present invention is contacted with usually 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg of the Tim protein of the present invention relative to 1 mg of the carrier-anti-Tim antibody complex of the present invention. When the carrier of the present invention is a microplate, the carrier-anti-Tim antibody complex of the present invention per well is contacted with usually 1.0 to 10 μg, preferably 1.0 to 5.0 μg, more preferably 1.0 to 2.0 μg of the Tim protein of the present invention, and the incubation is performed at a temperature of usually 4.0° C. to 37° C., preferably 11° C. to 30° C., more preferably 20° C. to 25° C., for usually 0.5 hour to 24 hours. The reaction is preferably carried out for 0.5 to 8.0 hours, more preferably for 0.5 to 2.0 hours, to allow the anti-Tim antibody in the vector-anti-Tim antibody complex of the present invention to bind to the Tim protein of the present invention, thereby obtaining the Tim4 vector of the present invention.

[0202] It should be noted that, usually, the contact between the carrier-anti-Tim antibody complex of the present invention and the Tim protein of the present invention in the method (c) is carried out by contacting the carrier-anti-Tim antibody complex of the present invention with a solution containing the Tim protein of the present invention.

[0203] As a solution containing the Tim protein of the present invention, any solution can be used as long as the Tim protein of the present invention is dissolved in a stable state, and examples thereof include purified water, for example, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (e.g., PBS, TBS, HBS, etc.). In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. In addition, in the solution, as long as it is an amount that does not hinder the binding of the Tim protein of the present invention to the carrier-anti-Tim antibody complex of the present invention after the solution containing the Tim protein of the present invention is contacted with the carrier-anti-Tim antibody complex of the present invention, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc. can be contained. Examples of surfactants include Tween 20, etc. The concentration of the surfactant in the solution containing the Tim protein of the present invention is usually 0.00001 to 0.2%, preferably 0.0005 to 0.1%. It should be noted that the solution in which the Tim protein of the present invention is dissolved may be referred to as "a solution containing the Tim protein of the present invention".

[0204] -Specific method for preparing the Tim carrier of the present invention using the method (c)-

[0205] The specific method for preparing the Tim carrier of the present invention in the method (c) can be carried out, for example, by the following method.

[0206] First, the Tim protein of the present invention is prepared according to the method for preparing the Tim protein of the present invention. Then, when the carrier of the present invention is a bead, usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 to 3.0 mg of the carrier-anti-Tim antibody complex of the present invention is brought into contact with usually 100 to 1000 μL, preferably 200 to 500 μL of a solution containing usually 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg of the Tim protein of the present invention relative to 1 mg of the carrier-anti-Tim antibody complex of the present invention (a solution containing the Tim protein of the present invention in, for example, purified water, a buffer solution of, for example, pH 7.0 to 8.0) and the mixture is stirred at a temperature of usually 4° C. to 37° C., preferably 11° C. to 30° C., more preferably 20° C. to 25° C., for usually 0.5 to 24 hours, preferably 0.5 to 3 hours. The Tim vector of the present invention is obtained by binding the antibody against the Tim protein of the present invention in the vector-anti-Tim antibody complex of the present invention to the Tim protein of the present invention through a reaction of 0.5 to 2.0 hours, more preferably 0.5 to 2.0 hours. When the carrier of the present invention is a microtiter plate, the carrier-anti-Tim antibody complex of the present invention is contacted with a solution containing 1.0 to 10 μg, preferably 1.0 to 5.0 μg, more preferably 1.0 to 2.0 μg of the Tim protein of the present invention (a solution containing the Tim protein-biotin complex of the present invention in, for example, purified water or a buffer having a buffering effect at pH 7.0 to 8.0, preferably 7.2 to 7.6) per well at a temperature of usually 4.0° C. to 37° C., preferably 11° C. to 30° C., more preferably 20° C. to 25° C., for usually 0.5 hour to 24 hours, preferably 0.5 hour to 8.0 hours. The Tim carrier of the present invention is obtained by reacting the affinity substance in the carrier-affinity substance complex of the present invention and the affinity tag in the Tim protein of the present invention having an affinity tag for 0.5 to 2.0 hours, more preferably for 0.5 to 2.0 hours.

[0207] <Binding method by physical adsorption>

[0208] As a method for binding the Tim protein of the present invention to the carrier of the present invention by physical adsorption, the Tim protein of the present invention and the carrier of the present invention can be brought into contact under conditions where the Tim protein of the present invention and the carrier of the present invention bind according to a method known per se.

[0209] The reaction temperature of the Tim protein of the present invention and the carrier of the present invention is usually 2°C to 37°C, preferably 4°C to 11°C.

[0210] The reaction time of the Tim protein of the present invention and the carrier of the present invention is usually 4 to 48 hours, preferably 12 to 24 hours.

[0211] As the Tim protein of the present invention, when the carrier of the present invention is a bead, the amount of the Tim protein of the present invention in contact with 1 mg of the carrier of the present invention is usually 5.0 to 50 μg, preferably 10 to 50 μg, and more preferably 20 to 50 μg. When the carrier of the present invention is a microplate, the amount of the Tim protein of the present invention in contact with one well is usually 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg.

[0212] It should be noted that, generally, the physical adsorption of the Tim protein of the present invention and the carrier of the present invention is performed by bringing a solution containing the Tim protein of the present invention into contact with the carrier of the present invention.

[0213] As a solution containing the Tim protein of the present invention, any solution can be used as long as the Tim protein of the present invention is dissolved in a stable state, and examples thereof include purified water, buffers having a buffering effect under conditions of, for example, pH 6.0 to 9.5, preferably 7.0 to 8.0 (e.g., zwitterionic buffers (Good's buffer) such as MOPS (3-(N-morpholino)propanesulfonic acid), carbonate buffer, PBS, TBS, HBS, etc.). In addition, the concentration of the buffer in the buffer is usually appropriately selected from the range of 5 to 100 mM, preferably from the range of 10 to 50 mM, and the concentration when NaCl is added is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. In addition, in the solution, salts such as sugars, NaCl, surfactants, preservatives, proteins, etc., can be contained in an amount that does not hinder the binding of the carrier of the present invention to the extracellular membrane vesicle or virus after the solution containing the Tim protein of the present invention is contacted with the carrier of the present invention. In addition, the solution obtained by dissolving the Tim protein of the present invention in the above solution may be simply referred to as "a solution containing the Tim protein of the present invention".

[0214] In the method for binding the Tim protein of the present invention to the carrier of the present invention, specific examples of the method of binding by physical adsorption include the following method.

[0215] First, the Tim protein of the present invention is prepared according to the method for preparing the Tim protein of the present invention. Then, when the carrier of the present invention is a bead, a solution (a solution containing the Tim protein of the present invention in, for example, purified water or a buffer solution of pH 7.0 to 8.0) of usually 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 50 μL to 100 μL containing usually 5.0 to 50 μg, preferably 10 to 50 μg, more preferably 20 to 50 μg of the Tim protein is brought into contact with 1 mg of the carrier of the present invention, or, when the carrier of the present invention is a microplate, a solution containing usually 0.1 μg to 10 μg, preferably 0.2 μg to 10 μg of the Tim protein is brought into contact with 1 mg of the carrier of the present invention. A solution of 5 μg, more preferably 0.5 μg to 2 μg of Tim protein (a solution containing the Tim protein of the present invention in, for example, purified water, a buffer solution of pH 7.0 to 8.0, etc.) is contacted with one well and reacted at a temperature of usually 2°C to 37°C, preferably 4°C to 11°C, for usually 4 hours to 48 hours, preferably 12 hours to 24 hours, to allow the carrier of the present invention to bind to the Tim protein of the present invention, thereby obtaining the Tim carrier of the present invention.

[0216] <Treatment of Tim carrier of the present invention>

[0217] The Tim vector of the present invention obtained in the above manner can be subjected to a blocking treatment commonly performed in the art.

[0218] If necessary, the Tim carrier of the present invention obtained in the above manner can be subjected to a purification treatment commonly performed in the field. As a purification treatment, any method can be used as long as the impurities attached to the carrier surface can be removed, for example, a method of washing the Tim carrier of the present invention using a washing solution (hereinafter, sometimes abbreviated as "washing operation") can be cited.

[0219] The present invention will be described by taking as an example the case where magnetic particles are used as the carrier.

[0220] First, a container containing a solution containing the Tim carrier of the present invention obtained in the above manner is placed on a magnetic rack, and the Tim carrier of the present invention is assembled on the tube wall using magnetic force, and the solution in the container is discarded. Then, a washing solution is added to the container and stirred. Then, the container is placed on a magnetic rack in the same manner as above, and the Tim carrier of the present invention is assembled on the tube wall using magnetic force, and the solution in the container is discarded. The washing operation can be repeated multiple times as needed. As the washing solution used in the washing operation, any solution that does not affect the binding of the Tim protein of the present invention in the Tim carrier of the present invention to the carrier of the present invention can be used, such as pure water, for example, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (such as PBS, TBS, HBS, etc.). In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. The solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc., as long as the amount does not hinder the binding of the Tim protein of the present invention to the carrier of the present invention.

[0221] Examples of the surfactant include Tween 20 and the like. The concentration of the surfactant in the washing solution is usually 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0222] If the Tim carrier of the present invention is used, extracellular membrane vesicles or viruses can be effectively obtained, and extracellular membrane vesicles or viruses in a sample can be obtained with high purity. In addition, extracellular membrane vesicles or viruses in a sample can be efficiently removed. Furthermore, extracellular membrane vesicles or viruses in a sample can be detected with high sensitivity.

[0223] <6. Method for obtaining extracellular membrane vesicles or viruses in a sample>

[0224] The method for obtaining extracellular membrane vesicles or viruses of the present invention (hereinafter, sometimes simply referred to as "the obtaining method of the present invention") is characterized by comprising the following steps.

[0225] (1) A step of forming a complex between the Tim protein bound to the carrier and the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions (hereinafter, sometimes referred to as a "complex formation step")

[0226] (2) A step of separating the complex from the sample (hereinafter, sometimes referred to as a "complex separation step")

[0227] (3) A step of separating the extracellular membrane vesicles or the virus from the complex to obtain the extracellular membrane vesicles or the virus (hereinafter, sometimes simply referred to as “obtaining step”).

[0228] <6-1. Regarding the composite forming step>

[0229] The complex formation step is a step of forming a complex between the Tim protein and the carrier and the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions.

[0230] <Sample of the present invention>

[0231] The sample of the present invention may be a sample containing the extracellular membrane vesicle or virus of the present invention in a liquid, or any sample among samples that may contain the extracellular membrane vesicle or virus of the present invention. The sample of the present invention may be any sample among samples from an organism, and samples obtained by dissolving or suspending the extracellular membrane vesicle or virus of the present invention in a solution such as a culture medium or a buffer. Specifically, the sample of the present invention includes body fluids such as blood, saliva, urine, milk, amniotic fluid, ascites, and cell culture supernatant.

[0232] As the solution containing (dissolving or suspending) the extracellular membrane vesicles or viruses of the present invention, any solution can be used as long as it dissolves or suspends the extracellular membrane vesicles or viruses of the present invention in a stable state and does not hinder the binding of the Tim protein bound to the carrier with the complex of the extracellular membrane vesicles or viruses in the sample. Examples thereof include water, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (e.g., TBS, HBS, etc.). It should be noted that phosphate buffer is not preferred because it generates a precipitate by binding to calcium. In addition, the buffer concentration in the buffer is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM.

[0233] In the solution, as long as the amount does not hinder the binding of the Tim protein bound to the carrier with the complex of the extracellular membrane vesicle or virus in the sample, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc. can be contained. As surfactants, for example, Tween 20 can be cited, and the concentration of the surfactant in the solution containing the extracellular membrane vesicle or virus of the present invention is usually 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0234] <Calcium ion concentration / source of calcium ions>

[0235] In the present invention, calcium ions are present when the Tim protein of the present invention forms a complex with a carrier and an extracellular membrane vesicle or virus in a sample. More specifically, calcium ions are present when the Tim protein of the present invention is brought into contact with an extracellular membrane vesicle in a sample.

[0236] The calcium ion concentration when the Tim protein of the present invention is brought into contact with the extracellular membrane vesicles or viruses in the sample of the present invention is generally 0.5 to 100 mM, preferably 1.0 to 10 mM, and more preferably 2.0 to 5.0 mM. It should be noted that the calcium ion concentration as described above is required in the solution containing the complex formed by the Tim protein of the present invention and the extracellular membrane vesicles or viruses in the sample of the present invention until the step of obtaining, that is, until the step of separating the complex.

[0237] The source of calcium ions is not particularly limited, and examples thereof include calcium chloride, calcium hydroxide, calcium hydrogen carbonate, calcium iodide, calcium bromide, and calcium acetate. Calcium chloride, calcium hydrogen carbonate, and calcium iodide are preferred, and calcium chloride and calcium hydrogen carbonate are more preferred.

[0238] It should be noted that as a method for causing calcium ions to be present when the Tim protein of the present invention is brought into contact with the extracellular membrane vesicles or viruses in the sample, generally, the solution containing the Tim carrier of the present invention, the sample, the solution containing the Tim protein of the present invention, or / and the solution containing the carrier of the present invention may contain calcium ions as described above, so that the calcium ion concentration when the Tim protein of the present invention and the carrier of the present invention are brought into contact with the extracellular membrane vesicles or viruses in the sample is within the above range. In addition, a solution containing calcium ions may be used and the solution may be mixed with the solution containing the Tim carrier of the present invention, the sample, the solution containing the Tim protein of the present invention, or / and the carrier of the present invention, so that the calcium ion concentration when the Tim protein of the present invention and the carrier of the present invention are brought into contact with the extracellular membrane vesicles or viruses in the sample is within the above range.

[0239] The solution containing calcium ions is the same as the solution containing (dissolving or suspending) the extracellular membrane vesicles or viruses of the present invention, and the specific examples are also the same.

[0240] <Sample quantity>

[0241] In the complex formation step, the amount of the sample to be brought into contact with 1 μg of the Tim protein of the present invention is usually 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0 ml.

[0242] <Temperature>

[0243] In the complex formation step, the temperature at which the Tim protein of the present invention is brought into contact with the extracellular membrane vesicles or viruses in the sample is generally 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0244] <Time>

[0245] In the complex formation step, the contact time between the Tim protein of the present invention and the sample is usually 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0246] The complex formation step is divided into the following two cases: (1-A) the case of using the pre-prepared Tim vector of the present invention (i.e., the case of using a vector bound to the Tim protein of the present invention), and (1-B) the case of using the Tim protein of the present invention and the vector of the present invention separately.

[0247] <About (1-A)>

[0248] (1-A) is a method in which the Tim vector of the present invention is brought into contact with an extracellular membrane vesicle or virus in a sample in the presence of calcium ions, thereby forming a complex of the Tim protein bound to the vector of the present invention and the extracellular membrane vesicle or virus in the sample (hereinafter, sometimes referred to as the "complex of the present invention"). In (1-A), calcium ions may be present when the Tim vector of the present invention is brought into contact with the extracellular membrane vesicle or virus in the sample. Specifically, a solution or / and a sample in which calcium ions are contained in a solution containing a vector bound with the Tim protein of the present invention or / and a sample may be used, or a solution containing a vector bound with the Tim protein of the present invention, a sample, and a solution containing calcium ions may be used.

[0249] -Amount of Sample of the Present Invention-

[0250] In (1-A), the amount of the sample to be brought into contact with 1 mg of the Tim carrier of the present invention is usually 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0 ml.

[0251] -Contact Temperature-

[0252] In (1-A), the temperature at which the Tim carrier of the present invention is brought into contact with a sample is usually 4.0 to 37°C, preferably 4.0 to 25°C, and more preferably 4.0 to 11°C.

[0253] -Contact time-

[0254] In (1-A), the contact time between the Tim carrier of the present invention and the sample (extracellular membrane vesicle or virus) is generally 0.5 to 24 hours, preferably 0.5 to 8.0 hours, and more preferably 0.5 to 4.0 hours.

[0255] -Amount of Tim carrier of the present invention-

[0256] In (1-A), the amount of the Tim carrier of the present invention is usually 0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6.0 mg per 1 mL of the solution for forming the complex of the present invention.

[0257] -1-Specific example of A-

[0258] (1-A) can be carried out, for example, by the following method. That is, 0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6.0 mg of the Tim carrier of the present invention, a solution containing calcium ions in an amount such that the calcium ion concentration in the solution when the complex of the present invention is formed becomes generally 0.5 to 100 mM, preferably 1.0 to 10 mM, and more preferably 2.0 to 5.0 mM, and 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0 ml of the sample, relative to 1 mg of the Tim carrier of the present invention, are mixed (the solution when the complex of the present invention is formed) at generally 4.0 to 37° C., preferably 4.0 to The contact is usually carried out at a temperature of 25°C, more preferably 4.0°C to 11°C, for 0.5 to 24 hours, preferably 0.5 to 8.0 hours, more preferably 0.5 to 4.0 hours, to form a complex between the Tim protein bound to the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample.

[0259] <About (1-B)>

[0260] (1-B) is a method of using the Tim protein of the present invention and the carrier of the present invention separately. For example, when the Tim protein of the present invention is bound to the carrier of the present invention by affinity binding, it can be carried out as shown in the following (1-Bi), (1-B-ii), or (1-B-iii).

[0261] (1-Bi) The Tim protein of the present invention, the carrier of the present invention, and the extracellular membrane vesicles or viruses in a sample are brought into contact with each other simultaneously in the presence of calcium ions to form the complex of the present invention.

[0262] (1-B-ii) In the presence of calcium ions, the Tim protein of the present invention is brought into contact with extracellular membrane vesicles or viruses in a sample, and after a complex of the Tim protein of the present invention and the extracellular membrane vesicles or viruses is formed, the complex is further brought into contact with the carrier of the present invention to form the complex of the present invention.

[0263] (1-B-iii) After the carrier of the present invention is brought into contact with extracellular membrane vesicles or viruses in a sample, the carrier is further brought into contact with the Tim protein of the present invention in the presence of calcium ions to form the complex of the present invention.

[0264] -About (1-Bi)-

[0265] (1-Bi) is a method of simultaneously contacting the Tim protein of the present invention with the carrier of the present invention and extracellular membrane vesicles or viruses in a sample in the presence of calcium ions to form the complex of the present invention consisting of [extracellular membrane vesicle or virus-(Tim protein of the present invention)-affinity substance-(carrier of the present invention)].

[0266] Specifically, for example, when two or more substances having affinity for each other are used as affinity substances, the Tim protein of the present invention bound to one of the affinity substances (affinity substance-bound Tim protein) is brought into contact simultaneously with a carrier bound to the remaining affinity substances (affinity substance-bound carrier) and extracellular membrane vesicles or viruses in the sample in the presence of calcium ions, so that the Tim protein of the present invention in the affinity substance-bound Tim protein is bound to the extracellular membrane vesicles or viruses, and the affinity substance in the affinity substance-bound Tim protein is bound to the affinity substance in the affinity substance-bound carrier, thereby forming a complex of the present invention consisting of [extracellular membrane vesicles or viruses in the sample-(Tim protein of the present invention)-affinity substance-(carrier of the present invention)].

[0267] In (1-Bi), calcium ions may be present when the Tim protein of the present invention is brought into contact with the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample. Specifically, a solution or sample in which calcium ions are contained in at least one of a solution containing the Tim protein of the present invention, a solution containing the carrier of the present invention, and a sample may be used, or the solution containing the Tim protein of the present invention, a solution containing the carrier of the present invention, a sample, and a solution containing calcium ions may be brought into contact.

[0268] -Amount of Sample of the Present Invention-

[0269] In (1-Bi), the amount of the sample to be brought into contact with 1 μg of the Tim protein of the present invention is usually 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0 ml.

[0270] -Amount of Tim protein of the present invention-

[0271] In (1-Bi), the amount of the Tim protein of the present invention is usually 0.01 to 200 μg, preferably 0.15 to 50 μg, and more preferably 0.5 to 24 μg per 1 mL of the solution for forming the complex of the present invention.

[0272] -Amount of the carrier of the present invention-

[0273] In (1-Bi), the amount of the carrier of the present invention is usually 0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6.0 mg, relative to 1 mL of the solution when the complex of the present invention is formed.

[0274] -Contact Temperature-

[0275] In (1-Bi), the temperature at which the Tim protein of the present invention is brought into contact with the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample is usually 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0276] -Contact time-

[0277] The contact time of the Tim protein of the present invention with the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample is usually 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0278] It should be noted that the contact of the Tim protein of the present invention with the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample is usually carried out by contacting a solution containing the Tim protein of the present invention, the carrier of the present invention, the extracellular membrane vesicles or viruses in the sample, and a solution containing calcium ions.

[0279] As a solution containing the Tim protein of the present invention, any solution can be used as long as the Tim protein of the present invention is dissolved in a stable state and does not hinder the binding of the Tim protein of the present invention to the carrier of the present invention and the extracellular membrane vesicle or virus. For example, purified water, for example, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (for example, PBS, TBS, HBS, etc.). In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. In the solution, as long as the amount is such that the Tim protein of the present invention is dissolved in a stable state and does not hinder the binding of the Tim protein of the present invention to the carrier of the present invention and the extracellular membrane vesicle or virus, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc. can be contained. Examples of the surfactant include Tween 20 and the like. The concentration of the surfactant in the solution containing the Tim protein of the present invention is usually 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0280] The solution containing calcium ions is the same as the solution containing (dissolving or suspending) the extracellular membrane vesicles or viruses of the present invention, and the specific examples are also the same.

[0281] The solution containing calcium ions is a solution containing calcium ions in an amount such that the calcium ion concentration in the solution when the complex of the present invention is formed is generally 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM.

[0282] - Specific example of (1-Bi) -

[0283] (1-Bi) can be carried out, for example, by the following method. That is, a solution containing usually 0.5 μL to 1 ml, preferably 0.5 μL to 100 μL, more preferably 0.5 μL to 10 μL of a solution containing usually 0.01 to 200 μg, preferably 0.15 to 50 μg, more preferably 0.5 to 24 μg of the Tim protein of the present invention relative to 1 mL of the solution when the complex of the present invention is formed (a solution in which the Tim protein of the present invention is contained in, for example, purified water, a buffer having a buffering effect at, for example, pH 7.0 to 8.0, preferably 7.2 to 7.6); usually 0.1 to 100 ml, preferably 0.5 to 10 μL, relative to 1 μg of the Tim protein of the present invention; A sample of preferably 0.1 to 10 ml, more preferably 0.1 to 1 ml; a solution containing calcium ions in an amount such that the calcium ion concentration in the solution when the complex of the present invention is formed becomes generally 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM; and a solution (solution when the complex of the present invention is formed) obtained by mixing a solution containing the Tim protein of the present invention, a sample, a solution containing calcium ions and a carrier of the present invention in an amount generally 0.1 to 20 mg, preferably 0.3 to 10 mg, more preferably 0.5 to 6 mg, contacting the carrier of the present invention for generally 0.5 to 24 hours, preferably 0.5 to 8 hours, more preferably 0.5 to 4 hours, to form the complex of the present invention.

[0284] -Regarding (1-B-ii)-

[0285] (1-B-ii) is a method of contacting the Tim protein of the present invention with extracellular membrane vesicles or viruses in a sample in the presence of calcium ions, forming a complex between the Tim protein of the present invention and the extracellular membrane vesicles or viruses, and then contacting the complex with the carrier of the present invention, thereby forming a complex consisting of [extracellular membrane vesicle or virus-(Tim protein of the present invention)-affinity substance-(carrier of the present invention)].

[0286] Specifically, for example, when two or more substances having mutual affinity are used as affinity substances, a Tim protein bound to one of the affinity substances (affinity substance-bound Tim protein) is brought into contact with extracellular membrane vesicles or viruses in a sample in the presence of calcium ions, so that the Tim protein in the affinity substance-bound Tim protein is bound to the extracellular membrane vesicle, thereby forming a [extracellular membrane vesicle or virus-(Tim protein of the present invention-affinity substance)] complex. Then, the [extracellular membrane vesicle-(Tim protein of the present invention-affinity substance)] complex is brought into contact with a carrier (affinity substance-bound carrier) bound to another affinity substance, so that the affinity substance in the [extracellular membrane vesicle or virus-(Tim protein of the present invention-affinity substance)] complex is bound to the affinity substance in the affinity substance-bound carrier, thereby forming a [extracellular membrane vesicle or virus-(Tim protein of the present invention-affinity substance)-(affinity substance-carrier of the present invention)] complex.

[0287] In addition, for example, when a substance having affinity for an affinity tag or an anti-Tim antibody is used as an affinity substance, the Tim protein of the present invention is brought into contact with the extracellular membrane vesicle or virus in the sample in the presence of calcium ions, and the Tim protein of the present invention is bound to the extracellular membrane vesicle or virus, thereby forming a [extracellular membrane vesicle or virus-Tim protein of the present invention] complex. Then, the [extracellular membrane vesicle or virus-Tim protein of the present invention] complex is brought into contact with a carrier (affinity substance-bound carrier) to which an affinity substance is bound, and the Tim protein of the present invention in the [extracellular membrane vesicle or virus-Tim protein of the present invention] complex is bound to the affinity substance in the affinity substance-bound carrier, thereby forming a [extracellular membrane vesicle or virus-Tim protein of the present invention-(affinity substance-carrier of the present invention)] complex.

[0288] In (1-B-ii), calcium ions may be present when the Tim protein of the present invention is brought into contact with the extracellular membrane vesicles or viruses in the sample. Specifically, a solution or / and a sample containing the Tim protein of the present invention may be used in which calcium ions are contained in a solution or / and a sample, or a solution or a sample containing the Tim protein of the present invention and a solution containing calcium ions may be used.

[0289] -Amount of Sample of the Present Invention-

[0290] In (1-B-ii), the amount of the sample to be brought into contact with 1 μg of the Tim protein of the present invention is usually 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0 ml.

[0291] -Amount of Tim protein of the present invention-

[0292] In (1-B-ii), the amount of the Tim protein of the present invention is usually 0.01 to 200 μg, preferably 0.15 to 50 μg, and more preferably 0.5 to 24 μg per 1 mL of the solution for forming the complex of the present invention.

[0293] -Amount of the carrier of the present invention-

[0294] In (1-B-ii), the amount of the carrier of the present invention is usually 0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6 mg per 1 mL of the solution for forming the complex of the present invention.

[0295] -Contact Temperature-

[0296] In (1-B-ii), the temperature at which the Tim protein of the present invention is brought into contact with the extracellular membrane vesicles or viruses in the sample is generally 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0297] In (1-B-ii), after the complex of the Tim protein of the present invention and the extracellular membrane vesicle or virus is formed, the temperature when the complex is contacted with the vector of the present invention is usually 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0298] -Contact time-

[0299] In (1-B-ii), the contact time of the Tim protein of the present invention and the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample of the present invention is generally 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0300] In (1-B-ii), after the complex of the Tim protein of the present invention and the extracellular membrane vesicle or virus is formed, the time for further contacting the complex with the vector of the present invention is usually 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0301] It should be noted that, usually, the contact of the Tim protein of the present invention with the extracellular membrane vesicles or viruses in a sample is performed by bringing a solution containing the Tim protein of the present invention into contact with the sample and a solution containing calcium ions.

[0302] As a solution containing the Tim protein of the present invention, any solution can be used as long as the Tim protein of the present invention is dissolved in a stable state and does not hinder the binding of the Tim protein of the present invention to the carrier of the present invention and the extracellular membrane vesicle or virus of the present invention. For example, purified water, for example, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (for example, TBS, HBS, etc.) can be mentioned. In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. In the solution, as long as the amount is such that the Tim protein of the present invention is dissolved in a stable state and does not hinder the binding of the Tim protein of the present invention to the carrier of the present invention and the extracellular membrane vesicle or virus, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc. can be contained. Examples of the surfactant include Tween 20 and the like. The concentration of the surfactant in the solution containing the Tim protein of the present invention is usually 0.00001 to 0.2%, and usually preferably 0.0005 to 0.1%.

[0303] The solution to be made to contain calcium ions is the same as the solution to be made to contain (dissolve or suspend) the extracellular membrane vesicles or viruses of the present invention, and the specific examples are also the same.

[0304] The solution containing calcium ions is a solution containing calcium ions in an amount such that the concentration of calcium ions in the solution when the complex of the present invention is formed is generally 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM.

[0305] - Specific example of (1-B-ii) -

[0306] (1-B-ii) can be carried out, for example, by the following method. Specifically, a solution containing usually 0.5 μL to 1 ml, preferably 0.5 μL to 100 μL, more preferably 0.5 μL to 10 μL of the Tim protein of the present invention, usually 0.01 to 200 μg, preferably 0.15 to 50 μg, more preferably 0.5 to 24 μg, relative to 1 mL of the solution for forming the complex of the present invention (a solution containing the Tim protein of the present invention in, for example, purified water, for example, a buffer having a buffering effect under the condition of pH 7.0 to 8.0, preferably 7.2 to 7.6); a sample containing usually 0.1 to 100 ml, preferably 0.1 to 10 ml, more preferably 0.1 to 1 ml, relative to 1 μg of the Tim protein of the present invention; and a solution containing calcium ions are stirred at a temperature of usually 4 to 37°C, preferably 4 to 25°C, more preferably The contact is carried out at a temperature of 4 to 11° C. for usually 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours, the amount of calcium ions in the solution containing calcium ions is such that the concentration of calcium ions in a solution obtained by mixing a solution containing a sample and calcium ions with a solution containing the Tim protein of the present invention, and in a solution after further contacting the solution with the carrier of the present invention becomes usually 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM, after a complex of the Tim protein of the present invention and an extracellular membrane vesicle or a virus is formed, usually 0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6 mg of the carrier of the present invention is further added relative to 1 mL of the solution in which the complex of the present invention is formed, and the mixture is stirred at a temperature of usually 4 to 37° C., preferably 4 to The obtained complex of the Tim protein of the present invention and the extracellular membrane vesicle or virus is contacted with the carrier of the present invention at a temperature of 25°C, more preferably 4 to 11°C, for usually 0.5 to 24 hours, preferably 0.5 to 8 hours, more preferably 0.5 to 4 hours to form the complex of the present invention.

[0307] -Regarding (1-B-iii)-

[0308] (1-B-iii) is a method in which the carrier of the present invention is brought into contact with extracellular membrane vesicles or viruses in a sample, and then the Tim protein of the present invention is brought into contact therewith in the presence of calcium ions, thereby forming a complex of the present invention consisting of [extracellular membrane vesicle or virus-(Tim protein of the present invention)-affinity substance-(carrier of the present invention)].

[0309] Specifically, for example, when two or more substances having affinity for each other are used as affinity substances, a carrier bound to one of the affinity substances (affinity substance bound carrier) is brought into contact with extracellular membrane vesicles or viruses in the sample, and then, in the presence of calcium ions, it is simultaneously brought into contact with Tim protein bound to another affinity substance (affinity substance bound Tim protein), so that the Tim protein in the affinity substance bound Tim protein is bound to the extracellular membrane vesicle or virus, and the affinity substance in the affinity substance bound Tim protein is bound to the affinity substance in the affinity substance bound carrier, thereby forming a [extracellular membrane vesicle or virus-(Tim protein of the present invention-affinity substance)-(affinity substance-carrier of the present invention)] complex.

[0310] In addition, for example, when a substance having an affinity for an affinity tag or an anti-Tim antibody is used as an affinity substance, a carrier bound with the affinity substance (affinity substance bound carrier) is brought into contact with the extracellular membrane vesicles or viruses in the sample, and then, in the presence of calcium ions, it is simultaneously brought into contact with the Tim protein of the present invention, so that the Tim protein of the present invention is bound to the extracellular membrane vesicles or viruses, and the Tim protein of the present invention is bound to the affinity substance in the affinity substance bound carrier, thereby forming a [extracellular membrane vesicle or virus-Tim protein of the present invention-(affinity substance-carrier of the present invention)] complex.

[0311] In (1-B-iii), calcium ions may be present when the Tim protein of the present invention is brought into contact with the extracellular membrane vesicles or viruses in the sample. Specifically, a solution containing the Tim protein of the present invention or / and the extracellular membrane vesicles or viruses in the sample, or / and a sample in which calcium ions are contained may be used, or a solution containing the Tim4 protein of the present invention, the extracellular membrane vesicles or viruses in the sample, and a solution containing calcium ions may be used.

[0312] -Amount of Sample of the Present Invention-

[0313] In (1-B-iii), the amount of the sample to be brought into contact with 1 μg of the Tim protein of the present invention is usually 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1 ml.

[0314] -Amount of Tim protein of the present invention-

[0315] In (1-B-iii), the amount of the Tim protein of the present invention is usually 0.01 to 200 μg, preferably 0.15 to 50 μg, and more preferably 0.5 to 24 μg per 1 mL of the solution for forming the complex of the present invention.

[0316] -Amount of the carrier of the present invention-

[0317] In (1-B-iii), the amount of the carrier of the present invention is usually 0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6 mg per 1 mL of the solution for forming the complex of the present invention.

[0318] -Contact Temperature-

[0319] In (1-B-iii), the temperature at which the carrier of the present invention is brought into contact with the extracellular membrane vesicles or viruses in a sample is usually 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0320] In (1-B-iii), the temperature at which the carrier of the present invention is brought into contact with extracellular membrane vesicles or viruses in a sample and then the Tim protein of the present invention is brought into contact therewith is generally 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0321] -Contact time-

[0322] In (1-B-iii), the contact time when the carrier of the present invention is brought into contact with the sample is usually 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0323] In (1-B-iii), after the carrier of the present invention is brought into contact with the extracellular membrane vesicles or viruses in the sample, the contact time when the Tim protein of the present invention is brought into contact therewith is generally 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0324] In general, after the carrier of the present invention is brought into contact with the extracellular membrane vesicles or viruses in a sample, the carrier is further brought into contact with the Tim protein of the present invention using a solution containing the Tim protein of the present invention and a solution containing calcium ions.

[0325] As a solution containing the Tim protein of the present invention, any solution can be used as long as the Tim protein of the present invention is dissolved in a stable state and does not hinder the binding of the Tim protein of the present invention to the carrier of the present invention and the extracellular membrane vesicle or virus. For example, purified water, for example, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (for example, PBS, TBS, HBS, etc.). In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. In the solution, as long as the Tim4 protein of the present invention is dissolved in a stable state and does not hinder the binding of the Tim protein of the present invention to the carrier of the present invention and the extracellular membrane vesicle or virus, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc. can be contained. Examples of the surfactant include Tween 20 and the like. The concentration of the surfactant in the solution containing the Tim protein of the present invention is usually 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0326] The solution to be made to contain calcium ions is the same as the solution to be made to contain (dissolve or suspend) the extracellular membrane vesicles or viruses of the present invention, and the specific examples are also the same.

[0327] The solution containing calcium ions is a solution containing calcium ions in an amount such that the calcium ion concentration in the solution when the complex of the present invention is formed is generally 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM.

[0328] - Specific example of (1-B-iii) -

[0329] (1-B-iii) can be carried out, for example, by the following method. That is, a sample of the present invention (usually 0.1 to 100 ml, preferably 0.1 to 10 ml, more preferably 0.1 to 1 ml) relative to 1 μg of the Tim protein of the present invention is brought into contact with a carrier of the present invention (usually 0.1 to 20 mg, preferably 0.3 to 10 mg, more preferably 0.5 to 6 mg) relative to 1 ml of the solution for forming the complex of the present invention at a temperature of usually 4 to 37° C., preferably 4 to 25° C., more preferably 4 to 11° C. for usually 0.5 to 24 hours, preferably 0.5 to 8 hours, more preferably 0.5 to 4 hours, and then a solution containing calcium ions and a solution containing calcium ions (usually 0.5 μL to 1 ml, preferably 0.5 μL to 100 μL, more preferably 0.5 μL to 10 μL) relative to 1 ml of the solution for forming the complex of the present invention are added. A solution of the Tim protein of the present invention (a solution containing the Tim protein of the present invention in, for example, purified water, for example, a buffer having a buffering effect under the condition of pH 7.0 to 8.0, preferably 7.2 to 7.6) is contacted at a temperature of usually 4 to 37° C., preferably 4 to 25° C., more preferably 4 to 11° C., for usually 0.5 to 24 hours, preferably 0.5 to 8 hours, more preferably 0.5 to 4 hours to form the complex of the present invention, wherein the amount of calcium ions in the solution containing calcium ions is such that the concentration of calcium ions in the solution obtained by contacting the solution containing the sample of the present invention and calcium ions with the carrier of the present invention and further contacting the solution containing the Tim protein of the present invention becomes usually 0.5 to 100 mM, preferably 1.0 to 10 mM, more preferably 2 to 5 mM.

[0330] <6-2. Complex separation step>

[0331] The complex separation step in the acquisition method of the present invention is a step of separating the obtained complex of the Tim protein bound to the carrier of the present invention (Tim carrier of the present invention) and the extracellular membrane vesicles or viruses in the sample (complex of the present invention) from the sample after the complex formation step, to obtain the separated complex of the present invention.

[0332] For the complex separation step in the obtaining method of the present invention, any method can be used as long as the complex of the present invention can be separated from the sample to obtain the complex of the present invention. For example, the following method can be mentioned.

[0333] (1) When a magnetic carrier is used as the carrier of the present invention, a container in which the complex formation step has been performed is placed on a magnetic stand as necessary, the complex of the present invention is aggregated on the tube wall by magnetic force, and the supernatant sample is removed to separate them.

[0334] (2) When the carrier of the present invention is in the form of beads, the container in which the complex formation step has been performed is centrifuged to collect the complex of the present invention as a precipitate, and then the supernatant sample is removed to separate them.

[0335] (3) When a non-bead-shaped plate or the like is used as the carrier of the present invention, a method of separating them by removing only the sample.

[0336] (4) A method of separating the complex of the present invention from a sample by filtration.

[0337] After the complex of the present invention is separated from the sample in this manner, the separated complex of the present invention can be obtained (recovered) by a method known per se.

[0338] - Specific example of complex separation step -

[0339] When a magnetic carrier is used as the carrier of the present invention, the container in which the complex formation step has been performed is placed on a magnetic stand as necessary, the complex of the present invention is aggregated on the tube wall by magnetic force, and the supernatant sample is removed.

[0340] <6-3. Washing operation>

[0341] After the complex formation step and the complex separation step, the obtained complex of the present invention can be washed with a washing solution containing calcium ions as needed (hereinafter, sometimes referred to as "washing operation"). By the washing operation, impurities in the sample of the present invention such as components from cells attached to the surface of the carrier of the present invention can be removed. As a washing method, in addition to using the washing solution containing calcium ions as described above, a washing method commonly performed in this field can also be used. As a washing solution containing calcium ions used in the washing operation, any solution containing calcium ions usually at 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM, and which does not affect the binding of the extracellular membrane vesicles or viruses of the present invention to the Tim4 protein of the present invention and the carrier of the present invention in the complex can be used. For example, a buffer solution containing calcium ions usually at 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM, which has a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6, and does not precipitate calcium (e.g., TBS, HBS) can be cited. It should be noted that phosphate buffer is not preferred because it generates precipitation due to the combination with calcium. In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. In the solution, as long as it is an amount that does not hinder the combination of the extracellular membrane vesicles or viruses of the present invention in the complex with the Tim protein of the present invention and the carrier of the present invention, it is possible to contain salts such as sugars and NaCl, surfactants, preservatives, proteins, etc. As a surfactant, for example, Tween 20 (Wako Pure Chemical Industries, Ltd.) can be cited, and the concentration of the surfactant in the washing solution is usually 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0342] For example, the case of using magnetic particles as the carrier of the present invention is described as an example. First, a washing solution containing calcium ions is added to a container containing the complex of the present invention obtained by the complex separation step, and stirred. Then, the container is placed on a magnetic stand, and the complex is assembled on the tube wall using magnetic force, and the solution in the container is discarded. The washing operation can be repeated as many times as needed.

[0343] - Specific example of washing operation -

[0344] First, a washing solution containing calcium ions (for example, a buffer solution containing calcium ions usually at 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM, having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6, and not causing calcium precipitation. However, since phosphate buffer combines with calcium to form a precipitate, it is not preferred.) is added to a container containing the complex of the present invention obtained through the complex separation step, and the mixture is stirred. Then, the container is placed on a magnetic stand, the complex is gathered on the tube wall using magnetic force, and the solution in the container is discarded. The washing operation can be repeated as many times as necessary.

[0345] <6-4. About the acquisition process>

[0346] The acquisition step is a step of separating the extracellular membrane vesicles or viruses from the obtained complex of the Tim protein bound to the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample after performing the complex formation step, the complex separation step, and the washing step (washing operation) as required, thereby obtaining the extracellular membrane vesicles or viruses of the present invention.

[0347] Thereby, the extracellular membrane vesicles or viruses of the present invention can be obtained at a high purity.

[0348] Specific examples of the acquisition step include the following (2-A) and (2-B).

[0349] (2-A) Method using a protein modifier

[0350] (2-B) Method for reducing calcium ion concentration

[0351] <(2-A): Method using a protein denaturing agent>

[0352] (2-A) is a method for separating extracellular membrane vesicles or viruses from the complex of the present invention by allowing a protein modifier to act on the obtained complex of the present invention after performing a complex formation step, a complex separation step, and a washing operation as required, thereby modifying the Tim protein of the present invention in the complex of the present invention. In this way, the extracellular membrane vesicles or viruses of the present invention can be obtained at a high purity.

[0353] -Protein Modifiers-

[0354] As the protein modifier used in (2-A), any substance can be used as long as it is a compound commonly used in the field for modifying proteins. For example, anionic surfactants such as SDS (sodium dodecyl sulfate) and N-lauroyl sarcosine; zwitterionic surfactants such as CHAPS (3-(3-cholamidopropyl) dimethylamino-1-propanesulfonate) and Zwittergent 3-12 (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate); Brij 35 (manufactured by Takara Bio Co., Ltd.), n-dodecyl-β-D-maltoside, ethylphenyl polyethylene glycol (Nonidet P-40), octyl-β-D-glucoside, Triton X-100 (polyoxyethylene (10) octylphenyl ether), Tween 20 (polyoxyethylene (20) sorbitan monolaurate) and the like; chaotropic agents such as urea, formamide, guanidine and the like; anionic surfactants are preferred, and SDS is particularly preferred.

[0355] In (2-A), in order to allow the protein modifier to act on the complex of the present invention, usually, a solution containing the protein modifier (hereinafter sometimes referred to as "solution containing the protein modifier") and the Tim protein bound to the carrier of the present invention are brought into contact with the complex of extracellular membrane vesicles or viruses in the sample, thereby allowing the solution containing the protein modifier to act on the complex of the present invention.

[0356] It should be noted that the contact between the solution containing the protein modifier and the complex of the present invention can be carried out by, for example, a method of suspending the complex in the solution (when the carrier is beads, etc.), a method of immersing the complex in the solution (when the carrier is a disc, a tube, etc.), a method of adding the complex (carrier) to the solution (when the carrier is a microplate, a tube, etc.), etc.

[0357] - A solution containing a protein modifier -

[0358] In (2-A), the solution containing the protein modifier may be purified water, a buffer capable of dissolving the protein modifier, or the like. The buffer may be a buffer having a buffering effect under conditions of pH 6 to 9, preferably pH 7 to 8 (e.g., Tris (trishydroxymethylaminomethane), HEPES, etc.). The concentration of the buffer in the buffer may be appropriately selected from a range of 5 to 100 mM, preferably from a range of 10 to 50 mM.

[0359] -Solution containing a protein modifier-

[0360] In (2-A), the pH of the solution containing the protein modifier is usually 6.0 to 9.0, preferably 7.0 to 8.0. The concentration of the protein modifier in the solution containing the protein modifier varies depending on the type of the protein modifier, but it can be any concentration range commonly used in this field. For example, in the case of SDS, it is usually 0.1 to 10%, preferably 0.3 to 4%, and more preferably 0.5 to 2%. In addition, the solution containing the protein modifier may contain, for example, sugars, salts such as NaCl, preservatives, proteins, etc. In (2-A), for the solution containing the protein modifier, 10 μL to 500 μL is usually used, preferably 20 μL to 200 μL, and more preferably 50 μL to 100 μL is used relative to 1 mg of the Tim carrier of the present invention.

[0361] -Action (contact) conditions-

[0362] In (2-A), the temperature and time for which the protein modifier is allowed to act (contact) on the complex are usually 4.0 to 37°C, preferably 10 to 30°C, more preferably 20 to 30°C, and the time is usually 5.0 to 60 seconds, preferably 10 to 30 seconds, more preferably 10 to 20 seconds.

[0363] -Tim Protein-

[0364] When a protein modifier is allowed to act (contact) on the complex (i.e., (2-A)), any protein can be used as the Tim protein of the present invention when obtaining extracellular membrane vesicles, as long as it is the Tim protein of the present invention, but the Tim4 protein of the present invention and the Tim1 protein of the present invention are preferred, and the Tim4 protein of the present invention is particularly preferred. In addition, when obtaining viruses, the Tim4 protein of the present invention and the Tim3 protein of the present invention are particularly preferred.

[0365] - Specific example of (2-A) -

[0366] (2-A) can be carried out, for example, by the following method. That is, after performing a complex formation step, a complex separation step, and a washing operation as required, a solution containing a protein modifier (a solution containing a protein modifier in purified water or a buffer having a buffering effect at a pH of usually 6 to 9, preferably 7 to 8) is added to the obtained complex of the present invention in an amount of usually 10 μL to 500 μL, preferably 20 μL to 200 μL, and more preferably 50 μL to 100 μL per 1 mg of the Tim carrier of the present invention, and the mixture is stirred using a vortex mixer or the like at a temperature of usually 4 to 37° C., preferably 10 to 30° C., The reaction is preferably carried out at a temperature of 20 to 30°C for usually 5 to 60 seconds, preferably 10 to 30 seconds, and more preferably 10 to 20 seconds, so that the Tim protein of the present invention in the complex of the present invention is brought into contact with the protein modifier so that the protein modifier acts and the extracellular membrane vesicles or viruses are separated from the complex of the present invention.

[0367] <(2-B): Method for reducing calcium ion concentration>

[0368] (2-B) is a method for separating the extracellular membrane vesicles or viruses of the present invention from the complex of the present invention by reducing the concentration of calcium ions bound to the obtained complex of the present invention and the concentration of calcium ions in a solution containing the complex after performing a complex formation step, a complex separation step, and a washing operation as required.

[0369] Thereby, the extracellular membrane vesicles or viruses of the present invention can be obtained in a highly pure and intact state.

[0370] In addition, calcium ions are known to be involved in the binding of Tim protein and phosphatidylserine (Immunity 2007 December; 27 (6): 941-951). After the complex formation step, the complex separation step, and the washing operation as required are performed, for the complex of the present invention obtained, in order to maintain the binding of the Tim protein of the present invention in the complex of the present invention and the extracellular membrane vesicles or viruses in the sample, calcium ions must be allowed to coexist in advance. For example, when the complex of the present invention is allowed to coexist in a solution, it is necessary for the solution of the complex of the present invention to contain more than 0.5 mM of calcium ions. Among them, when the complex of the present invention is allowed to coexist in a solution, it also includes a carrier in a particle state after the complex formation step, the complex separation step, and the washing operation as required are performed.

[0371] It should be noted that even if the complex of the present invention is dried after the complex formation step, the complex separation step, and the washing operation as required, calcium ions will not be eluted from the complex unless the acquisition step is performed, thereby maintaining the binding of the vector of the present invention to the extracellular membrane vesicle or virus.

[0372] (2-B) By making the concentration of calcium ions brought by calcium ions bound to the complex of the present invention lower than the concentration (effective concentration) required to maintain the binding of the Tim protein of the present invention and the extracellular membrane vesicle or virus as described above, the extracellular membrane vesicle or virus of the present invention can be separated from the complex of the present invention.

[0373] Specifically, the calcium ion concentration in the solution containing the complex of the present invention can be generally set to less than 0.5 mM, preferably less than 0.4 mM, and more preferably less than 0.2 mM.

[0374] Examples of methods for reducing the (effective) concentration of calcium ions include the following (2-Bi) and (2-B-ii).

[0375] (2-Bi) Method using calcium ion chelating agent

[0376] (2-B-ii) Method using a solution containing no calcium ions

[0377] -(2-Bi): Method using calcium ion chelating agent-

[0378] (2-Bi) is a method for separating extracellular membrane vesicles or viruses from the complex of the present invention by allowing a calcium ion chelator to act on calcium ions introduced by a solution containing calcium ions bound to the obtained complex of the present invention and the complex, and then reducing the (effective) concentration of calcium ions introduced by a solution containing calcium ions bound to the complex of the present invention and the complex (chelating the calcium ions), after performing a complex formation step, a complex separation step, and a washing operation as required.

[0379] Thereby, the extracellular membrane vesicles or viruses of the present invention can be obtained in a highly pure and intact state.

[0380] -Calcium ion chelator-

[0381] As the calcium ion chelating agent, any compound can be used as long as it is a compound that can chelate calcium ions, and examples thereof include EDTA (ethylenediaminetetraacetic acid), NTA (nitrilotriacetic acid), DTPA (diethylenetriaminepentaacetic acid), GLDA (L-glutamic acid diacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), GEDTA (ethylene glycol bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid), TTHA (triethylenetetraamine-N,N,N',N",N"',N"'-hexaacetic acid), HIDA (2-hydroxyethyliminobis(acetic acid)), DHEG (N,N-bis(2-hydroxyethyl)glycine), CyDTA (trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate), and PEG (PEG-2-hydroxyethylaminobis(2-hydroxyethyl)glycine). In order to make the calcium ion chelating agent act on the calcium ions bound to the complex of the present invention, usually, a solution containing the calcium ion chelating agent (hereinafter, sometimes referred to as "the solution containing the calcium ion chelating agent") is brought into contact with the granular complex of the present invention, thereby reacting the calcium ions bound to the complex of the present invention with the calcium ion chelating agent in the solution containing the calcium ion chelating agent.

[0382] It should be noted that the contact between the solution containing the calcium ion chelator and the complex of the present invention can be carried out by, for example, a method of suspending the complex in the solution (when the carrier is beads, etc.), a method of immersing the complex in the solution (when the carrier is a disk, a tube, etc.), a method of adding the solution to the complex (carrier) (when the carrier is a microplate, a tube, etc.), etc.

[0383] -Solution containing calcium ion chelator-

[0384] In (2-Bi), as a solution containing a calcium ion chelator, any solution that dissolves the calcium ion chelator may be used, and examples thereof include purified water and a buffer solution. As a buffer solution, a buffer solution having a buffering effect under the condition of a pH of usually 7.0 to 8.0, preferably 7.2 to 7.6 (e.g., PBS, TBS, HBS, etc.) is preferred. In addition, as a buffer concentration in the buffer solution, it is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. The solution containing a calcium ion chelator may contain, for example, sugars, salts such as NaCl, preservatives, proteins, etc.

[0385] In (2-Bi), the concentration of the calcium ion chelator in the solution containing the calcium ion chelator is usually 0.5 to 500 mM, preferably 0.5 to 100 mM, and more preferably 0.5 to 50 mM.

[0386] In (2-Bi), the pH of the solution containing the calcium ion chelating agent is usually 6.0 to 9.0, preferably 7.0 to 8.0, and more preferably 7.2 to 7.6.

[0387] In (2-Bi), the amount of the solution containing the calcium ion chelator to be mixed in the reaction solution may be an amount that makes the calcium ion concentration in the reaction solution lower than the effective concentration and allows the extracellular membrane vesicles or viruses to be separated from the complex of the present invention.

[0388] -Action (contact) conditions-

[0389] In (2-Bi), the temperature and time for allowing the calcium ion chelator to act (contact) on the complex are generally 4.0 to 37°C, preferably 10 to 30°C, more preferably 20 to 30°C, and the time is generally 5 to 60 seconds, preferably 10 to 30 seconds, more preferably 10 to 20 seconds.

[0390] -Tim Carrier-

[0391] In (2-Bi), as the binding mode of the Tim protein of the present invention and the carrier of the present invention, the binding mode is not limited as long as the carrier of the present invention binds to the Tim protein of the present invention. In the case of using a calcium ion chelator to dissolve extracellular membrane vesicles or viruses (i.e., the case of (2-Bi)), from the perspective of obtaining a large amount of extracellular membrane vesicles or viruses, the binding mode of the carrier of the present invention to the thiol group of the Tim protein of the present invention is preferred, and the binding mode of the carrier of the present invention to the thiol group of the Tim4 protein of the present invention is particularly preferred.

[0392] - Specific example of (2-Bi) -

[0393] (2-Bi) can be carried out, for example, by the following method. That is, after performing the complex formation step, the complex separation step, and the washing operation as required, a solution containing usually 0.5 to 500 mM, preferably 0.5 to 100 mM, and more preferably 0.5 to 50 mM of a calcium ion chelator (a solution containing a calcium ion chelator in purified water or a buffer having a buffering effect under the conditions of usually pH 7.0 to 8.0, preferably 7.2 to 7.6) is added to the obtained complex of the present invention relative to 1 mg of the Tim vector of the present invention, and the reaction is carried out at a temperature of usually 4.0 to 37°C, preferably 10 to 30°C, and more preferably 20 to 30°C for usually 5 to 60 seconds, preferably 10 to 30 seconds, and more preferably 10 to 20 seconds while stirring using a vortex mixer or the like, thereby separating the extracellular membrane vesicles or viruses from the complex of the present invention.

[0394] -(2-B-ii): Method using a solution containing no calcium ions-

[0395] (2-B-ii) is a method for separating extracellular membrane vesicles or viruses from the complex of the present invention by bringing the obtained complex of the present invention into contact with a solution not containing calcium ions after performing a complex formation step, a complex separation step, and a washing operation as required, thereby reducing (diluting) the (effective) concentration of calcium ions bound to the complex of the present invention.

[0396] That is, by bringing a solution containing no calcium ions into contact with the complex of the present invention, the (effective) concentration of calcium ions required to maintain the binding of the Tim protein of the present invention in the complex of the present invention with the extracellular membrane vesicles or viruses in the sample can be reduced (diluted), thereby separating the extracellular membrane vesicles from the complex of the present invention.

[0397] It should be noted that the contact between the solution not containing calcium ions and the complex of the present invention can be carried out by, for example, a method of suspending the complex in the solution (when the carrier is beads, etc.), a method of immersing the complex in the solution (when the carrier is a disc, a tube, etc.), a method of adding the solution to the complex (carrier) (when the carrier is a microplate, a tube, etc.), etc.

[0398] -Solution without calcium ions-

[0399] In (2-B-ii), the solution not containing calcium ions added to the obtained complex of the present invention can be any solution as long as it does not modify the extracellular membrane vesicles or viruses, and examples thereof include purified water, buffer, etc. As the buffer, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (such as PBS, TBS, HBS, etc.) is preferred. In addition, the concentration of the buffer in the buffer is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. The solvent not containing calcium ions may contain, for example, sugars, salts such as NaCl, preservatives, proteins, etc.

[0400] In (2-B-ii), the amount of the solution not containing calcium ions added to the obtained complex of the present invention may be an amount that can make the concentration of calcium ions lower than the effective concentration.

[0401] - Specific example of (2-B-ii) -

[0402] After performing the complex formation step, the complex separation step and the washing operation as required, a solution free of calcium ions is added to the obtained complex of the present invention so that the concentration of calcium ions in the solution containing the complex of the present invention is generally less than 0.5 mM, preferably less than 0.1 mM, and more preferably less than 0.01 mM.

[0403] It should be noted that after performing the complex formation step, the complex separation step and the washing operation as required, when the obtained complex of the present invention is present in a solution containing calcium ions (for example, the reaction solution after the complex formation step, the washing solution containing calcium ions), the solution containing calcium ions can be replaced with a solution not containing calcium ions, or / and diluted with a solution not containing calcium ions, so that the final calcium ion concentration becomes less than the above-mentioned effective concentration.

[0404] Since (2-B-ii) has a larger (volume increase) amount of the extracellular membrane vesicle-containing solution obtained in the obtaining step than (2-Bi), (2-Bi) is more preferred.

[0405] In the above, the solution (protein modifier-containing solution, calcium ion chelator-containing solution, calcium ion-free solution) that contacts / acts with the complex contains the carrier and the extracellular membrane vesicles or viruses separated (free) from the carrier (complex). Therefore, if the carrier is removed from the solution and only the solution is recovered, a solution containing extracellular membrane vesicles or viruses can be obtained.

[0406] By using the Tim carrier of the present invention, extracellular membrane vesicles or viruses can be efficiently removed from a sample, thereby obtaining a sample with few impurities.

[0407] The removal method of the present invention can accurately and effectively remove extracellular membrane vesicles or viruses in a sample that cannot be completely removed by conventional methods such as ultracentrifugation and polymer precipitation.

[0408] <7. Method for removing extracellular membrane vesicles or viruses from a sample>

[0409] The method for obtaining extracellular membrane vesicles or viruses of the present invention (hereinafter, sometimes simply referred to as "the removal method of the present invention") is characterized by comprising the following steps.

[0410] (1) A step of forming a complex between the Tim protein bound to the carrier and the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions (hereinafter, sometimes referred to as a "complex formation step")

[0411] (2) A step of separating the complex from the sample (hereinafter, sometimes simply referred to as a "complex separation step").

[0412] <7-1. Complex Formation Step>

[0413] The complex forming step in the removal method of the present invention is the same as the complex forming step in the acquisition method of the present invention, and various preferred conditions are also the same.

[0414] <7-2. Complex separation step>

[0415] The complex separation step in the removal method of the present invention is a step of separating the complex of the Tim protein bound to the carrier of the present invention (Tim carrier of the present invention) and the extracellular membrane vesicles or viruses in the sample (complex of the present invention) from the sample after the complex formation step and obtaining the separated sample.

[0416] In this way, a sample from which extracellular membrane vesicles or viruses have been removed can be obtained.

[0417] The complex separation step in the removal method of the present invention may be any method as long as it can remove the complex of the present invention from the sample, and the same method as the complex separation step in the acquisition method of the present invention can be cited. After the complex of the present invention is separated from the sample in this way, the separated sample can be obtained (recovered) by a method known per se.

[0418] This makes it possible to remove extracellular membrane vesicles and viruses in a sample that cannot be completely removed by conventional methods such as ultracentrifugation and polymer precipitation with high accuracy and efficiency.

[0419] In addition, by repeatedly performing the complex formation step and the complex separation step, extracellular membrane vesicles or viruses can be more effectively removed from the sample. When the complex formation step and the complex separation step are repeatedly performed, a new carrier can be used, and the carrier after use can be reused. For the reuse of the carrier, a solution containing a calcium ion chelator or a solution not containing calcium ions can be used to perform the same treatment as the acquisition step ((2-B) method for reducing calcium ion concentration) of the acquisition method of the present invention on the complex of the present invention removed by the complex separation step in the removal method of the present invention. Thus, the Tim carrier of the present invention and the extracellular membrane vesicles or viruses can be separated from the complex of the present invention. That is, since the Tim carrier of the present invention can be used again in the removal method of the present invention, the extracellular membrane vesicles or viruses in the sample can be removed with good accuracy and effectively by reusing the Tim carrier of the present invention.

[0420] By using the Tim carrier of the present invention, extracellular membrane vesicles or viruses in a sample can be detected with high accuracy and sensitivity.

[0421] <8. Method for detecting extracellular membrane vesicles or viruses in a sample>

[0422] The method for detecting extracellular membrane vesicles or viruses of the present invention (hereinafter, sometimes simply referred to as "the detection method of the present invention") is characterized by comprising the following steps.

[0423] (1) A step of forming a complex between the Tim protein bound to the carrier and the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions (complex formation step)

[0424] (2) Step of detecting the complex (detection step)

[0425] <8-1. Composite body forming step>

[0426] The complex formation step in the detection method of the present invention is a step of forming a complex between the Tim carrier of the present invention and the extracellular membrane vesicles or viruses in a sample in the presence of calcium ions.

[0427] - Sample of the present invention -

[0428] The sample of the present invention is the same as the sample in the obtaining method of the present invention.

[0429] -Calcium ion concentration / source of calcium ions-

[0430] In the detection method of the present invention, calcium ions are allowed to exist when forming a complex (complex of the present invention) between the Tim carrier of the present invention and the extracellular membrane vesicles or viruses in a sample.

[0431] In the detection method of the present invention, the calcium ion concentration when the Tim carrier of the present invention is brought into contact with the extracellular membrane vesicles or viruses in the sample of the present invention is generally 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM. It should be noted that it should be noted that the calcium ion concentration as described above is required in the solution containing the complex formed by the Tim carrier of the present invention and the extracellular membrane vesicles or viruses in the sample of the present invention until the detection step is performed, that is, until the step of detecting the complex is performed.

[0432] The source of calcium ions is not particularly limited, and the same specific examples as the source of calcium ions in the obtaining method of the present invention can be given.

[0433] It should be noted that as a method for making calcium ions present when the Tim carrier of the present invention is brought into contact with extracellular membrane vesicles or viruses in a sample, generally, the sample can contain calcium ions as described above, so that the calcium ion concentration when the Tim carrier of the present invention is brought into contact with extracellular membrane vesicles or viruses in the sample is within the above range.

[0434] The solution to be made to contain calcium ions is the same as the solution to be made to contain (dissolve or suspend) the extracellular membrane vesicles or viruses of the present invention, and the specific examples are also the same.

[0435] - Sample quantity -

[0436] In the complex formation step of the detection method of the present invention, the amount of the sample, when beads are used as the carrier of the present invention, is usually 0.1 to 1000 ml, preferably 0.1 to 500 ml, and more preferably 0.1 to 100 ml relative to 1 mg of the Tim carrier of the present invention. When a microplate is used as the carrier of the present invention, each well is usually 50 μL to 300 μL, preferably 100 μL to 200 μL.

[0437] -temperature-

[0438] In the complex formation step of the detection method of the present invention, the temperature when the Tim carrier of the present invention is brought into contact with the extracellular membrane vesicles or viruses in the sample is usually 2 to 37°C, preferably 2 to 30°C.

[0439] -time-

[0440] In the complex formation step of the detection method of the present invention, the contact time between the Tim protein of the present invention and the sample is generally 0.5 to 24 hours, preferably 1 to 20 hours, and more preferably 1 to 12 hours.

[0441] -Tim Protein-

[0442] As the Tim protein of the present invention in the detection method of the present invention, any protein can be used as long as it is the above-mentioned Tim protein of the present invention, but the Tim4 protein of the present invention is particularly preferred.

[0443] -Tim Carrier-

[0444] As for the binding mode of the Tim protein of the present invention and the vector of the present invention, there is no limitation as long as the vector of the present invention binds to the Tim protein of the present invention. However, from the perspective of being able to detect extracellular membrane vesicles or viruses with high sensitivity, a mode in which the vector of the present invention binds to the thiol group of the Tim protein of the present invention is preferred, and a mode in which the vector of the present invention binds to the thiol group of the Tim4 protein of the present invention is particularly preferred.

[0445] - Specific example of the complex formation step in the detection method of the present invention -

[0446] The complex formation step in the detection method of the present invention can be performed, for example, by the following method.

[0447] That is, when beads are used as the carrier of the present invention, the solution obtained by mixing the Tim carrier of the present invention, the sample, and the solution containing calcium ions (the solution for forming the complex of the present invention) is usually 0.001 to 20 mg, preferably 0.005 to 10 mg, and more preferably 0.01 to 6.0 mg of the Tim carrier of the present invention; the solution containing calcium ions is usually 0.1 to 1000 ml, preferably 0.1 to 500 ml, and more preferably 0.1 to 1000 ml of the Tim carrier of the present invention. 100 ml of a sample is contacted for usually 0.5 to 24 hours, preferably 1 to 20 hours, and more preferably 1 to 12 hours under temperature conditions of usually 2 to 37° C., preferably 2 to 30° C., so that the Tim protein bound to the carrier of the present invention forms a complex with the extracellular membrane vesicles or viruses in the sample, and the amount of calcium ions in the solution containing calcium ions is such that the calcium ion concentration in the solution when the complex of the present invention is formed becomes usually 0.5 to 100 mM, preferably 1.0 to 10 mM, and more preferably 2.0 to 5.0 mM.

[0448] When a microplate is used as the carrier of the present invention, a solution containing calcium ions and a sample of the present invention are added to each well of the microplate on which the Tim protein has been immobilized (the Tim carrier of the present invention) in an amount of usually 50 μL to 300 μL, preferably 100 μL to 200 μL per well, and the reaction is carried out under a temperature condition of usually 2° C. to 37° C., preferably 2° C. to 30° C., for usually 0.5 hour to 24 hours, preferably 1 hour to 20 hours, more preferably 1 hour to 12 hours, thereby forming a complex of the Tim carrier of the present invention and the extracellular membrane vesicle or virus in the sample (the complex of the present invention). The calcium ion concentration in the calcium ion-containing solution is an amount such that the calcium ion concentration in the solution when the complex of the present invention is formed becomes usually 0.5 to 100 mM, preferably 1.0 to 10 mM, more preferably 2.0 to 5.0 mM.

[0449] <8-2. Detection step in the detection method of the present invention>

[0450] The detection step in the detection method of the present invention is a step of detecting the complex (the complex of the present invention) of the Tim vector of the present invention and the extracellular membrane vesicles or viruses in the sample after performing the complex formation step, performing the complex separation step of the sample as needed, and further performing the washing operation as needed.

[0451] -Complex separation process-

[0452] The complex separation step in the detection method of the present invention is a step of removing the sample from the obtained complex of the Tim protein bound to the carrier of the present invention (Tim carrier of the present invention) and the extracellular membrane vesicles or viruses in the sample (complex of the present invention) after the complex formation step, as needed.

[0453] The complex separation step in the detection method of the present invention can be any method as long as the complex of the present invention can be separated from the sample, in other words, as long as the so-called B / F separation can be performed, and the B / F separation method used in the field can be used. This method is the same as the complex separation step in the acquisition method of the present invention.

[0454] - Specific example of complex separation step -

[0455] The complex separation step in the detection method of the present invention can be carried out according to conventional methods in the field. However, when magnetic beads are used as the carrier of the present invention, for example, the container in which the complex formation step has been carried out is placed on a magnetic stand as needed, and the complex of the present invention is aggregated on the tube wall using magnetic force, and the sample in the supernatant is removed.

[0456] When a microplate is used as the carrier of the present invention, for example, the sample is removed from the microplate on which the complex formation step has been performed using a micropipette or a plate washer as necessary.

[0457] <8-3. Washing operation>

[0458] After the complex formation step, a complex separation step may be performed as needed, and the obtained complex of the present invention may be washed with a washing solution containing calcium ions as needed (hereinafter, sometimes referred to as "washing operation"). By the washing operation, inclusions in the sample of the present invention such as cell-derived components attached to the surface of the carrier of the present invention can be removed. Various conditions such as the washing method are the same as those of the washing operation in the acquisition method of the present invention.

[0459] - Specific example of washing operation -

[0460] When magnetic beads are used as the carrier of the present invention and a tube with a capacity of 1.5 mL is used, first, a washing solution containing calcium ions (for example, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6, which does not precipitate calcium, and containing usually 100 μL to 1500 μL, preferably 200 μL to 1000 μL) is added to a container containing the complex of the present invention obtained by the complex separation step, and stirred. Then, the container is placed on a magnetic stand, the complex is assembled on the tube wall using magnetic force, and the solution in the container is discarded. The washing operation can be repeated as many times as needed.

[0461] When a microplate is used as a carrier of the present invention, first, a washing solution containing calcium ions (for example, a buffer having a buffering effect and not causing calcium precipitation under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6, containing usually 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2.0 to 5.0 mM calcium ions, is added to the well containing the complex of the present invention obtained by the complex separation step, and then the added washing solution is removed. The washing operation can be repeated as many times as necessary.

[0462] <8-4. Inspection process>

[0463] The detection step in the detection method of the present invention may be any method as long as it can detect the presence and / or amount of the complex of the present invention, and any method known per se for use in immunological assays may be used.

[0464] The detection step of the present invention is not particularly limited except that the Tim vector of the present invention prepared by the method described above is used. Examples of such immunological assays include: assays utilizing agglutination reactions, such as the reverse passive agglutination method (Tokyo Kagaku Dojin, Biochemistry Experiment Lecture Series 5, Immunobiochemical Research Methods, pp. 36-37; Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Summary, 30th edition, pp. 844-845, etc.); optical assays utilizing agglutination reactions, such as the nephelometry method (Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Summary, 30th edition, pp. 851-853, etc.) and the immunoturbidimetry method (Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Summary, 30th edition, pp. 853-854, etc.); and radioimmunoassay (RIA) (Tokyo Kagaku Dojin, Biochemistry Experiment Lecture Series 5, Immunobiochemical Research Methods, pp. 57-61; Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Summary, 30th edition, pp. 856-862). etc.), immunoradiometric assay (IRMA) (Kanehara Publishing Co., Ltd., "Summary of Clinical Examination Methods", 30th edition, p.856-862, etc.), enzyme-linked immunosorbent assay (EIA) (Tokyo 10 Chemical Association, Continued Biochemistry Experiment Lecture 5, Immunobiochemical Research Methods, p.62-65; Kanehara Publishing Co., Ltd., "Summary of Clinical Examination Methods", 30th edition, p.862-865; Japanese Patent Application Publication No. 56-106154; Japanese Patent Application Publication No. 58-23796 The present invention relates to immunological assay methods known in the art, such as the ELISA (Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Summary, 30th edition, p. 1145-1149, etc.), the fluorescence / luminescence immunoassay (Kanehara Publishing Co., Ltd., Clinical Laboratory Methods Summary, 30th edition, p. 865-867, etc.), and the flow cytometry method, preferably the ELISA or the flow cytometry method.

[0465] When the detection process of the present invention is performed by enzyme-linked immunosorbent assay (ELISA) or flow cytometry, it can be performed according to a method known per se within the scope of the complex of the present invention as the detection object. For example, a method using an anti-extracellular membrane vesicle antibody that binds to an extracellular membrane vesicle or a virus, a method using a labeled primary antibody formed by labeling a primary antibody with a marker such as an antiviral antibody, or a method using the primary antibody and a labeled secondary antibody that binds to the primary antibody. As the labeled primary antibody and the labeled secondary antibody, any antibody can be used as long as it is an antibody for labeling antibodies used in ELISA or flow cytometry, and examples thereof include, for example, fluorescent labeled antibodies labeled with fluorescent substances such as Cy3, Cy5, FITC, rhodamine, PE, etc.; enzyme labeled antibodies labeled with enzymes such as peroxidase and alkaline phosphatase; magnetic bead labeled antibodies; infrared labeled antibodies, etc. The fluorescence of the labeled antibody, etc. can be measured by a method known per se corresponding to the labeling method (label) of the labeled antibody.

[0466] -Dilution of labeled antibody-

[0467] In the detection method of the present invention, as a diluent of the labeled primary antibody reacting with the complex of the present invention, or a diluent of the primary antibody and the labeled secondary antibody, in addition to containing calcium ions, as long as it does not hinder the binding of the Tim protein with the complex of the extracellular membrane vesicle or virus in the sample and the antibody, for example, water, a buffer having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 (such as TBS, HBS, etc.), etc. It should be noted that phosphate buffer is not preferred because it generates precipitation due to the binding of calcium. In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5 to 50mM, preferably from the range of 10 to 30mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200mM, preferably from the range of 140 to 160mM. In the solution, as long as it is an amount that does not hinder the binding of the Tim carrier of the present invention with the complex of the extracellular membrane vesicle or virus in the sample, salts such as sugars, NaCl, surfactants, preservatives, proteins, etc. can be contained. As surfactant, for example, Tween 20 can be mentioned, and the concentration of the surfactant in the solution containing the extracellular membrane vesicle or virus of the present invention is usually 0.00001-0.2%, preferably 0.0005-0.1%. In addition, the concentration of calcium ions contained in the diluent is usually 0.5-100mM, usually preferably 1-10mM, and usually more preferably 2-5mM.

[0468] In the detection method of the present invention, the dilution ratio of the labeled primary antibody, or the primary antibody and the labeled secondary antibody that react with the complex of the present invention varies depending on the activity and concentration of the antibodies, but is usually 10-fold to 1,000,000-fold, preferably 1,000-fold to 100,000-fold.

[0469] In the detection method of the present invention, the amount of the diluted solution of the labeled primary antibody, or the primary antibody and the labeled secondary antibody solution that reacts with the complex of the present invention, when the carrier of the present invention is beads, is generally 0.1mL to 1000mL, preferably 0.1mL to 500mL, and more preferably 0.1mL to 100mL relative to 1 mg of the carrier of the present invention; when the carrier of the present invention is a microplate, each well is generally 50μL to 300μL, preferably 50μL to 200μL, and more preferably 50μL to 100μL.

[0470] -Reaction (contact) temperature-

[0471] In the detection method of the present invention, the reaction temperature of the complex of the present invention and the labeled primary antibody, or the primary antibody and the labeled secondary antibody is usually 2 to 37°C, preferably 11 to 337°C, more preferably 20 to 30°C.

[0472] - Reaction (contact) time -

[0473] In the detection method of the present invention, the reaction time of the complex of the present invention and the labeled primary antibody, or the primary antibody and the labeled secondary antibody is usually 0.5 to 12 hours, preferably 1 to 4 hours, more preferably 1 to 2 hours.

[0474] In the detection operation of the detection step in the detection method of the present invention, when a labeled primary antibody is used, after the complex of the present invention is reacted with the labeled primary antibody, unreacted labeled primary antibody can be removed by a washing operation. In addition, when a primary antibody and a labeled secondary antibody are used, after the complex of the present invention is reacted with the primary antibody, after the complex of the present invention and the primary antibody is reacted with the labeled secondary antibody, unreacted primary antibody and labeled secondary antibody can be removed by a washing operation. As a washing method, in addition to using a washing solution containing calcium ions as described above, a washing method commonly used in this field can be used. As the washing solution containing calcium ions used in the washing operation, it is sufficient that it contains calcium ions of usually 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM, and does not affect the binding of the extracellular membrane vesicle or virus of the present invention to the Tim protein of the present invention and the carrier of the present invention in the complex. For example, a buffer solution (such as TBS and HBS) containing calcium ions of usually 0.5 to 100 mM, usually preferably 1 to 10 mM, and usually more preferably 2 to 5 mM, which has a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6 and does not precipitate calcium. It should be noted that phosphate buffer is not preferred because it generates precipitation by binding with calcium. In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. In the solution, as long as it is an amount that does not hinder the binding of the extracellular membrane vesicles or viruses of the present invention in the complex with the Tim protein of the present invention and the carrier of the present invention, it is possible to contain salts such as sugars and NaCl, surfactants, preservatives, proteins, etc. As a surfactant, for example, Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) can be cited, and the concentration of the surfactant in the washing solution is usually 0.00001-0.2%, usually preferably 0.0005-0.1%. When the carrier of the present invention is a bead, it is usually 0.1 mL-1000 mL, preferably 0.1 mL-500 mL, and more preferably 0.1 mL-100 mL relative to 1 mg of the carrier of the present invention. When the carrier of the present invention is a microplate, the amount of washing solution used in one hole is usually 100 μL-300 μL, preferably 200 μL-300 μL, and the washing solution is removed after addition. As needed, the washing operation can be repeated multiple times.

[0475] -Detection-

[0476] When the detection step in the detection method of the present invention is colorimetric detection, examples of the labeling substance for labeling the primary antibody or the secondary antibody include peroxidase, alkaline phosphatase, etc. The labeling substance can be detected by a known method corresponding to each labeling substance.

[0477] As the colorimetric matrix liquid in colorimetric detection, any colorimetric matrix liquid may be used as long as it is a colorimetric matrix liquid commonly used in this field. For example, when peroxidase is used as a marker, TMB (tetramethylbenzidine) solution or OPD (o-phenylenediamine) solution can be mentioned, and TMB solution is preferred.

[0478] Regarding the amount of the colorimetric substrate in the colorimetric detection, when the carrier of the present invention is beads, it is usually 0.1mL to 1000mL, preferably 0.1mL to 500mL, and more preferably 0.1mL to 100mL relative to 1 mg of the carrier of the present invention. When the carrier of the present invention is a microplate, it is usually 50μL to 300μL per well, preferably 50μL to 200μL, and more preferably 50μL to 100μL.

[0479] When the detection step in the detection method of the present invention is colorimetric detection, the reaction time with the colorimetric substrate solution is usually 5 to 60 minutes, preferably 10 to 40 minutes.

[0480] When the detection step in the detection method of the present invention is colorimetric detection, the reaction temperature with the colorimetric substrate solution is usually 2°C to 37°C, preferably 20°C to 30°C.

[0481] When the detection step in the detection method of the present invention is colorimetric detection, in order to stop the colorimetric reaction, a strong acid such as 1 mol / L hydrochloric acid or 1 mol / L sulfuric acid is usually added as a reaction stopping solution in an amount equal to that of the colorimetric matrix solution to stop the colorimetric reaction.

[0482] When the detection step in the detection method of the present invention is fluorescence detection, and a fluorescent substance is used to label the primary antibody or the labeled secondary antibody, a fluorescence measuring liquid is added to the complex of the present invention and the labeled primary antibody, or to the complex of the present invention and the primary antibody and the labeled secondary antibody, and the fluorescence is measured. For the fluorescence measuring liquid, in addition to the fluorescence measuring liquid containing calcium ions, a fluorescence measuring liquid that can be generally used in this field (hereinafter, sometimes simply referred to as "measuring liquid") can be used. As the measuring solution containing calcium ions used as the fluorescence measuring solution, any solution containing calcium ions of usually 0.5 to 100 mM, usually preferably 1 to 10 mM, usually more preferably 2 to 5 mM, and not affecting the binding of the extracellular membrane vesicle or virus of the present invention to the Tim protein of the present invention and the carrier of the present invention in the complex can be used. For example, a buffer solution (such as TBS and HBS) containing calcium ions of usually 0.5 to 100 mM, usually preferably 1 to 10 mM, usually more preferably 2 to 5 mM, and having a buffering effect under the conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6, and not causing calcium precipitation can be mentioned. It should be noted that phosphate buffer is not preferred because it generates precipitation by binding with calcium. In addition, as the buffer concentration in the buffer, it is usually appropriately selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is usually appropriately selected from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. In the solution, as long as it is an amount that does not hinder the binding of the extracellular membrane vesicle or virus of the present invention in the complex with the Tim protein of the present invention and the carrier of the present invention, it is possible to contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins such as BSA, etc. As a surfactant, for example, Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) can be cited, and the concentration of the surfactant in the washing solution is usually 0.00001 to 0.2%, and usually preferably 0.0005 to 0.1%. Regarding the amount of the fluorescent assay solution used in the detection method of the present invention, when the detection method of the present invention is an ELISA method and the carrier of the present invention is a microplate, the amount of the assay solution used in one well is usually 50 μL to 300 μL, preferably 50 μL to 200 μL. When the detection method of the present invention is a flow cytometry method and the carrier of the present invention is a bead, the amount of the assay solution relative to 1 mg of the carrier of the present invention is usually 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, and more preferably 0.1 mL to 100 mL.

[0483] - Specific example of the detection step in the detection method of the present invention -

[0484] For example, when a microplate is used as the carrier of the present invention and the ELISA method is performed, after the complex formation step and the complex separation step as needed, and further after washing as needed, a dilution solution of usually 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 50 μL to 100 μL is added to each well of the obtained microplate on which the Tim protein of the present invention has been immobilized, and the reaction is carried out under the temperature conditions of usually 2 to 37° C., preferably 11 to 37° C., for usually 0.5 to 12 hours, preferably 1 to 4 hours. Then, each well is washed with a washing buffer containing calcium ions (a solution containing calcium ions of usually 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM, and which does not affect the binding of the extracellular membrane vesicle or virus of the present invention in the complex with the Tim protein of the present invention and the carrier of the present invention). The dilution solution is a solution containing usually 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM, and which does not affect the binding of the extracellular membrane vesicle or virus of the present invention to the Tim protein of the present invention and the carrier of the present invention). The primary antibody or non-labeled primary antibody against extracellular membrane vesicles or viruses labeled with peroxidase is diluted to a dilution solution generally 10 to 1,000,000 times, preferably 1,000 to 100,000 times, with a dilution solution containing 100 mM, usually preferably 1 to 10 mM, and usually more preferably 2 to 5 mM calcium ions. When using non-labeled primary antibodies, the secondary antibody labeled with peroxidase is reacted in the same manner as the primary antibody, and then each well is washed with a washing buffer containing calcium ions (containing calcium ions generally 0.5 to 100 mM, usually preferably 1 to 10 mM, usually more preferably 2 to 5 mM, and does not affect the binding of the extracellular membrane vesicle or virus of the present invention to the Tim protein of the present invention and the carrier of the present invention in the complex) of generally 100 μL to 300 μL, preferably 200 μL to 300 μL. Then, a color development substrate solution such as TMB solution or OPD solution is added to each well in an amount of usually 50 μL to 300 μL, preferably 50 μL to 200 μL, and more preferably 50 μL to 100 μL, and the reaction is carried out at a temperature of usually 2° C. to 37° C., preferably 20° C. to 30° C. for 5 minutes to 60 minutes, preferably 10 minutes to 40 minutes. Then, a reaction stop solution such as 1 mol / L hydrochloric acid or 1 mol / L sulfuric acid is added in an amount equal to that of the color development substrate solution to stop the color development reaction, and the absorbance is measured with an enzyme reader.

[0485] For example, when beads are used as the carrier of the present invention and flow cytometry is performed, after the complex formation step and the complex separation step as needed, and further after washing as needed, the obtained beads on which the Tim protein of the present invention has been immobilized are added with a dilution solution of usually 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, more preferably 0.1 mL to 100 mL, per 1 mg of the carrier of the present invention, and the reaction is carried out at a temperature of usually 2 to 37° C., preferably 11 to 37° C. for 0.5 to 12 hours, preferably 1 to 4 hours, and then the beads are washed with a washing buffer containing calcium ions (a solution containing calcium ions of usually 0.5 to 100 mM, usually preferably 1 to 10 mM, usually more preferably 2 to 5 mM, and which does not affect the binding of the extracellular membrane vesicle or virus of the present invention in the complex with the Tim protein of the present invention and the carrier of the present invention). The fluorescently labeled primary antibody or the non-labeled primary antibody is diluted to a dilution solution of 10 times to 1000000 times, preferably 1000 times to 100000 times, with a calcium ion dilution solution of 100mM, usually preferably 1 to 10mM, and usually more preferably 2 to 5mM. When using an unlabeled primary antibody, the fluorescently labeled secondary antibody is reacted in the same manner as the primary antibody, and then the beads are washed with a washing buffer containing calcium ions (containing usually 0.5 to 100mM, usually preferably 1 to 10mM, usually more preferably 2 to 5mM calcium ions, and a solution that does not affect the binding of the cell extracellular membrane vesicle or virus of the present invention to the Tim protein of the present invention and the vector of the present invention in the complex). Then, after adding a fluorescence measurement solution of usually 0.1mL to 1000mL, preferably 0.1mL to 500mL, and more preferably 0.1mL to 100mL to 1mg of the vector of the present invention to suspend the beads, the fluorescence intensity is measured by a flow cytometer.

[0486] It is believed that the affinity of antibodies for antigens is generally at the level of Kd of 10 nm to 100 pm. On the other hand, the binding force of Tim4 protein with phosphatidylserine has been reported to be Kd = about 2 nm (Nature (impact factor: 41.46). 12 / 2007; 450(7168): 435-9. DOI: 10.1038 / nature06307). Although the affinity of antibodies against surface antigens of extracellular membrane vesicles or viral envelopes for the surface antigens is the same as the affinity between phosphatidylserine on the surface of extracellular membrane vesicles or viral envelopes and the Tim protein of the present invention, it is unexpected that the detection method of the present invention for detecting extracellular membrane vesicles or viruses using the Tim protein of the present invention has higher sensitivity than the existing method for detecting extracellular membrane vesicles or viruses using antibodies.

[0487] The extracellular membrane vesicles or viruses obtained by the method of the present invention can be used for the analysis of nucleic acids such as proteins, small molecule RNA (microRNA) on the surface and inside of the particles, and can also be used in basic research such as functional analysis of extracellular membrane vesicles or viruses. In addition, they can also be used in diagnostic drugs, pharmaceuticals, vaccines, etc.

[0488] <9. Kit for capturing extracellular membrane vesicles or viruses of the present invention>

[0489] The extracellular membrane vesicle or virus capture kit of the present invention comprises the following components: 1. a reagent comprising the Tim protein of the present invention and a reagent comprising the vector of the present invention, or, 2. a reagent comprising the Tim vector of the present invention.

[0490] The kit may further include at least one selected from the group consisting of the washing solution containing calcium ions, the solution containing a protein modifier, and the solution containing a calcium ion chelating agent. Specific examples, preferred embodiments, etc. of each component are as described above.

[0491] In addition, the reagents contained in the kit may include reagents commonly used in the field, such as buffers, sensitizers, surfactants, preservatives (such as sodium azide, salicylic acid, benzoic acid, etc.), stabilizers (such as albumin, globulin, water-soluble gelatin, surfactants, sugars, etc.), activators, agents to avoid the influence of coexisting substances, and reagents used in other fields that do not hinder the stability of coexisting reagents, do not hinder the reaction or binding of the Tim protein of the present invention with the carrier, and do not hinder the reaction or binding of the Tim protein of the present invention bound to the carrier of the present invention with the extracellular membrane vesicles in the sample. In addition, in order to exert the effects of each reagent, the concentration range of the reagents, etc., can also be appropriately selected from the concentration range, etc., which are commonly used, for use.

[0492] Furthermore, the kit of the present invention may include instructions for the acquisition method of the present invention, the removal method of the present invention, the detection method of the present invention, etc. The "instructions" refer to instructions for use, accompanying documents, or manuals (leaflets) of the kit that substantially record the characteristics / principles / operation steps, determination steps, etc. of the method through text or diagrams.

[0493] Hereinafter, the present invention will be described in more detail with reference to experimental examples, embodiments and comparative examples, but the present invention is not limited thereto in any way.

[0494] The Tim protein of the present invention and the T-cell immunoglobulin and mucin domain-containing molecule 2 (Tim2) protein (hereinafter, sometimes abbreviated as “Tim2 protein”) may be collectively referred to as “Tim family protein”.

[0495] Example

[0496] Experimental Example 1. Preparation of Fc-tagged Tim4 protein

[0497] Fc tag-fused Tim4 protein was prepared by the following method.

[0498] <(1) Construction and cultivation of vector>

[0499] First, according to the method described in Miyanishi et al. Nature 2007, Vol. 450: 15, an Fc tag fusion Tim4 protein expression vector (hereinafter sometimes abbreviated as "pEF-Tim4-Fc") was constructed, which was formed by combining a cDNA (sequence number 26) encoding the N-terminal amino acid domain of the mouse Tim4 protein at positions 1 to 273 with the SalI-EcoRV site of the pEF-Fc vector.

[0500] On the other hand, 293T cells (RIKEN BRC) were cultured for 1 day in 25 150 mm cell culture dishes each using 20 mL of DMEM (Dulbecco's Modified Eagle Medium) (manufactured by Nakara Technologies) containing 10% FBS (manufactured by Biowest). Then, 25 mL of DMEM containing 10% FBS was replaced with 25 mL of DMEM not containing FBS for each dish.

[0501] Then, according to a conventional method, 20 μg of pEF-Tim4-Fc was gene-transfected into each 293T cell using polyethylenimine "MAX" (manufactured by Polyscience Co., Ltd.). After gene transfection, the cells were incubated at 37°C and 5% CO. 2 Under the same conditions, the gene-transferred 293T cells were cultured for 4 days.

[0502] <(2) Purification>

[0503] The culture fluid of the gene-transferred 293T cells was centrifuged (800×g, 5 minutes), and the culture supernatants were collected and pooled. The culture supernatants were filtered using a RF (trade name, Rapid Flow) 0.2 μm filter unit (manufactured by Thermo Fisher Scientific) to separate impurities and obtain a culture supernatant filtrate.

[0504] Then, 500 mL of the obtained culture supernatant filtrate was added to a Poly-Prep chromatography column (manufactured by Bio-Rad) filled with 700 μL of natural protein A Sepharose 4FF (nProtein A Sepharose 4Fast Flow) (manufactured by General Electric Healthcare Japan, protein A has affinity for Fc tag) washed with 20 mL of PBS, so that the Fc tag-fused Tim4 protein in the culture supernatant filtrate was bound to the natural protein A Sepharose 4FF. Then, the natural protein A Sepharose 4FF was washed with 20 mL of PBS. Then, elution was performed five times in 600 μL of 0.1 M glycine-hydrochloric acid buffer (pH 3.0) and 100 μL of 1 M Tris buffer (pH 8.0) as a neutralizing solution to obtain five elution fractions of 600 μL each.

[0505] For the obtained 5 dissolution components, the absorbance at 280 nm was measured respectively to determine the presence or absence of protein. After mixing the components containing protein into one, using an Amicon Ultra-0.5 mL 10K centrifugal filter column (product name and model) (manufactured by Millipore Corporation), it was concentrated by ultrafiltration method, and then using the column, the solvent was replaced with 40 μL of PBS. Then, after quantifying the protein amount in the eluate replaced with PBS by the bicinchoninic acid (BCA) method, the protein concentration was adjusted to 88 μg / mL with PBS to obtain a PBS solution containing Fc-tagged mouse-derived Tim4 protein (sometimes abbreviated as "Fc-tagged mTim4 protein") (sometimes abbreviated as "PBS solution containing Fc-tagged mTim4 protein").

[0506] Experimental Example 2. Preparation of FLAG-tagged Tim4 protein

[0507] The FLAG-tagged Tim4 protein was prepared by the following method.

[0508] <(1) Construction and culture of vector>

[0509] First, according to the conventional method, the FLAG-tagged mouse-derived Tim4 cDNA (cDNA encoding an amino acid sequence in which 1×FLAG was fused to the C-terminus of the N-terminal 1-273 amino acid domain of mouse-derived Tim4 protein, SEQ ID NO: 33 (with a stop codon (taa) at the end and a base sequence encoding 1×FLAG, but without restriction enzyme cleavage sites), manufactured by FASMAC Co., Ltd.) was combined with the XhoI / BamHI sites of the pCAG-Neo vector (manufactured by Wako Pure Chemical Industries, Ltd.) to construct a vector for expressing the FLAG-tagged Tim4 protein (hereinafter, sometimes abbreviated as "pCAG-Tim4-FLAG").

[0510] On the other hand, in a 225 cm 2 flask, 50 mL of DMEM (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% FBS (manufactured by Biosera) was used, and 293T cells (RIKEN BRC) were seeded at a cell confluence of 70 - 90% at the time of gene introduction and cultured for 1 day. Then, according to the conventional method, using Lipofectamine 2000 (product name, manufactured by Thermo Fisher Scientific), 60 μg of pCAG-Tim4-FLAG was introduced into the 293T cells cultured for 1 day. After gene introduction, at 37 °C, 5% CO 2The gene-transferred 293T cells were cultured for 1 day under the following conditions. Then, all the culture medium was removed, the gene-transferred 293T cells were washed twice with 10 mL of PBS, and the culture medium was replaced with 50 mL of Opti-MEM (trade name, manufactured by Thermo Fisher Scientific) and incubated at 37°C and 5% CO 2 Cultured for 3 days.

[0511] <(2) Purification>

[0512] The culture fluid of the gene-transferred 293T cells after culturing for 3 days was centrifuged (300×g, 5 minutes) to recover the culture supernatant. The recovered culture supernatant was then centrifuged three times (first time: 300×g, 3 minutes; second time: 1200×g, 20 minutes; third time: 10000×g, 20 minutes) to obtain a supernatant from which impurities were separated. The obtained supernatant was ultrafiltered using Vivaspin (trade name, molecular weight cutoff 30000, manufactured by General Electric Company (GE)) and concentrated to 10 times to obtain a culture supernatant concentrate. To the obtained culture supernatant concentrate 5 mL, 500 μL (1 / 10 of the culture supernatant concentrate) of ANTI-FLAG M2 affinity gel (manufactured by Sigma) washed with PBS was added, and the mixture was mixed by inversion for 3 hours to allow the FLAG tag fused Tim4 protein in the culture supernatant concentrate to react with the anti-FLAG M2 affinity gel, so that the FLAG tag fused Tim4 protein was bound to the anti-FLAG M2 affinity gel. Then, the anti-FLAG M2 affinity gel was washed 3 times with 5 μL of PBS.

[0513] Then, 250 μL (half the amount of the anti-FLAG M2 affinity gel used) of a 200 μg / mL FLAG peptide solution (a solution prepared by diluting DYKDDDDK peptide (manufactured by Wako Pure Chemical Industries, Ltd.) with PBS) was added to the anti-FLAG M2 affinity gel, mixed by inversion at 4°C for 30 minutes, and centrifuged (4°C, 8000×g, 1 minute), and the supernatant (eluate) was recovered. In order to remove the FLAG peptide in the recovered supernatant (eluate), 20 mL of PBS was added to the supernatant (eluate) for ultrafiltration to obtain a concentrated solution. PBS was added to the obtained concentrated solution, and the liquid volume was adjusted to 500 μL to obtain a PBS solution containing a FLAG tag-fused mouse-derived Tim4 protein (sometimes abbreviated as "FLAG tag-fused mTim4 protein") (sometimes abbreviated as "PBS solution containing a FLAG tag-fused mTim4 protein") (Experimental Example 2).

[0514] Experimental Example 3. Preparation of His-tag fused Tim4 protein

[0515] The His-tag fused Tim4 protein was prepared by the following method.

[0516] <(1) Construction and cultivation of vector>

[0517] According to conventional methods, His-tag fused mouse Tim4 cDNA (cDNA encoding an amino acid sequence formed by fusion of a 6×His tag to the C-terminus of the N-terminal 1 to 273 amino acid domain of the mouse Tim4 protein, sequence number 34 (cDNA encoding a 6×His tag with a stop codon (tga) at the end)) was combined with the XhoI / BamHI site of the pCAG-Neo vector (manufactured by Wako Pure Chemical Industries, Ltd.) to construct a His-tag fused Tim4 protein expression vector (hereinafter sometimes abbreviated as "pCAG-Tim4-His").

[0518] On the other hand, at 225cm 2 In a flask, 293T cells (RIKEN BRC) were cultured for 1 day in 50 mL of DMEM (Wako Pure Chemical Industries, Ltd.) containing 10% FBS (Biosera). Then, according to a conventional method, 60 μg of pCAG-Tim4-His was introduced into 293T cells using Lipfectamine 2000 (trade name, Thermo Fisher Scientific). After gene introduction, the cells were incubated at 37°C and 5% CO. 2 The gene-transferred 293T cells were cultured for 1 day under the following conditions. Then, all the culture medium was removed, the gene-transferred 293T cells were washed twice with 10 mL of PBS, and the culture medium was replaced with 50 mL of Opti-MEM (trade name, manufactured by Thermo Fisher Scientific) and incubated at 37°C and 5% CO 2 Cultured for 3 days.

[0519] <(2) Purification>

[0520] The culture fluid of the gene-transferred 293T cells after culturing for 3 days was centrifuged (300×g, 5 minutes) to recover the culture supernatant. The recovered culture supernatant was then centrifuged three times (first time: 300×g, 3 minutes; second time: 1200×g, 20 minutes; third time: 10000×g, 20 minutes) to obtain a supernatant from which impurities were separated. The obtained supernatant was ultrafiltered using Vivaspin (trade name, molecular weight cutoff 30000, manufactured by General Electric Company (GE)) and concentrated to 10 times to obtain a culture supernatant concentrate.

[0521] According to the scheme of being attached to nickel chelate sepharose 6FF (Ni Sepharose 6Fast Flow), prepare nickel chelate sepharose 6FF (General Electric Company (GE society) system) 2.7mL, transfer to post.Measure OD 280nm with the flow velocity of about 1mL / min on one side, add binding buffer (50mM Tris-HCl, 500mM NaCl, 20mM imidazoles) in post until the absorbance of OD280nm is stable.After the culture supernatant concentrate 5mL obtained is added to post, fully add binding buffer until the absorbance is stable, wash.Then, stripping buffer (50mM Tris-HCl, 500mM NaCl, 300mM imidazoles) is added to post, reclaim 10 stripping components of each 1mL.

[0522] <(3) Electrophoresis / Silver staining>

[0523] 15 μL of each eluted fraction was taken and mixed with 5 μL of 4× sample buffer (Wako Pure Chemical Industries, Ltd.), and heated at 98°C for 5 minutes to obtain 20 μL of each electrophoresis sample. 15 μL of each electrophoresis sample was added to SuperSep Ace 5-20% gel (trade name, Wako Pure Chemical Industries, Ltd.), and electrophoresis was performed at a current of 25 mA for 65 minutes.

[0524] The gel was stained with Wako Silver Staining II Kit (manufactured by Wako Pure Chemical Industries, Ltd.), and 5 fractions containing the target protein were selected and pooled (combined into one). In order to remove the imidazole contained in the eluate, 20 mL of PBS was added, and ultrafiltration was performed using Vivaspin (trade name, molecular weight cutoff 30000, manufactured by General Electric Company (GE)) to obtain a concentrated solution. PBS was added to the obtained concentrated solution, and the liquid volume was adjusted to 500 μL to obtain a PBS solution containing a His-tag fused mouse-derived Tim4 protein (sometimes abbreviated as "His-tag fused mTim4 protein") (sometimes abbreviated as "PBS solution containing a His-tag fused mTim4 protein").

[0525] Example 1-8. Obtaining extracellular membrane vesicles using the Tim4 vector of the present invention (obtaining method of the present invention)

[0526] The Tim4 vector of the present invention is prepared as follows, and the extracellular membrane vesicles of the present invention are obtained using the vector.

[0527] <(1) Preparation of culture supernatant sample containing calcium ions>

[0528] 80 mL of X-VIVO 15 medium (trade name, manufactured by Lonza) was used and incubated at 37°C and 5% CO 2 Under the condition of 1×10 7 After culturing the cells for 3 days, the cells were centrifuged (300 × g, 5 minutes) to precipitate the cells and the supernatant was removed. The precipitated cells were suspended in 60 mL of X-VIVO15 medium containing 10 μM monensin sodium (MP Biomedicals) and incubated at 37°C and 5% CO. 2 Cultured under the same conditions for 24 hours.

[0529] Then, the culture solution was centrifuged (300×g, 5 minutes) to recover the culture supernatant. The recovered culture supernatant 60 mL was further centrifuged three times (first time: 300×g, 3 minutes; second time: 1200×g, 20 minutes; third time: 10000×g, 20 minutes) to separate impurities and obtain a supernatant. The obtained supernatant was ultrafiltered using Vivaspin (trade name, molecular weight cutoff 30000, manufactured by General Electric (GE)) until it became less than 6 mL to obtain a concentrated solution. X-VIVO15 culture medium containing 10μM monensin sodium (manufactured by MP Biomedicals) was added to the obtained concentrated solution, and the liquid volume was adjusted to 6 mL to obtain a 10-fold concentrated K562 cell culture concentrate sample (hereinafter, sometimes referred to as "culture supernatant sample").

[0530] In addition, CaCl was added to the obtained culture supernatant sample. 2 , making the final concentration 2 mM, to obtain a solution containing 2 mM CaCl 2 A concentrated K562 cell culture supernatant sample (hereinafter, sometimes abbreviated as “calcium ion-containing culture supernatant sample”) was prepared.

[0531] <(2) Biotin labeling of Fc tag-fused mouse Tim4 protein>

[0532] For 114 μL of PBS solution containing Fc-tagged mTim4 protein fused by the same method as in Experimental Example 1 (containing 10 μg of Fc-tagged mTim4 protein), the amino group of the Fc-tagged mTim4 protein was biotin-labeled using the Biotin Labeling Kit-Amino (manufactured by Dojindo Chemical Research Institute) according to the protocol included in the kit, thereby obtaining 100 μL of PBS solution containing 3.8 μg of amino-biotin-labeled Fc-tagged mouse-derived Tim4 protein (sometimes abbreviated as "amino-biotin-labeled Fc-tagged mTim4 protein") (sometimes abbreviated as "PBS solution containing amino-biotin-labeled Fc-tagged mTim4 protein").

[0533] In addition, for 114 μL of PBS solution containing Fc-tagged mTim4 protein prepared by the same method as in Experimental Example 1 (containing 10 μg of Fc-tagged mouse-derived Tim4 protein), the thiol group of the Fc-tagged mouse-derived Tim4 protein was biotin-labeled using a biotin labeling kit-thiol (manufactured by Dojindo Chemical Research Institute) according to the protocol included in the kit, thereby obtaining 100 μL of PBS solution containing 5.9 μg of thiol-biotin-labeled Fc-tagged mouse-derived Tim4 protein (sometimes abbreviated as "thiol-biotin-labeled Fc-tagged mTim4 protein") (sometimes abbreviated as "PBS solution containing thiol-biotin-labeled Fc-tagged mTim4 protein").

[0534] <(3) Dilution of PBS solution containing Fc tag-fused mouse-derived Tim4 protein>

[0535] 11.4 μL of a PBS solution containing an Fc-tagged mTim4 protein prepared in the same manner as in Experimental Example 1 was mixed with 188.6 μL of PBS to obtain 200 μL of a PBS solution containing 1 μg of a biotin-unlabeled Fc-tagged mTim4 protein.

[0536] 26.3 μL of the PBS solution containing aminobiotin-labeled Fc-tagged mTim4 protein (containing 1 μg of aminobiotin-labeled Fc-tagged mTim4 protein) prepared above was mixed with 173.7 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of aminobiotin-labeled Fc-tagged mTim4 protein.

[0537] In addition, 16.9 μL of the PBS solution containing thiol-biotin labeled Fc tag fusion mTim4 protein (containing 1 μg of thiol-biotin labeled Fc tag fusion mTim4 protein) prepared above was mixed with 183.1 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of thiol-biotin labeled Fc tag fusion mTim4 protein.

[0538] <(4) Washing of beads>

[0539] 20 μL (containing 0.6 mg of Dynabeads G protein) of PBS-T solution (trade name, manufactured by Thermo Fisher Scientific) containing 30 μg / μL Dynabeads G protein was dispensed into three 1.5 mL tubes (manufactured by BM Instruments). Then, 500 μL of PBS was added to each 1.5 mL tube, and after stirring, the 1.5 mL tubes were placed on a magnetic stand, and the Dynabeads G protein was aggregated on the tube wall using a magnetic force, and the solution in the 1.5 mL tube was discarded with a pipette (hereinafter, sometimes referred to as "washing operation").

[0540] Separately, 60 μL of a PBS solution containing 10 μg / μL Dynabeads M-270 Streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific) (containing 0.6 mg of Dynabeads M-270 Streptavidin C1 beads) was dispensed into a 1.5 mL tube (manufactured by BM Equipment Co., Ltd.), and the washing operation was performed in the same manner as above with 500 μL of PBS.

[0541] <(5) Immobilization of Fc-tagged mouse Tim4 protein on beads>

[0542] In three 1.5 mL tubes containing Dynabeads G protein (0.6 mg), add a total of 200 μL of the PBS solution containing 1 μg of the biotin-unlabeled Fc-tagged mTim4 protein prepared above to one of them, and react at 8°C for 1 hour to obtain 200 μL of PBS solution containing a vector (mTim4 vector) bound to the biotin-unlabeled Fc-tagged mTim4 protein.

[0543] In the remaining two 1.5 mL tubes containing Dynabeads G protein (0.6 mg), add a total of 200 μL of PBS solution containing 1 μg of amino-biotin-labeled Fc-tagged mTim4 protein prepared above to one of them, react at 8°C for 1 hour, and obtain 200 μL of PBS solution containing a vector (mTim4 vector) containing amino-biotin-labeled Fc-tagged mTim4 protein.

[0544] To another, add a total of 200 μL of the PBS solution containing 1 μg of the Fc-tagged mTim4 protein fused to thiol-biotin labeled as above, and react at 8°C for 1 hour to obtain 200 μL of PBS solution containing a vector (mTim4 vector) containing the mTim4 protein fused to the Fc-tagged mTim4 protein labeled with thiol-biotin.

[0545] To a 1.5 mL tube containing Dynabeads M-270 streptavidin C1 beads, add 200 μL of PBS solution containing 1 μg of Fc-tagged mTim4 protein labeled with thiol-biotin prepared by the same method as above, and react at 8°C for 1 hour to obtain 200 μL of PBS solution containing a vector (mTim4 vector) containing mTim4 protein fused to Fc-tagged mTim4 protein labeled with thiol-biotin.

[0546] Thus, 200 μL of PBS solutions containing 0.6 mg of each of the four types of mTim4 vectors shown in Table 1 below were obtained.

[0547] Table 1

[0548]

[0549] <(6) Obtaining extracellular membrane vesicles using the obtaining method of the present invention>

[0550] 0.6 mg of each of the four mTim4 vectors listed in Table 1 was washed three times with 500 μL of PBS. Then, 200 μL of the culture supernatant sample containing calcium ions prepared in (1) was added to each mTim4 vector in a particle state, and the mixture was reacted at 8°C for 3 hours.

[0551] 2 mM CaCl 2 TBS-T (Tris Buffer), 0.05% Tween 20, 2 mM CaCl 2) 500 μL were used to wash the reacted mTim4 vectors three times. During the third washing operation, 250 μL of each of the four mTim4 vectors were dispensed into two 1.5 mL tubes.

[0552] To 0.3 mg of each of the four types of mTim4 vectors in particle state, 20 μL of 1% SDS aqueous solution or 1 mM EDTA aqueous solution was added as an eluent, and then mixed for 10 seconds at room temperature using a vortex mixer, and the rotation was stopped (spin down). 1.5 mL tubes were placed on magnetic racks, and the mTim4 vectors were aggregated on the tube wall using magnetic force, and the supernatant (eluent) was recovered.

[0553] The types of mTim4 protein and vector used in each example, the type of eluate used to obtain extracellular membrane vesicles from the mTim4 vector, and the lane numbers in the Western blotting described below are shown in Table 2 below.

[0554] Table 2

[0555]

[0556] <(7) Western blotting>

[0557] To 7.5 μL of each supernatant (eluate) obtained in Examples 1 to 8, 2.5 μL of 4× sample buffer (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was heated at 98°C for 5 minutes to obtain each sample for protein immunoblotting. To SuperSep Ace 5-20% gel (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), 10 μL of each sample for protein immunoblotting was added, and electrophoresis was performed at 25 mA for 65 minutes. A semi-dry blotting apparatus was used with discontinuous buffers (anode buffer 1: 0.3 M Tris / 20% methanol; anode buffer 2: 0.025 M Tris / 20% methanol; cathode buffer: 0.025 M Tris / 0.04 M aminocaproic acid / 20% methanol) at 1 mA / cm 2 The obtained electrophoresis gel was transferred to a PVDF membrane (manufactured by Millipore) for 60 minutes under the conditions of .

[0558] 3% skim milk diluted with PBS-T (PBS buffer, 0.1% Tween 20) was added to the PVDF membrane and reacted at room temperature for 1 hour for blocking. Then, 2 mL of anti-human Lamp-1 mouse monoclonal antibody (manufactured by BD Biosciences, hereinafter sometimes referred to as "anti-human Lamp-1 antibody") diluted 250 times with PBS-T was reacted at 8°C overnight.

[0559] Then, the PVDF membrane after the reaction was washed 3 times with PBS-T, and the secondary antibody {anti-mouse IgG (H+L), rabbit, IgG component, peroxidase-binding antibody} (Wako Pure Chemical Industries, Ltd.) diluted 10,000 times with PBS-T was reacted for 1 hour at room temperature. After washing 5 times with PBS-T, ImmunoStar Zeta (trade name, Wako Pure Chemical Industries, Ltd.) was added, and the luminescent signal was detected using LAS-4000 (General Electric Company (GE)). It should be noted that the anti-human Lamp-1 antibody is an antibody against Lamp-1, which is one of the marker proteins of exosomes.

[0560] <Results>

[0561] The obtained protein immunoblotting results are shown in Figure 1 . Figure 1 , the lanes are as follows.

[0562] Lane 1: Results of Example 1 (results when using an mTim4 vector in which a biotin-unlabeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1% SDS aqueous solution was used as an eluent);

[0563] Lane 2: Results of Example 2 (results when using an mTim4 vector in which a biotin-unlabeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1 mM EDTA solution was used as an eluent);

[0564] Lane 3: Results of Example 3 (results when using an mTim4 vector in which an aminobiotin-labeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1% SDS aqueous solution was used as an eluent);

[0565] Lane 4: Results of Example 4 (results when using an mTim4 vector in which an aminobiotin-labeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1 mM EDTA solution was used as an eluent);

[0566] Lane 5: Results of Example 5 (results when using an mTim4 vector in which a thiol-biotin-labeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1% SDS aqueous solution was used as an eluent);

[0567] Lane 6: Results of Example 6 (results when using an mTim4 vector in which a thiol-biotin-labeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1 mM EDTA solution was used as an eluent);

[0568] Lane 7: Results of Example 7 (results when using an mTim4 vector in which a thiol-biotin-labeled Fc tag-fused mTim4 protein was bound to streptavidin beads and a 1% SDS aqueous solution was used as an eluent);

[0569] Lane 8: Results of Example 8 (results when using an mTim4 vector in which a mouse-derived Tim4 protein fused with a thiol-biotin-labeled Fc tag was bound to streptavidin beads and a 1 mM EDTA solution was used as an eluent).

[0570] according to Figure 1 In any case in Examples 1-8, a band of Lamp-1, a marker of exosomes, was obtained at around 100 kDa, from which it can be seen that the extracellular membrane vesicles containing exosomes can be obtained by the acquisition method of the present invention (Example 1-8: Lanes 1-8).

[0571] In particular, by comparing Examples 2, 4, 6, and 8, it can be seen that when a calcium ion chelator is used as an eluent, more extracellular membrane vesicles can be obtained when the Tim4 protein is bound to the carrier via the sulfhydryl group of the Tim4 protein (Example 8: Lane 8), compared to the case where the Tim4 protein is bound to the carrier via an amino group (Example 4: Lane 4) and the case where the Tim4 protein is bound via an affinity tag (Example 2: Lane 2; Example 6: Lane 6).

[0572] Example 9-16. Obtaining extracellular membrane vesicles using the Tim4 vector of the present invention (obtaining method of the present invention)

[0573] The Tim4 vector of the present invention is prepared as shown below, and the extracellular membrane vesicles of the present invention are obtained using the vector.

[0574] <(1) Preparation of culture supernatant samples>

[0575] The same method as in "(1) Preparation of culture supernatant sample" of Example 1-8 was used.

[0576] <(2) Biotin labeling of Fc tag-fused mouse Tim4 protein>

[0577] For 114 μL of PBS solution containing Fc-tagged mTim4 protein (containing 10 μg of Fc-tagged mTim4 protein) prepared by the same method as in Experimental Example 1 "(2) Biotin labeling of Fc-tagged mouse-derived Tim4 protein", the thiol group of the Fc-tagged mTim4 protein was biotin-labeled using the same method as in Example 1 to obtain 100 μL of PBS solution containing 3.9 μg of Fc-tagged mTim4 protein labeled with thiol-biotin (hereinafter sometimes abbreviated as "PBS solution containing Fc-tagged mTim4 protein labeled with thiol-biotin").

[0578] <(3) Biotin labeling of FLAG-tag fused mouse Tim4 protein>

[0579] Using 99 μL of PBS solution containing FLAG-tagged mTim4 protein fused to a FLAG tag prepared by the same method as in Experimental Example 2 (containing 10 μg of FLAG-tagged mTim4 protein), the thiol group of the FLAG-tagged mTim4 protein was biotin-labeled using a biotin labeling kit - thiol (manufactured by Dojindo Chemical Research Institute Co., Ltd.) according to the protocol included in the kit to obtain 100 μL of a PBS solution containing 4.6 μg of FLAG-tagged mTim4 protein fused to a thiol-biotin-labeled FLAG tag (hereinafter sometimes referred to as "PBS solution containing thiol-biotin-labeled FLAG-tagged mTim4 protein").

[0580] <(4) Biotin labeling of His-tag fused mouse Tim4 protein>

[0581] For 54 μL of PBS solution containing His-tag fusion mTim4 protein prepared by the same method as in Experimental Example 3 (containing 10 μg of His-tag fusion mTim4 protein), the thiol group of the His-tag fusion mTim4 protein was biotin-labeled using a biotin labeling kit - thiol (manufactured by Dojindo Chemical Research Institute Co., Ltd.) according to the protocol included in the kit, to obtain 100 μL of PBS solution containing 7.2 μg of His-tag fusion mTim4 protein labeled with thiol-biotin (hereinafter sometimes referred to as "PBS solution containing His-tag fusion mTim4 protein labeled with thiol-biotin").

[0582] <(5) Dilution of tag-fused mouse-derived Tim4 protein>

[0583] 11.4 μL of PBS solution containing Fc-tagged mTim4 protein prepared by the same method as in Experimental Example 1 (containing 1 μg of Fc-tagged mouse Tim4 protein) was mixed with 188.6 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of biotin-unlabeled Fc-tagged mouse Tim4 protein.

[0584] 16.9 μL of the PBS solution containing the thiol-biotin labeled Fc tag fusion mTim4 protein prepared in (2) above (containing 1 μg of the thiol-biotin labeled Fc tag fusion mTim4 protein) was mixed with 183.1 μL of PBS to obtain 200 μL of a PBS solution containing 1 μg of the thiol-biotin labeled Fc tag fusion mTim4 protein.

[0585] In addition, 21.6 μL of the PBS solution containing thiol-biotin-labeled FLAG-tagged mTim4 protein (containing 1 μg of thiol-biotin-labeled FLAG-tagged mTim4 protein) prepared in (3) was mixed with 178.4 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of thiol-biotin-labeled FLAG-tagged mTim4 protein.

[0586] 13.9 μL of the PBS solution containing the His-tag fusion mTim4 protein labeled with thiol-biotin prepared in (4) (containing 1 μg of the His-tag fusion mTim4 protein labeled with thiol-biotin) was mixed with 186.1 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of the His-tag fusion mTim4 protein labeled with thiol-biotin.

[0587] <(6) Washing of beads>

[0588] 20 μL of a PBS-T solution (trade name, manufactured by Thermo Fisher Scientific) containing 30 μg / μL Dynabeads G protein (containing 0.6 mg of Dynabeads G protein) was dispensed into a 1.5 mL tube (manufactured by BM Equipment Co., Ltd.) and washed with 500 μL of PBS using the same method as in “(4) Washing of beads” in Example 1-8.

[0589] In addition, 60 μL of a PBS solution (containing 0.6 mg of Dynabeads M-270 streptavidin) containing 10 μg / μL of Dynabeads M-270 streptavidin (trade name, manufactured by Thermo Fisher Scientific) was dispensed into three 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.) and washed with 500 μL of PBS using the same method as in "(4) Washing of beads" of Example 1-8.

[0590] <(7) Immobilization of the tag-fused mouse Tim4 protein on beads>

[0591] Then, a total of 200 μL of PBS solution containing 1 μg of biotin-unlabeled Fc-tagged mTim4 protein fused to 0.6 mg of Dynabeads G protein after washing was added to a 1.5 mL tube, and the reaction was carried out at 8°C for 1 hour to obtain 200 μL of PBS solution containing a vector (mTim4 vector) bound to biotin-unlabeled Fc-tagged mTim4 protein.

[0592] Furthermore, a total amount of 200 μL of PBS solution containing 1 μg of thiol-biotin-labeled Fc-tagged fused mTim4 protein prepared above was added to one of three 1.5 mL tubes containing 0.6 mg of Dynabeads M-270 streptavidin in a particle state after the washing operation, and the reaction was carried out at 8°C for 1 hour to obtain 200 μL of PBS solution containing a vector (mTim4 vector) containing thiol-biotin-labeled Fc-tagged fused mTim4 protein.

[0593] In one of the remaining two 1.5 mL tubes containing 0.6 mg of Dynabeads M-270 streptavidin in a particle state after the washing operation, add a total of 200 μL of PBS solution containing 1 μg of thiol-biotin-labeled FLAG-tagged fused mTim4 protein prepared above, and react at 8°C for 1 hour to obtain 200 μL of PBS solution containing a vector (mTim4 vector) containing thiol-biotin-labeled FLAG-tagged fused mTim4 protein.

[0594] To another, add a total of 200 μL of the PBS solution containing 1 μg of the His-tagged mTim4 protein fused to the thiol-biotin label as prepared above, and react at 8°C for 1 hour to obtain 200 μL of a PBS solution containing a vector (mTim4 vector) containing the His-tagged mTim4 protein fused to the thiol-biotin label.

[0595] Thus, 200 μL of PBS solutions containing 0.6 mg of each of the four types of mTim4 vectors shown in Table 3 below were obtained.

[0596] Table 3

[0597]

[0598] <(8) Obtaining extracellular membrane vesicles using the obtaining method of the present invention>

[0599] The same method as in "(6) Obtaining extracellular membrane vesicles using the obtaining method of the present invention" of Examples 1-8 was used to obtain each supernatant (elution solution), except that "0.6 mg of the four types of mTim4 vectors described in Table 3" was used instead of "0.6 mg of the four types of mTim4 vectors described in Table 1". It should be noted that the types of mTim4 proteins and vectors used in each example, the types of eluates used to obtain extracellular membrane vesicles from mTim4 vectors, and the lane numbers in the protein immunoblotting described later are shown in Table 4 below.

[0600] Table 4

[0601]

[0602] <(7) Western blotting>

[0603] The same method as "(7) Protein immunoblotting" in Example 1-8 was used except that "7.5 μL of each supernatant (eluate) obtained in Example 9-16 (described (8))" was used instead of "7.5 μL of each supernatant (eluate) obtained in Example 1-8".

[0604] <Results>

[0605] The obtained protein immunoblotting results are shown in Figure 2 middle. Figure 2 , the lanes are as follows.

[0606] They show respectively:

[0607] Lane 1: Results of Example 9 (results when using an mTim4 vector in which a biotin-unlabeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1% SDS aqueous solution was used as an eluent);

[0608] Lane 2: Results of Example 10 (results when using an mTim4 vector in which a biotin-unlabeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1 mM EDTA solution was used as an eluent);

[0609] Lane 3: Results of Example 11 (results when using an mTim4 vector in which a thiol-biotin-labeled Fc tag-fused mTim4 protein was bound to streptavidin beads and a 1% SDS aqueous solution was used as an eluent);

[0610] Lane 4: Results of Example 12 (results when an mTim4 vector in which a thiol-biotin-labeled Fc tag-fused mTim4 protein was bound to streptavidin beads was used and a 1 mM EDTA solution was used as an eluent);

[0611] Lane 5: Results of Example 13 (results when an mTim4 vector in which a thiol-biotin-labeled FLAG tag fused to an mTim4 protein was bound to streptavidin beads was used and a 1% SDS aqueous solution was used as an eluent);

[0612] Lane 6: Results of Example 14 (results when an mTim4 vector in which a thiol-biotin-labeled FLAG tag fused to an mTim4 protein was bound to streptavidin beads was used and a 1 mM EDTA solution was used as an eluent);

[0613] Lane 7: Results of Example 15 (results when an mTim4 vector in which a thiol-biotin-labeled His-tag fused mTim4 protein was bound to streptavidin beads was used and a 1% SDS aqueous solution was used as an eluent);

[0614] Lane 8: Results of Example 16 (results when using an mTim4 vector in which a thiol-biotin-labeled His-tag fused mTim4 protein was bound to streptavidin beads and a 1 mM EDTA solution was used as an eluent).

[0615] according to Figure 2 It can be seen that in any case of Examples 9-16, a band of Lamp-1, a marker protein of exosomes, was obtained at around 100 kDa. Therefore, the acquisition method of the present invention can obtain extracellular membrane vesicles containing exosomes (Example 9-16: Lanes 1-8).

[0616] Furthermore, it was found that when the Tim4 vector of the present invention was used, extracellular membrane vesicles could be obtained regardless of the type, length, or presence of the tag (Example 9-16: Lanes 1-8).

[0617] By comparing Examples 10, 12, 14, and 16, it can be seen that when EDTA, a calcium ion chelator, is used as an eluent, more extracellular membrane vesicles can be obtained when the Tim4 protein is bound to the carrier via the thiol group of the Tim4 protein (Example 12: Lane 4; Example 14: Lane 6; Example 16: Lane 8) compared to the case where the Tim4 protein is bound to the carrier via the affinity tag (Example 10: Lane 2).

[0618] Example 17-18. Obtaining extracellular membrane vesicles using the Tim4 vector of the present invention (obtaining method of the present invention)

[0619] The Tim4 vector of the present invention is prepared as follows, and the extracellular membrane vesicles of the present invention are obtained using the vector.

[0620] <(1) Preparation of culture supernatant samples>

[0621] The same method as in "(1) Preparation of culture supernatant sample" of Example 1-8 was used.

[0622] <(2) Dilution of biotin-unlabeled FLAG-tagged mouse Tim4 protein>

[0623] Mix 47 μL of PBS solution containing 5 μg of biotin-unlabeled FLAG-tagged mTim4 protein fused to mouse source Tim4 protein (PBS solution containing biotin-unlabeled FLAG-tagged fused to mouse source Tim4 protein) prepared by the same method as in Experimental Example 2 with 153 μL of PBS to obtain 200 μL of PBS solution containing 5 μg of biotin-unlabeled FLAG-tagged mTim4 protein fused to mouse source.

[0624] <(3) Washing of beads>

[0625] 50 μL of a 50% glycerol TBS solution (Wako Pure Chemical Industries, Ltd.) containing 10 μg / μL of anti-DYKDDDDK tag antibody magnetic beads (containing 0.5 mg of anti-DYKDDDDK tag antibody magnetic beads) was dispensed into a 1.5 mL tube (BM Equipment Co., Ltd.), and washed with 500 μL of PBS.

[0626] <(4) FLAG tag fused mouse Tim4 protein fixed to the vector>

[0627] Then, a total of 200 μL of a PBS solution containing 5 μg of FLAG tag-fused mTim4 protein was added to the anti-DYKDDDDK tag antibody magnetic beads after washing, and the reaction was carried out at 8° C. for 1 hour to obtain 200 μL of a PBS solution containing mTim4 vector.

[0628] As described above, 200 μL of a PBS solution containing 0.5 mg of the mTim4 vector shown in Table 5 below was obtained.

[0629] Table 5

[0630]

[0631] <(5) Obtaining extracellular membrane vesicles using the obtaining method of the present invention>

[0632] Except for using "0.5 mg of mTim4 vector described in Table 5" instead of "0.6 mg of four types of mTim4 vectors described in Table 1", the same method as "(6) Obtaining extracellular membrane vesicles using the obtaining method of the present invention" in Example 1-8 was used to obtain each supernatant (eluate).

[0633] The types of mTim4 protein and vector used in each example, the type of eluate used to obtain extracellular membrane vesicles from the mTim4 vector, and the lane numbers in the Western blotting described below are shown in Table 6 below.

[0634] Table 6

[0635]

[0636] <(6) Western blotting>

[0637] The same method as "(7) Protein immunoblotting" in Example 1-8 was used except that "7.5 μL of each supernatant (eluate) obtained in Example 17-18 (described (5))" was used instead of "7.5 μL of each supernatant (eluate) obtained in Example 1-8".

[0638] <Results>

[0639] The obtained protein immunoblotting results are shown in Figure 3 . Figure 3 , the lanes are as follows.

[0640] Lane 1: Results of Example 17 (using an mTim4 vector in which a biotin-unlabeled FLAG-tag-fused mTim4 protein was bound to anti-DYKDDDDK tag antibody beads, and using a 1% SDS aqueous solution as an eluent);

[0641] Lane 2: Results of Example 18 (results when using an mTim4 vector in which a biotin-unlabeled FLAG-tag-fused mTim4 protein was bound to anti-DYKDDDDK tag antibody beads and a 1 mM EDTA solution was used as an eluent).

[0642] according to Figure 3 It can be seen that in any case in Examples 17-18, a band of Lamp-1, which is an exosome marker, was observed near 100 kDa. Therefore, even if a carrier in which the Tim4 protein is immobilized by an anti-tag antibody is used, the extracellular membrane vesicles containing exosomes can be obtained by the acquisition method of the present invention (Examples 17-18: Lanes 1-2).

[0643] Examples 19-20 and Comparative Examples 1-3. Comparison of the purity of extracellular membrane vesicles obtained by the acquisition method of the present invention and the conventional method

[0644] As described below, the purity of the extracellular membrane vesicles obtained by the method of the present invention using SDS elution (Example 19), the method of the present invention using EDTA elution (Example 20), ultracentrifugation separation (Comparative Example 1), Exo Quick (Comparative Example 2) and total exosome isolation (Total Exosome Isolation) (Comparative Example 3) were compared.

[0645] <Obtaining extracellular membrane vesicles using the method of the present invention (Examples 19-20)>

[0646] The mTim4 protein was reacted with the extracellular membrane vesicles in the culture supernatant sample containing calcium ions by the same method as in Example 13-14, except that "500 μL of the culture supernatant sample containing calcium ions" was used instead of "200 μL of the culture supernatant sample containing calcium ions", and as the eluent, "50 μL of a 1% SDS aqueous solution" was used instead of "20 μL of a 1% SDS aqueous solution", and "50 μL of a 1 mM EDTA aqueous solution" was used instead of "20 μL of a 1 mM EDTA aqueous solution". The extracellular membrane vesicles were eluted using 1% SDS aqueous solution and 1 mM EDTA aqueous solution as eluents, respectively, to obtain supernatants (eluents), respectively. The mTim4 protein was obtained by binding the FLAG tag-fused mTim4 protein labeled with thiol biotin to Dynabeads The beads were formed by using M-270 Streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific).

[0647] The obtained supernatants (eluates) were designated as sample 1 (when eluted with a 1% SDS aqueous solution) and sample 2 (when eluted with a 1 mM EDTA aqueous solution).

[0648] In addition, the types of mTim4 protein and carrier used in Examples 19-20, and the types of eluates used to obtain extracellular membrane vesicles from the mTim4 carrier are shown in Table 7 below.

[0649] Table 7

[0650]

[0651] <Obtaining extracellular membrane vesicles by ultracentrifugation (Comparative Example 1)>

[0652] 1 mL of the culture supernatant sample prepared by the same method as Example 1 was centrifuged (20000×g, 30 minutes) to separate impurities and obtain a supernatant. Then, 1 mL of the obtained supernatant was subjected to ultracentrifugation (110000×g, 70 minutes) to obtain a precipitate component. Then, the obtained precipitate component was suspended with 1 mL of PBS. After the suspension of the precipitate component was subjected to ultracentrifugation again (110000×g, 70 minutes), the obtained precipitate component was suspended in 50 μL of PBS. The obtained suspension was used as Sample 3 (Comparative Example 1).

[0653] <Obtaining extracellular membrane vesicles by centrifugation using commercially available reagents (Obtaining extracellular membrane vesicles using ExoQuick) (Comparative Example 2)>

[0654] 1 mL of the culture supernatant sample prepared by the same method as in Example 1 was centrifuged (20000×g, 30 minutes) to separate impurities and obtain a supernatant. Then, 1 mL of the obtained supernatant was mixed with 0.2 mL of ExoQuick-TC Reagent (trade name, System Biosciences), and the mixture was allowed to stand overnight at 8°C. Then, the mixture that had been allowed to stand overnight was centrifuged (1500×g, 30 minutes), and the obtained precipitate fraction was suspended in 100 μL of PBS. The obtained suspension was used as Sample 4 (Comparative Example 2).

[0655] <Obtaining extracellular membrane vesicles by centrifugation using commercially available reagents (Obtaining extracellular membrane vesicles by total exosome isolation) (Comparative Example 3)>

[0656] 1 mL of the K562 cell culture supernatant concentrate prepared by the same method as in Example 1 was centrifuged (20,000 × g, 30 minutes) to separate impurities and obtain a supernatant. Then, 1 mL of the obtained supernatant was mixed with 0.5 mL of Total Exosome Isolation Reagent (manufactured by Thermo Fisher Scientific) and allowed to stand at 8°C for 1 day. The precipitate fraction obtained by centrifuging the mixed solution (10,000 × g, 1 hour) was suspended in 100 μL of PBS. The obtained suspension was used as Sample 5 (Comparative Example 3).

[0657] In addition, the method used in each Example and Comparative Example, the obtained sample, and the lane number in the western blotting and silver staining described later are shown in the following Table 8.

[0658] Table 8

[0659]

[0660] <Western blotting>

[0661] The protein amount in samples 1-5 obtained by each method was measured by the BCA method, and based on the measurement results, the protein amount was used as the reference amount for electrophoresis, and protein immunoblotting was performed. That is, 37.5 μL of PBS solution containing 0.25 μg of each protein in samples 1-5 was mixed with 12.5 μL of 4× sample buffer (Wako Pure Chemical Industries, Ltd.), and then heated at 98° C. for 5 minutes to obtain 50 μL of each sample for protein immunoblotting.

[0662] Then, 20 μL of each sample for protein immunoblotting was added to two gels in SuperSep Ace 5-20% gel (trade name, Wako Pure Chemical Industries, Ltd.), and electrophoresis was performed at 25 mA for 60 minutes. A semi-dry blotting apparatus and discontinuous buffers (anode buffer 1: 0.3 M Tris / 20% methanol; anode buffer 2: 0.025 M Tris / 20% methanol; cathode buffer: 0.025 M Tris / 0.04 M aminocaproic acid / 20% methanol) were used at 1 mA / cm 2Under the conditions of , one of the two gels obtained was transferred to a PVDF membrane (Millipore) for 60 minutes. 3% skim milk diluted with PBS-T was added to the PVDF membrane, and the reaction was carried out at room temperature for 1 hour to block. 2 mL of anti-human Lamp-1 mouse monoclonal antibody (BD Biosciences) diluted 250 times with PBS-T was reacted at room temperature for 1 hour. After washing with PBS-T three times, secondary antibodies {anti-mouse IgG (H+L), rabbit, IgG component, peroxidase-conjugated antibody (Wako Pure Chemical Industries, Ltd.)} diluted 10,000 times with PBS-T were reacted at room temperature for 1 hour. After washing with PBS-T five times, ECL prime (trade name, General Electric (GE)) was added, and the luminescent signals were detected using LAS-4000 (trade name, General Electric (GE)).

[0663] Separately, another gel was subjected to silver staining using Wako Silver Staining II Kit (trade name, manufactured by Wako Pure Chemical Industries, Ltd.).

[0664] <Results>

[0665] The obtained protein immunoblotting results are shown in Figure 4 -A. The results of silver staining are shown in Figure 4 -B. Figure 4 -A and Figure 4 -B, the lanes are as follows.

[0666] Lane 1: Results of Example 19 (results when the acquisition method of the present invention was carried out using an mTim4 vector in which a thiol-biotin-labeled FLAG tag fused to an mTim4 protein was bound to streptavidin beads and a 1% SDS aqueous solution was used as an eluent);

[0667] Lane 2: Results of Example 20 (results when the acquisition method of the present invention was carried out using an mTim4 vector in which a thiol-biotin-labeled FLAG tag fused to an mTim4 protein was bound to streptavidin beads and a 1 mM EDTA solution was used as an eluent);

[0668] Lane 3: the result of Comparative Example 1 (the result when extracellular membrane vesicles were obtained by ultracentrifugation separation);

[0669] Lane 4: the result of Comparative Example 2 (the result when extracellular membrane vesicles were obtained by ExoQuick);

[0670] Lane 5: Results of Comparative Example 3 (results when extracellular membrane vesicles were obtained by the total exosome isolation method).

[0671] according to Figure 4 -A, in any of Examples 19-20 of the acquisition method of the present invention, a band of Lamp-1, an exosome marker, was observed near 100 kDa, indicating that the acquisition method of the present invention can obtain extracellular membrane vesicles containing exosomes (Examples 19-20: Lanes 1-2).

[0672] On the other hand, according to Figure 4 -A, for any of the existing methods (ultracentrifugation separation (Comparative Example 1), ExoQuick (Comparative Example 2), and Total Exsome Isolation (Comparative Example 3)), only a faint and trace band of the exosome marker Lamp-1 was observed around 100 kDa (Comparative Example 1-3: Lanes 3-5).

[0673] In addition, according to Figure 4 -B. The acquisition method of the present invention (Examples 19-20) has fewer bands derived from included proteins, etc. (Examples 19-20: Lanes 1-2, Comparative Example 1-3: Lanes 3-5) compared with any existing methods (ultracentrifugation separation (Comparative Example 1), ExoQuick (Comparative Example 2) and Total Exsome Isolation (Comparative Example 3)).

[0674] According to the above results, the acquisition method of the present invention (Examples 19 and 20) can obtain extracellular membrane vesicles (lanes 1-5) with better purity than any existing method (ultracentrifugation separation method (Comparative Example 1), ExoQuick (Comparative Example 2) and total exosome separation method (Total ExsomeIsolation) (Comparative Example 3)).

[0675] Example 21. Observation of the extracellular membrane vesicles obtained by the acquisition method of the present invention using an electron microscope

[0676] In order to examine the state of the extracellular membrane vesicles obtained by the obtaining method of the present invention, observation was performed using an electron microscope.

[0677] <(1) Preparation of mouse macrophage culture supernatant samples>

[0678] In order to obtain peritoneal macrophages, 2 mL of a 3% thioglycolate solution (manufactured by Fruka Reagent Co., Ltd.) was injected into the peritoneal cavity of six 8-week-old female C57BL / 6J mice (purchased from Japan SLC Co., Ltd.). Three days later, macrophages were recovered from the peritoneal cavity, and the recovered macrophages were cultured for 2 days in four 150 mm cell culture dishes with 80 mL of DMEM (manufactured by Nakara Tech Co., Ltd.) containing 10% FBS (manufactured by Biowest Co., Ltd.), and the culture supernatant was recovered to obtain a mouse macrophage culture supernatant sample.

[0679] <(2) Preparation of Tim4 vector>

[0680] 1 mg of Dynabeads MyOne Streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific) was used instead of 0.6 mg of Dynabeads MyOne Streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific), and 100 μL of a PBS solution containing 100 μg of thiol-biotin-labeled Fc-tag fused mTim4 protein was used instead of 200 μL of a PBS solution containing 1 μg of thiol-biotin-labeled Fc-tag fused mTim4 protein. The reaction time between Dynabeads and the PBS solution containing the mTim4 protein fused to the Fc tag labeled with thiol-biotin was adjusted to 2 hours instead of 1 hour. In addition, the mTim4 carrier in which the mTim4 protein fused to the Fc tag labeled with thiol-biotin was bound to the Dynabeads MyOne streptavidin C1 beads was prepared by the same method as in Example 11-12.

[0681] <(3) Obtaining extracellular membrane vesicles using Tim4 vector>

[0682] 32 mL of mouse macrophage culture supernatant sample was centrifuged (first time: 800 × g, 10 minutes; second time: 12000 × g, 30 minutes) to obtain the supernatant. CaCl was added to the obtained supernatant. 2 After the final concentration was 2 mM, 1 mg of the mTim4 vector prepared above was added and stirred at room temperature for 1 hour. Then, the mTim4 vector stirred at room temperature for 1 hour was recovered and the final concentration was 2 mM CaCl 2 The mixture was washed three times with 5 mL of TBS-T and then washed twice with 1 mL of TBS-T. Thereafter, elution was performed three times with 100 μL of TBS containing 1 mM EDTA to obtain 300 μL of an extracellular membrane vesicle fraction.

[0683] Then, in order to reliably recover the extracellular membrane vesicles in the mouse macrophage culture supernatant sample, the extracellular membrane vesicles were recovered again from the mouse macrophage culture supernatant sample after elution three times with 100 μL of TBS containing 1 mM EDTA by the following method to obtain 300 μL of extracellular membrane vesicle fraction. That is, CaCl was added to the mouse macrophage culture supernatant sample after elution three times with 100 μL of TBS containing 1 mM EDTA. 2 After the final concentration was 2 mM, 1 mg of mTim4 vector was eluted three times with 100 μL of TBS containing 1 mM EDTA, and the mixture was stirred at room temperature for 1 hour. Then, the mTim4 vector was recovered after stirring at room temperature for 1 hour and eluted with 100 μL of TBS containing 1 mM EDTA at a final concentration of 2 mM. 2 The mixture was washed three times with 5 mL of TBS-T and then washed twice with 1 mL of TBS-T. Elution was then performed three times with 100 μL of TBS containing 1 mM EDTA to obtain 300 μL of an extracellular membrane vesicle fraction.

[0684] Then, in order to more reliably recover the extracellular membrane vesicles in the mouse macrophage culture supernatant sample, the above operation was repeated to recover the extracellular membrane vesicles from the mouse macrophage culture supernatant sample after elution six times with 100 μL of TBS containing 1 mM EDTA, to obtain 300 μL of the extracellular membrane vesicle fraction.

[0685] A total of 900 μL of the extracellular membrane vesicle fraction obtained through the above 9 times of elution operation was concentrated to 60 μL using an Amicon Ultra-0.5 mL 10K centrifugal filter column to obtain a sample for electron microscopic observation.

[0686] <(4) Observation of the extracellular membrane vesicles obtained by the obtaining method of the present invention using an electron microscope>

[0687] 10 μL of the sample for electron microscopic observation was adsorbed on the grid, and the remaining sample for electron microscopic observation was sucked with filter paper. The grid was then washed with water, stained twice with an aqueous uranyl acetate solution, and observed using a transmission electron microscope by negative staining.

[0688] In addition, the types of mTim4 protein and carrier used in Example 21, and the types of eluates used to obtain extracellular membrane vesicles from the mTim4 carrier are shown in Table 9 below.

[0689] Table 9

[0690]

[0691] <Results>

[0692] The electron microscope observation images obtained are shown in Figure 5 .according to Figure 5 According to the obtaining method of the present invention, extracellular membrane vesicles with a diameter of about 50 to 150 nm can be obtained while maintaining a spherical shape.

[0693] As can be seen from the above, the acquisition method of the present invention can obtain extracellular membrane vesicles in an intact state.

[0694] Example 22-25. Obtaining extracellular membrane vesicles using the Tim4 vector of the present invention (obtaining method of the present invention)

[0695] The extracellular membrane vesicles of the present invention are obtained using a carrier in which human Tim4 protein is immobilized on beads (hereinafter, sometimes abbreviated as "hTim4 carrier") and an mTim4 carrier.

[0696] <(1) Preparation of culture supernatant samples>

[0697] The same method as in "(1) Preparation of culture supernatant sample" of Example 1-8 was used.

[0698] <(2) Dilution of Fc-tag fused Tim4 protein>

[0699] 100 μg of a freeze-dried product of a biotin-unlabeled Fc-tag-fused human Tim4 protein (Wako Pure Chemical Industries, Ltd., sequence number 7 (human Tim4 protein in which an Fc tag is fused to the amino acid domain at positions 25 to 315 of the N-terminus of the human Tim4 protein (GenBank: GenBank NP_612388.2))) was dissolved in 1 mL of PBS to prepare a PBS solution containing 100 μg / mL of biotin-unlabeled Fc-tag-fused hTim4 protein. 10 μL of the PBS solution containing the biotin-unlabeled Fc-tag-fused hTim4 protein was mixed with 190 μL of PBS to obtain 200 μL of a PBS solution containing 1 μg of the biotin-unlabeled Fc-tag-fused hTim4 protein.

[0700] In addition, 100 μg of a freeze-dried product of a biotin-unlabeled Fc tag-fused mouse-derived Tim4 protein (produced by Wako Pure Chemical Industries, Ltd., sequence number 6 (mTim4 protein fused with an Fc tag at the amino acid domain at positions 22 to 279 of the N-terminal of the mouse-derived Tim4 protein (GenBank: GenBank NP_848874.3)) was dissolved in 1 mL of PBS to prepare a PBS solution containing 100 μg / mL of biotin-unlabeled Fc tag-fused mTim4 protein. 10 μL of the PBS solution containing biotin-unlabeled Fc tag-fused mTim4 protein was mixed with 190 μL of PBS to obtain 200 μL of a PBS solution containing 1 μg of biotin-unlabeled Fc tag-fused mTim4 protein.

[0701] <(3) Washing of beads>

[0702] 20 μL of a PBS-T solution (trade name, manufactured by Thermo Fisher Scientific) containing 30 μg / μL of Dynabeads protein G (containing 0.6 mg of Dynabeads protein G) was dispensed into two 1.5 mL tubes (manufactured by BM Equipment Co.), and washing was performed with 500 μL of PBS.

[0703] <(4) Immobilization of biotin-unlabeled Fc-tagged Tim4 protein on beads>

[0704] In two 1.5 mL tubes containing Dynabeads G protein (0.6 mg), a total of 200 μL of the PBS solution containing 1 μg of the biotin-unlabeled Fc-tagged hTim4 protein prepared above was added to one of the tubes, and the reaction was carried out at 8°C for 1 hour to obtain 200 μL of a PBS solution containing a vector (hTim4 vector) bound to the biotin-unlabeled Fc-tagged hTim4 protein.

[0705] In the remaining 1.5 mL tube containing Dynabeads G protein (0.6 mg), add a total of 200 μL of the PBS solution containing 1 μg of the biotin-unlabeled Fc-tagged mTim4 protein prepared above, and react at 8°C for 1 hour to obtain 200 μL of PBS solution containing a vector (mTim4 vector) bound to the biotin-unlabeled Fc-tagged mTim4 protein.

[0706] <(5) Obtaining extracellular membrane vesicles using the obtaining method of the present invention>

[0707] 0.6 mg of the hTim4 vector and mTim4 vector obtained above were washed three times with 500 μL of PBS, and then 200 μL of the culture supernatant sample containing calcium ions was added to the hTim4 vector and mTim4 vector in the particle state, and the reaction was carried out at 8°C for 3 hours. 2 The hTim4 vector and mTim4 vector were washed three times with 500 μL of TBS-T.

[0708] When washing for the third time, 250 μL of hTim4 vector and mTim4 vector (0.3 mg of vector) were respectively dispensed into two 1.5 mL tubes. 20 μL of 1% SDS aqueous solution or 1 mM EDTA aqueous solution as an eluent was added to 0.3 mg of mTim4 vector and hTim4 vector in a particle state, and then mixed for 10 seconds at room temperature using a vortex mixer and spun down. The 1.5 mL tubes were placed on magnetic racks, and the hTim4 vector and mTim4 vector were aggregated on the tube wall using magnetic force, and the supernatant (eluent) was recovered respectively.

[0709] The types of Tim4 proteins and vectors used in Examples 22 to 25, the types of eluates used to obtain extracellular membrane vesicles from the Tim4 vectors, and lane numbers in the Western blotting described below are shown in Table 10 below.

[0710] Table 10

[0711]

[0712] <(6) Western blotting>

[0713] The same method as "(7) Protein immunoblotting" in Example 1-8 was used except that "7.5 μL of each supernatant (eluate) obtained in Examples 22-25 (described (5))" was used instead of "7.5 μL of each supernatant (eluate) obtained in Examples 1-8".

[0714] <Results>

[0715] The obtained protein immunoblotting results are shown in Figure 6 . Figure 6 , the lanes are as follows.

[0716] Lane 1: Results of Example 22 (when hTim4 vector in which hTim4 protein fused to a biotin-unlabeled Fc tag was bound to protein G beads was used and 1% SDS aqueous solution was used as an eluent);

[0717] Lane 2: Results of Example 23 (results when hTim4 vector in which hTim4 protein fused to a biotin-unlabeled Fc tag was bound to protein G beads was used and 1 mM EDTA aqueous solution was used as an eluent);

[0718] Lane 3: Results of Example 24 (results when using an mTim4 vector in which a biotin-unlabeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1% SDS aqueous solution was used as an eluent);

[0719] Lane 4: Results of Example 25 (results when using an mTim4 carrier in which a biotin-unlabeled Fc-tag-fused mTim4 protein was bound to protein G beads and a 1 mM EDTA aqueous solution was used as an eluent).

[0720] Depend on Figure 6 It can be seen that in any of Examples 22-25, a band of Lamp-1, an exosome marker, was observed near 100 kDa. Therefore, the acquisition method of the present invention can obtain extracellular membrane vesicles containing exosomes (Examples 22-25: Lanes 1-4).

[0721] Furthermore, it is known that the Tim4 protein can be used in the Tim4 vector and the acquisition method of the present invention regardless of the biological species from which it comes.

[0722] Examples 26-27 / Comparative Example 4-9. Comparison of the amount of extracellular membrane vesicles obtained using the Tim4 carrier and the carrier with immobilized PS protein

[0723] The extracellular membrane vesicles of the present invention are obtained using the Tim4 vector and a vector in which PS proteins (Annexin V from humans, MFG-E8 from humans, and MFG-E8 from mice) are immobilized on beads.

[0724] <(1) Dilution of PBS solution containing PS protein>

[0725] 20 μg of His-tag fused human membrane adhesion protein-5 (produced by Creative BioMart, sometimes abbreviated as "His-tag fused h-membrane adhesion protein-5") was dissolved in 100 μL of PBS to a concentration of 200 μg / mL to prepare a PBS solution containing His-tag fused h-membrane adhesion protein-5. 5 μL of the PBS solution containing 1 μg of His-tag fused h-membrane adhesion protein-5 protein was mixed with 195 μL of PBS for dilution.

[0726] 50 μg of His-tag fused human MFG-E8 (manufactured by R&D Systems, sometimes abbreviated as "His-tag fused hMFG-E8") was dissolved in 500 μL of PBS to a concentration of 100 μg / mL to prepare a PBS solution containing His-tag fused hMFG-E8. 10 μL of a PBS solution containing 1 μg of His-tag fused hMFG-E8 protein was mixed with 190 μL of PBS for dilution. 50 μg of His-tag fused mouse MFG-E8 (manufactured by R&D Systems, sometimes abbreviated as "His-tag fused mMFG-E8") was dissolved in 500 μL of PBS to a concentration of 100 μg / mL to prepare a PBS solution containing His-tag fused mMFG-E8. 10 μL of a PBS solution containing 1 μg of His-tag fused mouse MFG-E8 protein was mixed with 190 μL of PBS for dilution.

[0727] <(2) Preparation of anti-His tag antibody-immobilized beads>

[0728] 100 μL (containing 3 mg of Dynabeads M-270 carboxylic acid) of a solution containing 30 μg / μL of Dynabeads M-270 carboxylic acid (trade name, manufactured by Thermo Fisher Scientific) was dispensed into a 1.5 mL tube (manufactured by BM Equipment Co., Ltd.), and a washing operation was performed using a reaction buffer (0.1 M MES, pH 5.0).

[0729] Then, 60 μL of a 6×His antibody (Wako Pure Chemical Industries, Ltd.) solution diluted with 490 μL of a reaction buffer (containing 60 μg of a 6×His antibody) was added, and the mixture was mixed by inversion at room temperature for 30 minutes. Then, 50 μL of 6 mg / mL WSC (Dojindo Chemical Research Institute) was added, and the mixture was mixed by inversion at room temperature for 4 hours. After the inversion mixing, the Dynabeads M-270 carboxylic acid was washed with TBS-T, and then diluted with 100 μL of PBS to obtain 3 mg of anti-His tag antibody-immobilized beads.

[0730] <(3) Preparation of Tim4 carrier and carriers having various PS proteins immobilized thereon>

[0731] Then, 100 μL of the obtained PBS solution containing anti-His tag antibody-immobilized beads (containing 3 mg of anti-His tag antibody-immobilized beads) was dispensed into four 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.) in 20 μL portions (containing 0.6 mg of anti-His tag antibody-immobilized beads), and each tube was washed with 500 μL of PBS.

[0732] Then, 200 μL of the PBS solution containing the His-tag fusion mTim4 protein prepared above (containing 1 μg of the His-tag fusion mTim4 protein) was added to one of the four 1.5 mL tubes containing 0.6 mg of anti-His-tag antibody-immobilized beads in the particle state after the washing operation, and the reaction was carried out at 8°C for 1 hour.

[0733] 200 μL of the PBS solution containing the His-tag fused h-nectin-5 prepared above (containing 1 μg of His-tag fused h-nectin-5) was added to one of the plates, and the reaction was carried out at 8° C. for 1 hour.

[0734] 200 μL of the PBS solution containing His-tag fusion hMFG-E8 prepared above (containing 1 μg of His-tag fusion hMFG-E8) was added to one of the tubes, and the reaction was carried out at 8° C. for 1 hour.

[0735] 200 μL of the PBS solution containing His-tag fusion mMFG-E8 prepared above (containing 1 μg of His-tag fusion mMFG-E8) was added to one of the tubes, and the reaction was carried out at 8° C. for 1 hour.

[0736] According to the above contents, 200 μL of PBS solutions containing 0.6 mg of each of the four carriers shown in Table 11 below were obtained.

[0737] Table 11

[0738]

[0739] <(4) Obtaining extracellular membrane vesicles>

[0740] After washing 200 μL of the PBS solution containing 0.6 mg of the four carriers obtained above three times with 500 μL of PBS, 200 μL of the culture supernatant sample containing calcium ions prepared by the same method as in Examples 1-8 was added to each of the five carriers in a granular state, and the reactions were carried out at 8°C for 3 hours.

[0741] Then, the mixture was washed with 2 mM CaCl 2 The four types of carriers reacted with the culture supernatant samples containing calcium ions were washed three times with 500 μL of TBS-T.

[0742] When washing for the third time, 250 μL of each of the four carriers was dispensed into two 1.5 mL tubes. 20 μL of 1% SDS aqueous solution or 1 mM EDTA aqueous solution was added to 0.3 mg of each of the four carriers in the particle state, and then the mixture was vortexed at room temperature for 10 seconds and then spun down. The 1.5 mL tubes were placed on magnetic racks, and the four carriers were assembled on the tube wall using magnetic force, and the supernatant (eluent) was recovered.

[0743] The types of PS proteins and carriers used in each example, the types of eluates used to obtain extracellular membrane vesicles from the carriers, and lane numbers in the Western blotting described below are shown in Table 12 below.

[0744] Table 12

[0745]

[0746] <(5) Western blotting>

[0747] Instead of "7.5 μL of each supernatant (eluate) obtained in Examples 1-8", the same method as "(7) Western blotting" in Example 1-8 was used, using "7.5 μL of each supernatant (eluate) obtained in Examples 26-27 and Comparative Examples 4-9 (described in (4))".

[0748] <Results>

[0749] The obtained protein immunoblotting results are shown in Figure 7 . Figure 7 , the lanes are as follows.

[0750] Lane 1: Results of Example 26 (results when a carrier in which the His-tag-fused mTim4 protein was bound to anti-His-tag antibody-immobilized beads was used and a 1% SDS aqueous solution was used as an eluent);

[0751] Lane 2: Results of Example 27 (results when a carrier in which the His-tag-fused mTim4 protein was bound to anti-His-tag antibody-immobilized beads was used and a 1 mM EDTA aqueous solution was used as an eluent);

[0752] Lane 3: Results of Comparative Example 4 (results when a carrier in which His-tag-fused h-nectin-5 was bound to anti-His-tag antibody-immobilized beads was used and a 1% SDS aqueous solution was used as an eluent);

[0753] Lane 4: Results of Comparative Example 5 (results when a carrier in which His-tag-fused h-nectin-5 was bound to anti-His-tag antibody-immobilized beads was used and a 1 mM EDTA aqueous solution was used as an eluent);

[0754] Lane 5: Results of Comparative Example 6 (results when a carrier in which His-tag fused hMFG-E8 was bound to anti-His-tag antibody-immobilized beads was used and a 1% SDS aqueous solution was used as an eluent);

[0755] Lane 6: Results of Comparative Example 7 (results when a carrier in which His-tag fused hMFG-E8 was bound to anti-His-tag antibody-immobilized beads was used and a 1 mM EDTA aqueous solution was used as an eluent);

[0756] Lane 7: Results of Comparative Example 8 (results when a carrier in which His-tag fused mMFG-E8 was bound to anti-His-tag antibody-immobilized beads was used and a 1% SDS aqueous solution was used as an eluent);

[0757] Lane 8: Results of Comparative Example 9 (results when a carrier in which His-tag fused mMFG-E8 was bound to anti-His-tag antibody-immobilized beads was used and a 1 mM EDTA aqueous solution was used as an eluent).

[0758] Depend on Figure 7 It can be seen that in any of Examples 26-27, a band of Lamp-1, an exosome marker, was obtained at around 100 kDa. Therefore, the acquisition method of the present invention can obtain extracellular membrane vesicles containing exosomes (Examples 26-27: Lanes 1-2).

[0759] On the other hand, by Figure 7 It was found that no Lamp-1 band was observed near 100 kDa in any of Comparative Examples 4-9, and therefore, extracellular membrane vesicles containing exosomes could not be obtained for carriers on which PS proteins other than Tim4 protein (fibronectin-5, MFG-E8) were immobilized (Comparative Example 4-9: Lanes 3-8).

[0760] Example 28-33. Obtaining extracellular membrane vesicles using the obtaining method of the present invention (discussion of complex formation step)

[0761] The extracellular membrane vesicles were obtained by the obtaining methods of the present invention as described below.

[0762] In Examples 28-29, the mouse-derived Tim4 protein fused with a FLAG tag and labeled with thiol-biotin prepared by the same method as in Examples 13-14 was reacted with the vector of the present invention, and then a sample was added to obtain the extracellular membrane vesicles of the present invention.

[0763] In Examples 30-31, the mouse-derived Tim4 protein fused with a FLAG tag and labeled with thiol-biotin prepared by the same method as in Examples 13-14 was allowed to react with a sample, and then the vector of the present invention was added to obtain the extracellular membrane vesicles of the present invention.

[0764] For Examples 32-33, the mouse-derived Tim4 protein fused with a FLAG tag labeled with thiol-biotin prepared by the same method as in Examples 13-14, the sample, and the vector of the present invention were added simultaneously to obtain the extracellular membrane vesicles of the present invention.

[0765] <Washing of beads>

[0766] 60 μL (containing 0.6 mg of Dynabeads M-270 Streptavidin C1 beads) of a PBS solution containing 10 μg / μL Dynabeads M-270 Streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific) was dispensed into three 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.), and each tube was washed with 500 μL of PBS.

[0767] By the same method as in Example 13-14, 270 μL of PBS solution containing His-tag fusion mTim4 protein (containing 50 μg of His-tag fusion mTim4 protein) was biotin-labeled to obtain 200 μL of PBS solution containing 39.2 μg of FLAG-tag fusion mTim4 protein labeled with thiol biotin. 194.9 μL of PBS solution was added to 5.1 μL of PBS solution containing 1 μg of FLAG-tag fusion mTim4 protein labeled with thiol biotin to obtain 200 μL of PBS solution containing 1 μg of FLAG-tag fusion mTim4 protein labeled with thiol biotin.

[0768] <Case in which the FLAG-tag-fused mTim4 protein labeled with thiol-biotin is allowed to react with the carrier of the present invention, and then the extracellular membrane vesicles in the sample of the present invention are allowed to react with the carrier (Examples 28-29)>

[0769] In a 1.5 mL tube containing 0.6 mg of washed granular Dynabeads M-270 streptavidin C1 beads, 200 μL of PBS solution containing 1 μg of thiol-biotin labeled FLAG tag fused mTim4 protein was added, and the Dynabeads M-270 streptavidin C1 beads were brought into contact with the thiol-biotin labeled FLAG tag fused mTim4 protein, and reacted at 8°C for 1 hour to obtain the mTim4 vector. Then, the obtained mTim4 vector was washed three times with 500 μL of PBS, and 200 μL of the culture supernatant sample containing calcium ions prepared by the same method as in Example 1 was added to a 1.5 mL tube containing 0.6 mg of the washed mTim4 vector, and the reaction was carried out at 8°C for 3 hours. Then, the supernatant sample was added with a final concentration of 2 mM CaCl 2 The mTim4 vector obtained by reacting the culture supernatant sample containing calcium ions was washed three times with 500μL of TBS-T. In the third wash, 250μL of the mTim4 vector was dispensed into two 1.5mL tubes. 20μL of a 1% SDS aqueous solution or a 1mM EDTA aqueous solution as an eluent was added to 0.3mg of each mTim4 vector in a particle state, and then mixed at room temperature for 10 seconds using a vortex mixer and spun down. The 1.5mL tubes were placed on magnetic stands, and the mTim4 vectors were aggregated on the tube wall using magnetic force, and the supernatants (eluents) were recovered respectively.

[0770] <Case in which the FLAG tag-fused mTim4 protein labeled with thiol-biotin is allowed to react with the sample of the present invention, and then the vector of the present invention is allowed to react with the sample (Examples 30-31)>

[0771] 200 μL of the culture supernatant sample containing calcium ions prepared by the same method as in Example 1 was dispensed into a 1.5 mL tube, and 5.1 μL of a PBS solution containing 1 μg of thiol-biotin-labeled FLAG-tag fusion mTim4 protein was added to allow the extracellular membrane vesicles in the culture supernatant sample containing calcium ions to contact the thiol-biotin-labeled FLAG-tag fusion mTim4 protein, and the reaction was carried out at 8°C for 3 hours.

[0772] A solution obtained by reacting the extracellular membrane vesicles in the culture supernatant sample containing calcium ions with the FLAG tag fused mTim4 protein labeled with thiol biotin at 8°C for 3 hours was added to a 1.5 mL tube containing 0.6 mg of washed granular Dynabeads M-270 streptavidin C1 beads, and the reaction was carried out at 8°C for 1 hour to obtain a complex of extracellular membrane vesicles and mTim4 carrier. Then, a solution containing 2 mM CaCl was added to the solution.2 The obtained complex of extracellular membrane vesicles and mTim4 vector was washed three times with 500 μL of TBS-T. During the third wash, 250 μL of the complex of extracellular membrane vesicles and mTim4 vector was dispensed into two 1.5 mL tubes. 20 μL of 1% SDS aqueous solution or 1 mM EDTA aqueous solution as an eluent was added to 0.3 mg of each complex of extracellular membrane vesicles and mTim4 vector in a particle state, and then mixed at room temperature for 10 seconds using a vortex mixer and spun down. The 1.5 mL tubes were placed on magnetic stands respectively, and the mTim4 vectors were aggregated on the tube wall by magnetic force, and the supernatant (eluent) was recovered respectively.

[0773] <A case where the mTim4 protein fused to the FLAG tag labeled with thiol biotin, the sample of the present invention, and the vector of the present invention are reacted simultaneously (Examples 32-33)>

[0774] 200 μL of the culture supernatant sample containing calcium ions prepared by the same method as in Example 1 and 5.1 μL of a PBS solution containing 1 μg of FLAG-tagged mTim4 protein fused to thiol-biotin-labeled were added to a 1.5 mL tube containing 0.6 mg of washed granular Dynabeads M-270 streptavidin C1 beads, and the reaction was carried out at 8°C for 4 hours. 2 The granular Dynabeads M-270 Streptavidin C1 beads after the reaction were washed three times with 500 μL of TBS-T. During the third wash, 250 μL of mTim4 vector was dispensed into two 1.5 mL tubes, and 20 μL of 1% SDS aqueous solution or 1 mM EDTA aqueous solution as an eluent was added to 0.3 mg of mTim4 vector, and then mixed at room temperature for 10 seconds using a vortex mixer, and then spun down. The 1.5 mL tubes were placed on magnetic racks, and the Dynabeads M-270 Streptavidin C1 beads were assembled on the tube wall by magnetic force, and the supernatant (eluent) was recovered.

[0775] In addition, the order of contacting the carrier, the mTim4 protein, and the culture supernatant sample containing calcium ions, and the type of the eluate used to obtain the extracellular membrane vesicles from the carrier in each example are shown in Table 13 below.

[0776] Table 13

[0777]

[0778] <Western blotting>

[0779] The same method as in "(7) Western blotting" of Example 1-8 was used except that "7.5 μL of each supernatant (eluate) obtained in Examples 28 to 33" was used instead of "7.5 μL of each supernatant (eluate) obtained in Examples 1-8".

[0780] <Results>

[0781] The results of the obtained protein immunoblotting are shown in Figure 8 middle. Figure 8 , the lanes are as follows.

[0782] Lane 1: Results of Example 28 (results when extracellular membrane vesicles were obtained by contacting the carrier with the mTim4 protein and then with a calcium ion cell culture supernatant sample, and a 1% SDS aqueous solution was used as an eluent);

[0783] Lane 2: Results of Example 29 (results when extracellular membrane vesicles were obtained by contacting the carrier with the mTim4 protein and then with a calcium ion cell culture supernatant sample, and a 1 mM EDTA aqueous solution was used as an eluent);

[0784] Lane 3: Results of Example 30 (the results when extracellular membrane vesicles were obtained by contacting the mTim4 protein with a calcium ion cell culture supernatant sample and then contacting it with a carrier, and a 1% SDS aqueous solution was used as an eluent);

[0785] Lane 4: Results of Example 31 (results when extracellular membrane vesicles were obtained by contacting the mTim4 protein with a calcium ion cell culture supernatant sample and then contacting it with a carrier, and a 1 mM EDTA aqueous solution was used as an eluent);

[0786] Lane 5: Results of Example 32 (results when extracellular membrane vesicles were obtained by a method in which the carrier was brought into contact with a calcium ion cell culture supernatant sample and mTim4 protein at the same time, and a 1% SDS aqueous solution was used as an eluent);

[0787] Lane 6: Results of Example 33 (results when extracellular membrane vesicles were obtained by a method in which the carrier was brought into contact with a calcium ion cell culture supernatant sample and mTim4 protein at the same time, and a 1 mM EDTA aqueous solution was used as an eluent).

[0788] according to Figure 8 In any of Examples 28-33, a band of Lamp-1 as an exosome marker was obtained at around 100 kDa (Examples 28-33: Lanes 1-6).

[0789] In addition, according to the results of Examples 28-33, it can be seen that for the step of forming a complex between the Tim4 protein bound to the carrier and the extracellular membrane vesicles in the sample (complex formation step), the order of contact between the Tim4 protein of the present invention, the carrier of the present invention, and the extracellular membrane vesicles in the sample is irrelevant, and as a result, it is sufficient to form a complex between the mTim4 protein bound to the carrier and the extracellular membrane vesicles in the sample.

[0790] Examples 34-35 / Comparative Examples 10-13 Comparison of the acquisition method of the present invention with the existing method

[0791] As described below, extracellular membrane vesicles were obtained by the method of the present invention using SDS elution (Example 34), the method of the present invention using EDTA elution (Example 35), the anti-CD63 antibody immobilization method (Comparative Examples 10-11), and the exosome-human CD63 isolation / detection (Exosome-Human CD63 Isolation / Detection) (Comparative Examples 12-13).

[0792] <Immobilization of Tim4 protein on a carrier>

[0793] 15 μL of a solution (containing 1×10 7 Dynabeads M-270 Streptavidin) were dispensed into 1.5 mL tubes (manufactured by BM Instruments Co., Ltd.), and washed with PBS.

[0794] Then, a total of 200 μL of PBS solution containing 0.25 μg of mouse-derived Tim4 protein fused with a FLAG tag labeled with thiol biotin prepared by the same method as in Example 11-12 was added to a 1.5 mL tube containing Dynabeads M-270 streptavidin after the washing operation, and the mixture was reacted for 1 hour under refrigerated conditions to obtain a PBS solution containing the mTim4 vector.

[0795] <Preparation of anti-CD63 antibody (H5C6) immobilized carrier>

[0796] 50 μL of a solution (containing 1×10 8Each Dynabeads M-270 carboxylic acid) was dispensed into a 1.5 mL tube (manufactured by BM Equipment Co., Ltd.), and a washing operation was performed with a reaction buffer (0.1 M MES, pH 5.0).

[0797] On the other hand, 30 g (60 μL) of anti-CD63 antibody (H5C6) (manufactured by BD Pharmingen) was diluted with 490 μL of reaction buffer (0.1 M MES, pH 5.0) to obtain a reaction buffer-diluted anti-CD63 antibody solution.

[0798] Then, 550 μL of the anti-CD63 antibody solution diluted with the reaction buffer was added to the 1.5 mL tube containing the washed Dynabeads M-270 carboxylic acid, and the mixture was mixed by inversion at room temperature for 30 minutes. Then, 50 μL of 3 mg / mL WSC (manufactured by Dojindo Chemical Research Institute) was further added, and the mixture was mixed by inversion at room temperature for 4 hours. After washing with TBS-T, the mixture was diluted with 50 μL of PBS to obtain a PBS solution containing the anti-CD63 antibody (H5C6) carrier.

[0799] Based on the above contents, two vectors shown in the following Table 14 were obtained.

[0800] Table 14

[0801]

[0802] <Obtaining extracellular membrane vesicles using the obtaining method of the present invention>

[0803] 500 μL of PBS was used to treat 200 μL of the PBS solution containing the mTim4 vector obtained above (containing 1×10 7 After washing three times, 50 μL of a culture supernatant sample containing calcium ions prepared in the same manner as in Example 1-8 was added to the mTim4 vector in a particle state, and the mixture was reacted at 8° C. for 3 hours.

[0804] Then, the mixture was washed with 2 mM CaCl 2 Wash the reacted mTim4 vector three times with 500μL of TBS-T. During the third wash, dispense 250μL of mTim4 vector into two 1.5mL tubes. Add 20μL of 1% SDS aqueous solution and 1mM EDTA aqueous solution as eluent to 0.3mg of mTim4 vector in particle state, then mix at room temperature for 10 seconds using a vortex mixer and spin down. Place the 1.5mL tubes on magnetic racks, use magnetic force to aggregate the mTim4 vectors on the tube wall, and recover the supernatant (eluent).

[0805] <Isolation of extracellular membrane vesicles using anti-CD63 antibody immobilization method>

[0806] 5 μL of PBS solution containing anti-CD63 antibody (H5C6) carrier (containing 1×10 7 The extracellular membrane vesicles in the culture supernatant sample containing calcium ions are obtained by the same method as in the <Obtaining extracellular membrane vesicles by the obtaining method of the present invention>, the extracellular membrane vesicles are dissolved by 1% SDS aqueous solution or 1 mM EDTA aqueous solution, and the supernatant (eluate) is recovered.

[0807] <Exosome-Human CD63 Isolation / Detection to Obtain Extracellular Membrane Vesicles>

[0808] 1 mL (containing 1×10 7 The extracellular membrane vesicles in the culture supernatant sample containing calcium ions are obtained by the same method as in the <Obtaining extracellular membrane vesicles by the obtaining method of the present invention>, the extracellular membrane vesicles are dissolved by 1% SDS aqueous solution or 1 mM EDTA aqueous solution, and the supernatant (eluate) is recovered.

[0809] The types of carriers used in the Examples and Comparative Examples, the types of eluates used to obtain extracellular membrane vesicles from the carriers, and lane numbers in the Western blotting described below are shown in Table 15 below.

[0810] Table 15

[0811]

[0812] <Western blotting>

[0813] The same method as in “(7) Protein immunoblotting” of Example 1-8 was used except that “7.5 μL of each supernatant (eluate) obtained in Examples 34-35 and Comparative Examples 10-13” was used instead of “7.5 μL of each supernatant (eluate) obtained in Examples 1-8”.

[0814] <Results>

[0815] The obtained protein immunoblotting results are shown in Fig. 9 . Fig. 9 , the lanes are as follows.

[0816] Lane 1: results of Example 34 (results when extracellular membrane vesicles were obtained by the method of the present invention and a 1% SDS aqueous solution was used as an eluent);

[0817] Lane 2: results of Example 35 (results when extracellular membrane vesicles were obtained by the method of the present invention and 1 mM EDTA aqueous solution was used as an eluent);

[0818] Lane 3: Results of Comparative Example 10 (results when extracellular membrane vesicles were obtained using the anti-CD63 antibody method and a 1% SDS aqueous solution was used as an eluent);

[0819] Lane 4: Results of Comparative Example 11 (results when extracellular membrane vesicles were obtained using the anti-CD63 antibody method and a 1 mM EDTA aqueous solution was used as an eluent);

[0820] Lane 5: Results of Comparative Example 12 (results when extracellular membrane vesicles were obtained by Exosome-Human CD63 Isolation / Detection and 1% SDS aqueous solution was used as an eluent);

[0821] Lane 6: Results of Comparative Example 13 (results when extracellular membrane vesicles were obtained by Exosome-Human CD63 Isolation / Detection and a 1 mM EDTA aqueous solution was used as an eluent).

[0822] according to Fig. 9 In Example 34-35 in which extracellular membrane vesicles were obtained by the method of the present invention, a band of CD63, one of the exosome markers, was obtained at around 25-60 kDa (Example 34-35: Lanes 1-2).

[0823] On the other hand, according to Fig. 9 In Comparative Examples 10-11 in which extracellular membrane vesicles were obtained using the anti-CD63 antibody method and Comparative Examples 12-13 in which extracellular membrane vesicles were obtained using Exosome-Human CD63 Isolation / Detection, only a relatively light CD63 band was obtained near 25-60 kDa (Comparative Examples 10-13: Lanes 3-6).

[0824] Based on the above content, it can be seen that compared with the anti-CD63 antibody method, exosome-human CD63 isolation / detection, etc., which use antibodies against the surface antigen protein of extracellular membrane vesicles and obtain extracellular membrane vesicles by the affinity between the surface antigen protein and the antibody, the acquisition method of the present invention can recover more extracellular membrane vesicles.

[0825] Examples 36-38. Obtaining residual extracellular membrane vesicles from a sample after ultracentrifugation using the method of the present invention

[0826] As described below, the acquisition method of the present invention is carried out on a sample obtained by removing and acquiring extracellular membrane vesicles by ultracentrifugation (hereinafter, sometimes simply referred to as ultracentrifugation treatment), which is a conventional method.

[0827] <(1) Removal of extracellular membrane vesicles by ultracentrifugation>

[0828] 15 mL of FBS (manufactured by Corning) was centrifuged (10,000 × g, 20 minutes) to separate impurities, and the supernatant was transferred to a new tube to obtain centrifuged FBS. Then, 5 mL of the obtained centrifuged FBS was subjected to ultracentrifugation (110,000 × g, overnight) or ultracentrifugation (450,000 × g, overnight), and the supernatant was transferred to a new tube to obtain 5 mL of ultracentrifugation (110,000 × g) FBS and 5 mL of ultracentrifugation (450,000 × g) FBS, respectively.

[0829] <(2) Washing of beads>

[0830] 180 μL of a PBS solution (containing 1.8 mg of Dynabeads MyOne Streptavidin C1 beads) containing 10 μg / μL of Dynabeads MyOne Streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific) was dispensed into three tubes. Then, 1500 μL of PBS was added to each tube, and after stirring, the tubes were placed on a magnetic stand, and the Dynabeads MyOne Streptavidin C1 beads were aggregated on the tube wall by magnetic force, and the solution in the tube was discarded with a pipette (hereinafter, sometimes referred to as a washing operation).

[0831] <(3) Immobilization of Fc-tag fused mouse Tim4 protein on beads>

[0832] To 1.8 mg of Dynabeads MyOne streptavidin C1 beads in the particle state after the washing operation, 1500 μL of PBS solution containing 3 μg of mouse-derived Tim4 protein fused with a thiol-biotin-labeled Fc tag was added, and the mixture was reacted at 8°C for 10 minutes to obtain 1500 μL of PBS solution containing a vector (mTim4 vector) containing mTim4 protein fused with a thiol-biotin-labeled Fc tag.

[0833] <(4) Obtaining extracellular membrane vesicles using the obtaining method of the present invention>

[0834] The obtained mTim4 vector (1.8 mg) was washed three times with 1500 μL of PBS to obtain the mTim4 vector in particle state. Then, 5 mL of each of the FBS after centrifugation, the FBS after ultracentrifugation (110,000 × g), and the FBS after ultracentrifugation (450,000 × g) were dispensed into three 15 mL centrifuge tubes (Corning), 1.8 mg of the mTim4 vector in particle state was added, and the reaction was carried out at 8 ° C for 2 hours. After the reaction, the 15 mL centrifuge tubes were placed on a magnetic stand, and the mTim4 vector was aggregated on the tube wall by magnetic force, and each supernatant (each FBS) was recovered into a new 15 mL centrifuge tube. After washing with 2 mM CaCl 2 TBS-T (Tris buffer, 0.0005% Tween 20, 2 mM CaCl 2 )3 mL, respectively, to wash the mTim4 vector particles remaining in the centrifuge tube, and then add 2 mM CaCl 2 The cells were suspended in 1 mL of TBS-T, all of the suspension was transferred to a 1.5 mL tube, and then washed twice.

[0835] To 1.8 mg of the mTim4 vector in the particle state, 50 μL of TBS solution containing 1 mM EDTA was added as an eluent, and then mixed at room temperature for 10 seconds using a vortex mixer and spun down. 1.5 mL tubes were placed on a magnetic rack, and the mTim4 vectors were assembled on the tube wall by magnetic force, and the eluent was recovered. 50 μL of TBS solution containing 1 mM EDTA was added again, and the eluent was recovered in the same manner using the mTim4 vector used above.

[0836] Then, the beads used for recovering the eluate twice were added again to each supernatant (each FBS) recovered in the 15 mL centrifuge tube, and the operation of recovering the extracellular membrane vesicles in each supernatant (each FBS) was repeated twice by the same method as above to obtain each eluate.

[0837] <(5) Western blotting>

[0838] 100 μL of each obtained elution was ultrafiltered using VIVASPIN 500 (MVCO 10000) (trade name, manufactured by Sartorius) to reduce it to 30 μL. 5 μL of 4× sample buffer (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 15 μL of each elution after ultrafiltration, and heated at 98°C for 5 minutes to obtain each sample for protein immunoblotting. 20 μL of each sample for protein immunoblotting was added to SuperSep Ace 5-20% gel (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), and electrophoresis was performed at 25 mA for 65 minutes. The blotting was performed using a semi-dry blotting apparatus with discontinuous buffers (anode buffer 1: 0.3 M Tris / 20% methanol; anode buffer 2: 0.025 M Tris / 20% methanol; cathode buffer: 0.025 M Tris / 0.04 M aminocaproic acid / 20% methanol) at 1 mA / cm 2Under the conditions of , the obtained electrophoresis gel was transferred to a PVDF membrane (Millipore) for 60 minutes. 3% skim milk diluted with PBS-T (PBS buffer, 0.1% Tween 20 (Tween20)) was added to the transferred PVDF membrane, and reacted at room temperature for 1 hour for blocking. 2 mL of anti-bovine CD9 antibody (Novus Biologicals, sometimes referred to as "anti-bovine CD9 antibody") diluted 1000 times with PBS-T was reacted at room temperature for 1 hour. The reacted PVDF membrane was washed 3 times with PBS-T, and the secondary antibody {anti-mouse IgG (H+L), rabbit, IgG component, peroxidase-conjugated antibody} (Wako Pure Chemical Industries, Ltd.) diluted 10000 times with PBS-T was reacted at room temperature for 1 hour. After washing 5 times with PBS-T, ImmunoStar Basic (trade name, Wako Pure Chemical Industries, Ltd.) was added to the membrane after reacting the anti-bovine CD9 antibody, and ImmunoStar Zeta (trade name, Wako Pure Chemical Industries, Ltd.) was added to the membrane after reacting the anti-flotillin-2 antibody, and the luminescent signal was detected using LAS-4000 (trade name, General Electric Company (GE)). It should be noted that the anti-bovine CD9 antibody and the anti-flotillin-2 antibody are antibodies that recognize CD9 and Flotillin-2, which are marker proteins of exosomes, respectively.

[0839] <(6) Measurement of average particle number and average particle size>

[0840] 100 μL of each obtained eluate was treated with a centrifugal filter (0.45 μm, PVDF) (manufactured by Millipore), and then diluted 5-fold with water. The particles contained in the dilution were measured 3 times each using NanoSight LM10 (trade name, manufactured by NanoSight) in accordance with the instruction manual of NanoSight, and the average particle number (Particles) and average particle size (Mean Size) were detected.

[0841] In addition, the sample processing method used in each example, the method of obtaining the extracellular membrane vesicles remaining in the sample, etc. are shown in the following Table 16.

[0842] Table 16

[0843]

[0844] <Results>

[0845] The obtained protein immunoblotting results are shown in Fig.10 The results of the average number of particles and average particle size measured using NanoSight are shown in Table 17. Fig.10 In the figure, each lane shows the following results.

[0846] Lane 1: Results of Example 36 (results when the sample was subjected to centrifugal separation and extracellular membrane vesicles were obtained using the method of the present invention);

[0847] Lane 2: results of Example 37 (the sample was subjected to centrifugal separation treatment, ultracentrifuged at 110,000×g, and extracellular membrane vesicles were obtained using the method of the present invention);

[0848] Lane 3: Results of Example 38 (the sample was centrifuged and ultracentrifuged at 450,000×g, and then extracellular membrane vesicles were obtained by the method of the present invention).

[0849] according to Fig.10 It can be seen that after the sample was centrifuged and ultracentrifuged at 110,000×g or 450,000×g, when extracellular membrane vesicles were obtained using the acquisition method of the present invention (Examples 37-38), bands of CD9 and vesicle protein-2, which are exosome markers, were also confirmed, and the extracellular membrane vesicles contained in the sample could not be completely removed (obtained) by ultracentrifugation, and a large amount of extracellular membrane vesicles remained in the sample (Examples 37-38: Lanes 2-3). It can be further seen that the acquisition method of the present invention can obtain (remove) extracellular membrane vesicles that cannot be completely removed (obtained) by the ultracentrifugation separation method and remain in the sample (Examples 37-38: Lanes 2-3).

[0850] According to Table 17, when the sample was subjected to centrifugal separation, ultracentrifuged at 110000×g or 450000×g, and extracellular membrane vesicles were obtained by the method of the present invention (Examples 37-38), and when the sample was subjected to centrifugal separation, and extracellular membrane vesicles were obtained by the method of the present invention (Example 36), the size (particle size) of the extracellular membrane vesicles obtained was almost the same. Therefore, the extracellular membrane vesicles that could not be completely removed by ultracentrifugation and remained in the sample did not form fragments, but existed as particles.

[0851] Table 17

[0852]

[0853] Examples 39-40. Obtaining residual extracellular membrane vesicles from a sample after total exosome isolation (polymer precipitation) using the method of the present invention

[0854] As described below, the acquisition method of the present invention is implemented for a sample in which extracellular membrane vesicles contained in a sample are removed (obtained) by total exosome isolation (polymer precipitation) as a conventional method (hereinafter sometimes referred to as "polymer precipitation treatment").

[0855] <(1) Removal of extracellular membrane vesicles by total exosome isolation (polymer precipitation) method>

[0856] 10 mL of FBS (manufactured by Corning) was centrifuged (10,000 × g, 20 minutes) to separate impurities and obtain FBS after centrifugation. Then, 1 mL of Total Exosome Isolation reagent (from serum) (manufactured by Thermo Fisher Scientific) was added to 5 mL of FBS after centrifugation, and the mixture was allowed to stand for 30 minutes under refrigeration conditions, and then centrifuged (10,000 × g, 20 minutes). The supernatant was recovered to obtain 6 mL of FBS after total exosome isolation.

[0857] <(2) Washing of beads>

[0858] 180 μL of a PBS solution (containing 1.8 mg of Dynabeads MyOne Streptavidin C1 beads) containing 10 μg / μL of Dynabeads MyOne Streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific) was dispensed into two tubes. Then, 1500 μL of PBS was added to each tube and stirred. Then, each tube was placed on a magnetic stand, and the Dynabeads MyOne Streptavidin C1 beads were aggregated on the tube wall by magnetic force, and the solution in the tube was discarded with a pipette (hereinafter, sometimes referred to as a washing operation).

[0859] <(3) Immobilization of Fc-tagged mouse Tim4 protein on beads>

[0860] To 1.8 mg of Dynabeads MyOne streptavidin C1 beads in a particle state after the washing operation, add 1500 μL of PBS solution containing 3 μg of mouse-derived Tim4 protein fused with a thiol-biotin-labeled Fc tag prepared by the same method as Example 5-8, and react at 8°C for 10 minutes to obtain 1500 μL of PBS solution containing a vector (mTim4 vector) containing mTim4 protein fused with a thiol-biotin-labeled Fc tag.

[0861] <(4) Obtaining extracellular membrane vesicles using the obtaining method of the present invention>

[0862] 1.8 mg of the obtained mTim4 vector was washed three times with 1500 μL of PBS to obtain the mTim4 vector in a particle state. Then, 1.8 mg of the mTim4 vector in a particle state was added to 5 mL of FBS after centrifugation or 6 mL of FBS after total exosome isolation in a 15 mL centrifuge tube (Corning), and the reaction was carried out at 8°C for 2 hours.

[0863] After the reaction, 15 mL centrifuge tubes were placed on magnetic racks to aggregate mouse Tim4 vectors on the tube wall using magnetic force, and each supernatant (each FBS) was collected in a new 15 mL centrifuge tube. 2 TBS-T (Tris buffer, 0.0005% Tween 20, 2 mM CaCl 2 )3 mL were used to wash the mTim4 vector particles remaining in the centrifuge tube, and then 1 mL of 2 mM CaCl was added to the mTim4 vector particles. 2 The cells were suspended in TBS-T, all the suspension was transferred to a 1.5 mL tube, and then washed twice.

[0864] To each mTim4 vector 1.8 mg in a particle state, 50 μL of TBS solution containing 1 mM EDTA was added as an eluent, and then mixed at room temperature for 10 seconds using a vortex mixer and spun down. 1.5 mL tubes were placed on a magnetic rack, and the mTim4 vectors were assembled on the tube wall by magnetic force, and the eluent was recovered. 50 μL of TBS solution containing 1 mM EDTA was added again, and the eluent was recovered by the same method using the mTim4 vector used above.

[0865] Then, the mTim4 vector (beads) used to recover the eluate twice was added again to each supernatant (each FBS) recovered in the 15 mL centrifuge tube. Furthermore, the operation of recovering the extracellular membrane vesicles in each supernatant (each FBS) was repeated twice using the same method as above to obtain each eluate.

[0866] <(5) Western blotting>

[0867] 300 μL of each eluate obtained was ultrafiltered using VIVASPIN 500 (MVCO 10000) (trade name, manufactured by Sartorius) to reduce it to 30 μL. 10 μL of 4× sample buffer (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 30 μL of each eluate after ultrafiltration, and heated at 98°C for 5 minutes to obtain each sample for protein immunoblotting. 20 μL of each sample for protein immunoblotting was added to two locations in SuperSep Ace 5-20% gel (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), and electrophoresis was performed at 25 mA for 65 minutes. The blotting was performed using a semi-dry blotting apparatus and discontinuous buffers (anode buffer 1: 0.3 M Tris / 20% methanol; anode buffer 2: 0.025 M Tris / 20% methanol; cathode buffer: 0.025 M Tris / 0.04 M aminocaproic acid / 20% methanol) at 1 mA / cm 2The obtained electrophoresis gel was transferred to a PVDF membrane (Millipore) under the conditions of 60 minutes. 3% skim milk diluted with PBS-T (PBS buffer, 0.1% Tween 20) was added to the PVDF membrane after transfer, and the mixture was reacted at room temperature for 1 hour for blocking. 2 mL of anti-bovine CD9 antibody (Novus Biologicals, hereinafter sometimes referred to as "anti-bovine CD9 antibody") diluted 1000 times with PBS-T or anti-human vesperin-2 antibody (BD Biosciences, hereinafter sometimes referred to as "anti-human vesperin-2 antibody") diluted 250 times with PBS-T was reacted at room temperature for 1 hour. The PVDF membrane after the reaction was washed 3 times with PBS-T, and the secondary antibody {anti-mouse IgG (H+L), rabbit, IgG component, peroxidase-bound antibody} (Wako Pure Chemical Industries (Strain)) diluted 10,000 times with PBS-T was reacted at room temperature for 1 hour. After washing 5 times with PBS-T, ImmunoStar Basic (trade name, Wako Pure Chemical Industries (Strain)) was added to the membrane after the anti-bovine CD9 antibody was reacted, and ImmunoStar Zeta (trade name, Wako Pure Chemical Industries (Strain)) was added to the membrane after the anti-floating flank protein-2 antibody was reacted, and the luminescent signal was detected using LAS-4000 (trade name, General Electric Company (GE)). It should be noted that the anti-bovine CD9 antibody is an antibody against CD9, which is one of the marker proteins of exosomes, and the anti-human floating flank protein-2 antibody is an antibody against floating flank protein-2, which is one of the marker proteins of exosomes.

[0868] The sample treatment metho...

Claims

1. A method for detecting extracellular membrane vesicles in a sample, characterized in that: It includes the following steps: (1) a complex formation step, which is a step of forming a complex between the T cell immunoglobulin and mucin domain molecule 4 (Tim4) protein bound to a carrier and the extracellular membrane vesicles in the sample in the presence of calcium ions; (2) a detection step of detecting the complex using an antibody that binds to the extracellular membrane vesicles, The antibody that binds to extracellular membrane vesicles is labeled.

2. The method of claim 1, wherein: The method for detecting the cell extracellular membrane vesicles is ELISA or flow cytometry.

3. The method of claim 1, wherein: The Tim4 protein comprises an IgV domain.

4. The method of claim 1, wherein: The carrier and the Tim4 protein are bound via the sulfhydryl group of the Tim4 protein.

5. Use of a kit for detecting extracellular membrane vesicles in the method according to any one of claims 1 to 4, wherein: The kit comprises a carrier bound to Tim4 protein and an antibody bound to extracellular membrane vesicles.

6. Use of a kit for detecting extracellular membrane vesicles in the method according to any one of claims 1 to 4, wherein: The kit comprises Tim4 protein, a carrier and an antibody binding to cell extracellular membrane vesicles.

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