Tim protein-binding carrier, extracellular membrane vesicle using the carrier, and virus acquisition method, removal method, detection method, and kit including the carrier

Abstract

The present application provides a carrier and a method for obtaining or removing, or detecting, an extracellular membrane vesicle or a virus present in a sample with high purity, in an intact state, easily and efficiently. The present application relates to: 1. a carrier (Tim carrier) combined with a protein (Tim protein) selected from among a protein included in a molecule 4 (Tim4) protein, a Tim3 protein, and a Tim1 protein; 2. a method for obtaining an extracellular membrane vesicle or a virus in a sample; 3. a method for removing an extracellular membrane vesicle or a virus in a sample; 4. a method for detecting an extracellular membrane vesicle or a virus in a sample; 5. a capture kit for an extracellular membrane vesicle or a virus, which is formed by containing a Tim carrier; and 6. a capture kit for an extracellular membrane vesicle or a virus, which is formed by containing a reagent including a Tim protein and a reagent including a carrier.

Description

Technical Field

[0001] This invention relates to a Tim protein binding vector, extracellular membrane vesicles using the vector, methods for obtaining, removing, and detecting viruses, and a kit including the vector. Background Technology

[0002] As is well known, extracellular membrane vesicles contain proteins, microRNAs, and other nucleic acids within their particles, responsible for intercellular transport. Extracellular membrane vesicles are also secreted into bodily fluids such as blood. The proteins and microRNAs within these vesicles have attracted attention as diagnostic markers for diseases. Furthermore, their application as a delivery tool for nucleic acid drugs is also noteworthy.

[0003] In addition, a large number of viruses are enveloped viruses with a membrane-like envelope on their surface. Enveloped viruses are mostly diseases caused by influenza viruses, human immunodeficiency virus (HIV), etc., and therefore attract considerable attention as research subjects. Furthermore, enveloped viruses are inactivated and used as vaccines and vectors for disease prevention and treatment.

[0004] Therefore, in basic research involving the application of extracellular membrane vesicles in diagnostics or pharmaceuticals, the use of enveloped viruses as vaccines or vectors, and the functional analysis of extracellular membrane vesicles or enveloped viruses, obtaining extracellular membrane vesicles and enveloped viruses with high purity is essential. There is a strong desire for a simple method to obtain high-purity extracellular membrane vesicles and enveloped viruses. Furthermore, to prevent contamination from biological extracellular membrane vesicles and enveloped viruses, there is a need to develop a method to effectively remove extracellular membrane vesicles and enveloped viruses from materials derived from bodily fluids. Moreover, there is a strong desire for a method with high sensitivity to detect obtained extracellular membrane vesicles and enveloped viruses, as well as extracellular membrane vesicles and enveloped viruses in the sample.

[0005] The most common known method for obtaining extracellular membrane vesicles is to perform ultracentrifugation on the sample to obtain extracellular membrane vesicles as a precipitate (Non-Patent Literature 1). However, with this method, in addition to extracellular membrane vesicles, protein complexes or globules, lipoproteins such as HDL, etc., contained in the sample also co-precipitate, making it difficult to obtain extracellular membrane vesicles with high purity. By using the sucrose density gradient method to perform density fractionation on the precipitate obtained by ultracentrifugation, protein complexes and globules can be separated, but it is difficult to separate them from HDL, which has the same density. Furthermore, this method requires ultracentrifugation, making it difficult to process multiple samples simultaneously. Moreover, ultracentrifugation requires expensive equipment.

[0006] In addition, there is a method that involves adding commercially available reagents, such as ExoQuick (manufactured by SystemBiosciences) and Total Exosome Isolation Reagent (manufactured by Thermo Fisher Scientific), and centrifuging to obtain extracellular membrane vesicles as a precipitate (Non-Patent Document 1). However, this method has the problem that the purity of the extracellular membrane vesicles obtained is lower than that obtained by the ultracentrifugation method described above.

[0007] In addition to the existing methods, there are other methods that use antibodies against surface antigen proteins of extracellular membrane vesicles to obtain extracellular membrane vesicles through the affinity between the surface antigen protein and the antibody (anti-CD63 antibody immobilization method, exosome-human CD63 isolation / detection (Thermo Fisher Scientific Co., Ltd.) etc.) (Non-Patent Literature 1). While these methods can obtain high-purity extracellular membrane vesicles, they have the following problems: only extracellular membrane vesicles with surface antigen proteins against the antibody can be obtained; the yield of extracellular membrane vesicles is low; surfactants, acidic buffers, etc., are required to dissolve the extracellular membrane vesicles from the antibody; and it is difficult to obtain intact extracellular membrane vesicles (i.e., in a state that retains the original function of the extracellular membrane vesicles).

[0008] The most common known method for obtaining viruses is to perform ultracentrifugation on samples to obtain the virus as a precipitate (Non-Patent Literature 1). However, with this method, in addition to the virus, protein complexes or lectins, lipoproteins such as HDL, etc., contained in the sample also co-precipitate, making it difficult to obtain viruses of high purity. By using the sucrose density gradient method to perform density fractionation on the precipitate obtained by ultracentrifugation, protein complexes and lectins can be separated; however, it is difficult to separate them from lectins of equal density. Furthermore, there is a problem that the activity of the virus decreases due to ultracentrifugation (Non-Patent Literature 2). In addition, since the method requires ultracentrifugation, it is difficult to process multiple samples simultaneously. Moreover, ultracentrifugation requires expensive equipment.

[0009] Besides the method described above, methods for purifying viruses via ion exchange chromatography are known (Non-Patent Document 3). However, this method requires setting optimal conditions for various viruses. Furthermore, setting these conditions can sometimes be difficult, making it hard to apply to the purification of all viruses.

[0010] The most common known method for removing extracellular membrane vesicles is to perform ultracentrifugation on the sample to separate the vesicles into a supernatant. However, this method is difficult to completely remove extracellular membrane vesicles, leaving some incompletely removed vesicles in the sample.

[0011] Furthermore, another method involves adding commercially available reagents, such as ExoQuick (manufactured by SystemBiosciences) and Total Exosome Isolation Reagent (manufactured by Thermo Fisher Scientific), and centrifuging to precipitate and fractionate extracellular membrane vesicles, obtaining the supernatant as a residue to remove the sample. However, even with this method, extracellular membrane vesicles cannot be completely removed, remaining in the sample, making complete removal difficult. Additionally, the added reagents can sometimes cause problems with the biological activity of the extracellular membrane vesicles due to contamination of the sample.

[0012] As a method for removing viruses, there are known similar methods that have the same problems as methods for removing extracellular membrane vesicles.

[0013] As a method for detecting extracellular membrane vesicles, sandwich ELISA using antibodies against surface antigens of extracellular membrane vesicles is known as a routine method. However, it has been reported that the lowest detection sensitivity of sandwich ELISA systems using antibodies that detect colorimetric signals is approximately 3 μg based on purified exosomes, which is insufficient for measuring bodily fluid samples such as serum (Non-Patent Literature 4). Similarly, as a method for detecting viruses, the same method is known, but it is also not sufficient for detecting viruses.

[0014] In addition, flow cytometry is known as another method for detecting extracellular membrane vesicles. However, for conventional methods that use carriers with immobilized antibodies against the surface antigens of extracellular membrane vesicles to capture extracellular membrane vesicles, sufficient sensitivity cannot be obtained, and it is difficult to detect them directly from samples such as culture supernatants or body fluids. Therefore, it is necessary to concentrate or purify the extracellular membrane vesicles from the sample before detection.

[0015] Existing technical documents

[0016] Non-patent literature

[0017] Non-patent literature 1: Kenneth W. Witwer et al. Journal of Extracellular Vesicles 2013 May 27; 2. doi:10.3402;

[0018] Non-patent literature 2: GYChen et al. Biotechnol. Prog. 25 (2009) 1669;

[0019] Non-patent literature 3: Petra Gerster et al. Journal of Chromatography A, 1290 (2013) 36-45;

[0020] Non-patent literature 4: Mariatonia Logozzi et al. PLoS ONE 4 (2009) 5219. Summary of the Invention

[0021] The problem that the invention aims to solve

[0022] As mentioned above, existing methods for obtaining extracellular membrane vesicles or viruses are difficult to use simply and effectively to obtain high-purity extracellular membrane vesicles or viruses in their intact state. Furthermore, existing methods for removing extracellular membrane vesicles or viruses are ineffective in removing them from samples. Moreover, existing methods for detecting extracellular membrane vesicles or viruses lack sufficient sensitivity.

[0023] Therefore, the objective of this invention is to obtain or remove extracellular membrane vesicles or viruses present in a sample with good purity and in an intact state, and to detect extracellular membrane vesicles or viruses with high sensitivity.

[0024] Technical solutions to the problem

[0025] The present invention is made to solve the aforementioned problem and consists of the following technical solution.

[0026] 1. A vector (Tim vector) wherein a protein (Tim protein) selected from a protein containing T cell immunoglobulin and mucin domain molecule 4 (Tim4), a protein containing T cell immunoglobulin and mucin domain molecule 3 (Tim3), and a protein containing T cell immunoglobulin and mucin domain molecule 1 (Tim1) is bound.

[0027] 2. A method for obtaining extracellular membrane vesicles or viruses from a sample, characterized in that it comprises the following steps:

[0028] (1) The process of forming a complex of Tim protein bound to a carrier and extracellular membrane vesicles or viruses in a sample in the presence of calcium ions (complex formation process).

[0029] (2) The process of separating the composite from the sample (composite separation process);

[0030] (3) The process of isolating extracellular membrane vesicles or viruses from the complex and obtaining extracellular membrane vesicles or viruses (obtaining process).

[0031] 3. A method for removing extracellular membrane vesicles or viruses from a sample, characterized in that it comprises the following steps:

[0032] (1) The process of forming a complex of Tim protein bound to a carrier and extracellular membrane vesicles or viruses in a sample in the presence of calcium ions (complex formation process).

[0033] (2) The process of separating the composite from the sample (composite separation process).

[0034] 4. A method for detecting extracellular membrane vesicles or viruses in a sample, characterized by comprising the following steps:

[0035] (1) The process of forming a complex of Tim protein bound to a carrier and extracellular membrane vesicles or viruses in a sample in the presence of calcium ions (complex formation process).

[0036] (2) The testing procedure for the composite material (testing procedure)

[0037] 5. A kit for capturing extracellular membrane vesicles or viruses, wherein the vesicles are formed containing a Tim vector.

[0038] 6. A reagent kit for capturing extracellular membrane vesicles or viruses, wherein the kit comprises a reagent containing a Tim protein and a reagent containing a carrier.

[0039] In view of the above, the 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 with phosphatidylserine on their surface can be obtained with high purity, the extracellular membrane vesicles or viruses can be obtained in an intact state, the extracellular membrane vesicles or viruses can be effectively removed, and the extracellular membrane vesicles or viruses can be detected with high sensitivity, thus completing the present invention.

[0040] Phosphatidylserine, a phospholipid, is present (exposed) on the surface of extracellular membrane vesicles. Proteins that bind to phosphatidylserine (hereinafter sometimes referred to as "phosphatidylserine-binding proteins" or "PS proteins") include, for example, Annexin V (membrane adhesion protein-5), MFG-E8, Tim1 (containing T-cell immunoglobulin-mucin domain 1), Tim3 (containing T-cell immunoglobulin-mucin domain 3), and Tim4 (containing T-cell immunoglobulin-mucin domain 4) (The Journal of Biochemistry 265, 4923-4928 (25 March 1990), Nature 417, 182-187 (9 May)). 2002), Nature450,435-439(15November 2007).

[0041] However, it has been found that, when using PS proteins other than Tim1, Tim3 and Tim4, it is difficult to obtain, remove or detect extracellular membrane vesicles or viruses.

[0042] The effects of the invention

[0043] This invention enables the simple and efficient acquisition, removal, and high-sensitivity detection of extracellular membrane vesicles or viruses present in a sample, while maintaining high purity and in their intact state. Attached Figure Description

[0044] Figure 1 In Examples 1-8, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0045] Figure 2 In Examples 9-16, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0046] Figure 3 In Examples 17-18, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0047] Figure 4A is the confirmation by Western blotting in Examples 19-20 and Comparative Examples 1-3 of whether electrophoretic images of extracellular membrane vesicles were obtained. Figure 4 -B is the electrophoretic image of extracellular membrane vesicles obtained by silver staining in Examples 19-20 and Comparative Examples 1-3.

[0048] Figure 5 These are extracellular membrane vesicles obtained using the method of the present invention, as observed by electron microscopy in Example 21.

[0049] Figure 6 In Examples 22-25, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0050] Figure 7 In Examples 26-27 and Comparative Examples 4-9, silver staining was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0051] Figure 8 In Examples 28-33, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0052] Figure 9 In Examples 34-35 and Comparative Examples 10-13, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0053] Figure 10 In Examples 36-38, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0054] Figure 11 In Examples 39-40, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0055] Figure 12 In Examples 41-47, the electrophoretic images of whether extracellular membrane vesicles were obtained (removed) were confirmed by Western blotting.

[0056] Figure 13 In Examples 48-55, the electrophoretic images of whether (removed) extracellular membrane vesicles were obtained were confirmed by Western blotting.

[0057] Figure 14 In Examples 56-67 and Comparative Examples 14-15, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0058] Figure 15 In Examples 68-79, Western blotting was used to confirm whether electrophoretic images of extracellular membrane vesicles were obtained.

[0059] Figure 16 In Examples 80-83 and Comparative Examples 16-19, the electrophoretic images of extracellular membrane vesicles were confirmed by Western blotting.

[0060] Figure 17 In Examples 84-95 and Comparative Examples 20-21, it was confirmed by Western blotting whether an electrophoretic image of the virus had been obtained.

[0061] Figure 18 In Examples 96-99 and Comparative Examples 22-25, it was confirmed by Western blotting whether an electrophoretic image of the virus was obtained.

[0062] Figure 19 The results are obtained by detecting extracellular membrane vesicles using the ELISA method in Examples 100-105 and Comparative Examples 26-33.

[0063] Figure 20 The results are from the detection of extracellular membrane vesicles by ELISA in Examples 106-109.

[0064] Figure 21 The results are from the detection of the virus by ELISA in Examples 110-115 and Comparative Examples 34-39.

[0065] Figure 22 In Examples 116-119, it was confirmed by Western blotting whether an electrophoretic image of the virus had been obtained.

[0066] Figure 23 The results are obtained by flow cytometry analysis of extracellular membrane vesicles in Examples 120-121 and Comparative Examples 40-41.

[0067] Figure 24 In Examples 122-123 and Comparative Examples 42-43, it was confirmed by Western blotting whether an electrophoretic image of the virus had been obtained.

[0068] Figure 25 In Examples 124-125 and Comparative Examples 44-45, it was confirmed by Western blotting whether an electrophoretic image of the virus had been obtained. Detailed Implementation

[0069] <1. Extracellular membrane vesicles of the present invention>

[0070] The extracellular membrane vesicles of the present invention are small membrane vesicles secreted by cells in vivo or cultured cells, consisting of a lipid bilayer membrane and having phosphatidylserine on the membrane surface. The diameter of these vesicles is typically 20 nm to 1000 nm, preferably 50 nm to 500 nm, and more preferably 50 nm to 200 nm.

[0071] Regarding the extracellular membrane vesicles of this invention, as described in the articles *Nature Reviews Immunology* (9, 581-593 (August 2009)) and *Hypertrophy Research* (Vol. 13, No. 2 2007) by Naoto Aoki et al., various types of vesicles can be classified according to their origin of formation and the size of small membrane vesicles. Specifically, examples include exosomes, microvesicles, ectosomes, membrane granules, exosome-like vesicles, apoptotic vesicles, and adiposomes.

[0072] Exosomes are small membrane vesicles derived from late endosomes, composed of a lipid bilayer membrane with phosphatidylserine residues on the membrane surface. The diameter of these vesicles is typically 50 nm to 200 nm, preferably 50 nm to 150 nm, and more preferably 50 nm to 100 nm. Exosomes are known to contain proteins such as tetraspanins (CD63, CD9, etc.), Alix, TSG101, Lamp-1, and Flotillin.

[0073] Microvesicles are small membrane vesicles derived from the cell membrane (plasma membrane), composed of a lipid bilayer, and having phosphatidylserine residues on the membrane surface. Microvesicles are typically 100 nm to 1000 nm in size, preferably 100 nm to 800 nm, and more preferably 100 nm to 500 nm. It is well known that microvesicles contain proteins such as integrins, selectins, and CD40 ligands.

[0074] Extranuclear granules are small membrane vesicles derived from the cell membrane (cytoplasmic membrane), composed of a lipid bilayer, and having phosphatidylserine residues on the membrane surface. Extranuclear granules are typically 50 nm to 200 nm in size, preferably 50 nm to 150 nm, and more preferably 50 nm to 100 nm. Extranuclear granules are known to contain CR1 and proteolytic enzymes, but not CD63.

[0075] Membrane particles are small membrane vesicles derived from the cell membrane (cytoplasmic membrane), composed of a lipid bilayer, and containing phosphatidylserine residues on their surface. Membrane particles typically range in size from 50 nm to 80 nm. They are known to contain CD133 but not CD63.

[0076] Exosome-like vesicles are small membrane vesicles derived from early endosomes, composed of a lipid bilayer membrane with phosphatidylserine residues on its surface. Exosome-like vesicles typically range in size from 20 nm to 50 nm. They are known to contain TNFRI (tumor necrosis factor receptor I).

[0077] Apoptotic vesicles are small membrane vesicles derived from apoptotic cells, composed of a lipid bilayer membrane with phosphatidylserine residues on its surface. Apoptotic vesicles are typically 50 nm to 500 nm in size, preferably 50 nm to 300 nm, and more preferably 50 nm to 200 nm. Apoptotic vesicles are known to contain histones.

[0078] Fat bodies are small membrane vesicles derived from adipocytes, composed of a lipid bilayer membrane with phosphatidylserine residues on the membrane surface. Fat bodies typically range in size from 100 nm to 1000 nm, preferably from 100 nm to 800 nm, and more preferably from 100 nm to 500 nm. Fat bodies are known to contain MFG-E8 (milk fat globule-EGF factor 8).

[0079] <2. The virus of this invention>

[0080] The virus of the present invention is a virus whose capsid (outer shell) has an envelope composed of a lipid bilayer membrane derived from the host cell membrane, nuclear membrane, Golgi apparatus, endoplasmic reticulum, etc., and has phosphatidylserine residues on its surface (hereinafter referred to as "enveloped virus"). The diameter of the virus of the present invention is typically 20 nm to 320 nm. Examples of enveloped viruses of the present invention include those belonging to the families described in the 2nd edition of the Biochemical Dictionary (Tokyo Chemical Dojin, 1990, 1503p-1505p). Specifically, examples include the families Poxviridae, Baculoviridae, Rhabdoviridae, Bunyaviridae, Togaviridae, Herpesviridae, Paramyxoviridae, Orthomyxoviridae, Retrovirus, Arenaviridae, and Coronaviridae.

[0081] <3. The Tim protein of the present invention>

[0082] The Tim protein of the present invention is selected from at least one of the following: the T cell immunoglobulin and mucin domain molecule 1 (Tim1) protein of the present invention (hereinafter, sometimes referred to as "Tim1 protein of the present invention"), the T cell immunoglobulin and mucin domain molecule 3 (Tim3) protein of the present invention (hereinafter, sometimes referred to as "Tim3 protein of the present invention"), and the T cell immunoglobulin and mucin domain molecule 4 (Tim4) protein of the present invention (hereinafter, sometimes referred to as "Tim4 protein of the present invention").

[0083] As the Tim4 protein (containing T cell immunoglobulin and mucin domain 4) of the present invention, any protein capable of binding to the extracellular membrane vesicles or viruses of the present invention is acceptable, and animal-derived Tim4 proteins are preferred. Among them, human or mouse-derived Tim4 proteins are preferred (hereinafter, human-derived Tim4 proteins are sometimes referred to as "human-derived Tim4 proteins", and mouse-derived Tim4 proteins are sometimes referred to as "mouse-derived Tim4 proteins").

[0084] More specifically, any protein that has at least an amino acid sequence with a binding domain (IgV domain) relative to phosphatidylserine can be used. It can be a protein with the full-length amino acid sequence of the Tim4 protein or a part of the Tim4 protein.

[0085] Examples of amino acid sequences relative to the binding domain (IgV domain) of the phosphatidylserine include, for example, sequence number 1 (the N-terminal amino acid domain of mouse Tim4 protein, positions 22-135 (RefSeq NP_848874.3) and sequence number 2 (the N-terminal amino acid domain of human Tim4 protein, positions 25-137 (RefSeq NP_612388.2)).

[0086] Examples of full-length amino acid sequences of the Tim4 protein include sequence number 3 (amino acid domains 1-343 of the full-length sequence of the mouse Tim4 protein (RefSeq NP_848874.3)) and sequence number 4 (amino acid domains 1-378 of the full-length sequence of the human Tim4 protein (RefSeq NP_612388.2)).

[0087] Examples of sequences containing an amino acid sequence with a binding domain (IgV domain) and a mucin domain relative to phosphatidylserine, such as sequence number 5 (the N-terminal amino acid domain of mouse Tim4 protein (RefSeq NP_848874.3)), sequence number 6 (the N-terminal amino acid domain of mouse Tim4 protein (RefSeq NP_848874.3)), and sequence number 7 (the N-terminal amino acid domain of human Tim4 protein (RefSeq NP_612388.2)). These sequences may, if desired, include a signal sequence.

[0088] As the Tim1 protein (containing T-cell immunoglobulin and mucin domain molecule 1) of the present invention, any protein capable of binding to the extracellular membrane vesicles or viruses of the present invention is acceptable, and animal-derived Tim1 proteins are preferred. Among them, human or mouse-derived Tim1 proteins are preferred (hereinafter, human-derived Tim1 proteins are sometimes referred to as "human-derived Tim1 proteins", and mouse-derived Tim1 proteins are referred to as "mouse-derived Tim1 proteins").

[0089] More specifically, it can be any protein that has at least an amino acid sequence with a binding domain (IgV domain) relative to phosphatidylserine, and can be a protein with the full-length amino acid sequence of the Tim1 protein or a part of the Tim1 protein.

[0090] Examples of amino acid sequences that represent the binding domain (IgV domain) relative to phosphatidylserine include, for example, sequence number 8 (the N-terminal amino acid domain of mouse Tim1 protein, positions 22-131 (RefSeq NP_001160104.1) and sequence number 9 (the N-terminal amino acid domain of human Tim1 protein, positions 21-130 (RefSeq NP_036338.2)).

[0091] Examples of the full-length amino acid sequences of the Tim1 protein include sequence number 10 (amino acid domains 1-282 of the full-length sequence of the mouse Tim1 protein (RefSeq NP_001160104.1)) and sequence number 11 (amino acid domains 1-364 of the full-length sequence of the human Tim1 protein (RefSeq NP_036338.2)).

[0092] Examples of sequences that are part of the Tim1 protein include sequence number 12 (the N-terminal amino acid domain of mouse Tim1 protein (RefSeq NP_001160104.1)) and sequence number 13 (the N-terminal amino acid domain of human Tim1 protein (RefSeq NP_612388.2)), which contain amino acid sequences with a binding domain (IgV domain) and a mucin domain relative to phosphatidylserine. If necessary, the sequence may include a signal sequence.

[0093] As the Tim3 protein (containing T-cell immunoglobulin and mucin domain molecule 3) of the present invention, any protein capable of binding to the extracellular membrane vesicles or viruses of the present invention is acceptable, and animal-derived Tim3 proteins are preferred. Among them, human or mouse-derived Tim3 proteins are preferred (hereinafter, human-derived Tim3 proteins are sometimes referred to as "human-derived Tim3 proteins", and mouse-derived Tim3 proteins are sometimes referred to as "mouse-derived Tim3 proteins").

[0094] More specifically, any protein that has at least an amino acid sequence with a binding domain (IgV domain) relative to phosphatidylserine can be a protein with the full-length amino acid sequence of the Tim3 protein or a part of the Tim3 protein.

[0095] Examples of amino acid sequences that represent the binding domain (IgV domain) relative to phosphatidylserine include, for example, sequence number 14 (the N-terminal amino acid domain of mouse Tim3 protein, positions 22-134 (RefSeq NP_599011.2) and sequence number 15 (the N-terminal amino acid domain of human Tim3 protein, positions 22-135 (RefSeq NP_116171.3)).

[0096] Examples of the full-length amino acid sequences of the Tim3 protein include sequence number 16 (the domain of amino acids 1 to 281 of the full-length sequence of the mouse Tim3 protein (RefSeq NP_599011.2)) and sequence number 17 (the domain of amino acids 1 to 301 of the full-length sequence of the human Tim3 protein (RefSeq NP_116171.3)).

[0097] Examples of sequences that are part of the Tim3 protein include sequence number 18 (the N-terminal amino acid domain of mouse Tim3 protein (RefSeq NP_599011.2)) and sequence number 19 (the N-terminal amino acid domain of human Tim3 protein (RefSeq NP_116171.3)), which contain amino acid sequences with a binding domain (IgV domain) and a mucin domain relative to phosphatidylserine. If necessary, these sequences may include a signal sequence.

[0098] In addition, the Tim protein of the present invention can be a variant formed by the deletion, substitution, insertion and / or addition of one or more amino acids, as long as it can bind to the extracellular membrane vesicles or viruses of the present invention.

[0099] The Tim protein of the present invention can be any protein that possesses the above-described properties, including proteins extracted from cells (e.g., immune cells such as macrophages) and tissues of organisms containing Tim proteins such as mice or humans, plants, etc., as well as proteins prepared by gene recombination technology based thereon.

[0100] When using a protein prepared by gene recombination technology as the Tim protein of the present invention, a protein having one or more affinity tags is preferred from the viewpoint of facilitating purification.

[0101] As an affinity tag, any tag can be used as long as it is used when preparing proteins through gene recombination technology. Examples of affinity tags include Fc tag, FLAG tag, His tag, GST tag, MBP tag, HA tag, Myc tag, Strep(II) tag, PA tag, etc.

[0102] The affinity tag fuses with the C-terminal side of the Tim protein of the present invention.

[0103] Therefore, the Tim protein of the present invention includes not only proteins consisting solely of the amino acid sequence (full-length or partial sequence) of the Tim protein, but also proteins having the amino acid sequence (full-length or partial sequence) of the Tim protein and the amino acid sequence of the affinity tag as described above.

[0104] In addition, the affinity tag can bind directly to the Tim protein, or can bind through a spacer such as described in "Protein Expression Using a Cell-Free Protein Synthesis Kit (Transdirect insect cell) Derived from Insect Cultured Cells" ("Protein Expression Using a Cell-Free Protein Synthesis Kit Trans direct insect cell Derived from Insect Cultured Cells") (Kawase Tetsu, Suzuki Takashi, Ito Shoji, Yoshimitsu Shibata (Shimadzu Corporation / Analytical and Measuring Instrument Division), publication date 2008 / 6 / 9, Protein Science Society Archives, 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 partial sequence), the amino acid sequence of the affinity tag as described above, and the amino acid sequence of the spacer.

[0105] <4. Preparation Method of the Tim Protein of the Present Invention>

[0106] The Tim protein of the present invention can be produced by conventional chemical production methods according to its amino acid sequence. For example, the Tim protein of the present invention can be obtained by usual chemical production methods (chemical synthesis methods) such as the fluorenylmethyloxycarbonyl method (Fmoc method) and the tert-butoxycarbonyl method (tBoc method). In addition, chemical synthesis can be carried out using a commercially available peptide synthesizer.

[0107] Furthermore, the Tim protein of the present invention can also be obtained by the following well-known method using genetic recombination technology, that is, combining a nucleic acid molecule encoding the Tim protein of the present invention into an appropriate expression vector such as a plasmid or a phage, transforming (or transducing) a host cell with the recombinant expression vector, amplifying the obtained host cell, and secreting it into or outside the cell.

[0108] Regarding the preparation method of the Tim protein of the present invention, the case of preparation by genetic recombination technology is described below.

[0109] <The Expression Vector of the Present Invention>

[0110] 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 simply referred to as "the Tim coding sequence of the present invention").

[0111] In the Tim coding sequence of the present invention, examples of nucleic acid sequences encoding the Tim4 protein include: for example, sequence number 20 (the base sequence of cDNA encoding the amino acid domains 1 to 343 of the full-length sequence of the mouse Tim4 protein (RefSeq No. NM_178759.4), with the last three bases containing a stop codon (tga)), and sequence number 21 (the base sequence of cDNA encoding the amino acid domains 1 to 378 of the full-length sequence of the human Tim4 protein (RefSeq No. NM_138379.2), with the last three bases containing a stop codon (taa)), etc.

[0112] 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 domain of amino acids 1 to 282 of the full-length sequence of the mouse Tim1 protein (RefSeq No. NM_001166632.1, with the last three bases containing a stop codon (tga)) and sequence number 23 (the base sequence of cDNA encoding the domain of amino acids 1 to 364 of the full-length sequence of the human Tim1 protein (RefSeq No. NM_012206.3, with the last three bases containing a stop codon (taa)), etc.

[0113] 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 domains 1 to 281 of the full-length sequence of the mouse Tim3 protein (RefSeq No. NM_134250.2, with the last three bases containing a stop codon (tga)) and sequence number 25 (the base sequence of cDNA encoding the amino acid domains 1 to 301 of the full-length sequence of the human Tim3 protein (RefSeq No. NM_032782.4, with the last three bases containing a stop codon (tag)), etc.

[0114] As expression vectors for the present invention, the Tim coding sequence of the present invention can be introduced into commercially available vectors using conventional cloning methods. Examples of expression vectors for the present invention include, for instance, cDNA encoding the N-terminal domains 1-273 of mouse Tim4 protein (with a stop codon (tga) at the last 3 bases), 27 (cDNA encoding the N-terminal domains 1-279 of mouse Tim4 protein (with a stop codon (tga) at the last 3 bases), 28 (cDNA encoding the N-terminal domains 1-315 of human Tim4 protein (with a stop codon (tga) at the last 3 bases), and 29 (cDNA encoding the N-terminal domains 1-212 of mouse Tim1 protein) using conventional cloning techniques. The following are examples of vectors: DNA, cDNA encoding the N-terminal 1-295 amino acid domain of human Tim1 protein, with the last 3 bases containing a stop codon (tga)); cDNA encoding the N-terminal 1-189 amino acid domain of mouse Tim3 protein, with the last 3 bases containing a stop codon (tga)); or cDNA encoding the N-terminal 1-200 amino acid domain of human Tim3 protein, with the last 3 bases containing a stop codon (tga)); or cDNA encoding the N-terminal 1-200 amino acid domain of human Tim3 protein, with the last 3 bases containing a stop codon (tga); and so on, combined into an expression vector such as the commercially available pCAG-Neo vector (manufactured by Wako Pure Chemical Industries, Ltd.). As for the vector for introducing the Tim coding sequence, any vector capable of expressing and producing the Tim protein of this invention in host cells is acceptable, and commercially available vectors are convenient to use. Examples of commercially available vectors for the stated purpose, when the host is an animal cell, include pCAG-Neo vectors and pcDNA vectors.

[0115] <Host>

[0116] As a host, any host capable of expressing the Tim protein of the present invention can be used, such as Escherichia coli, insect cells, mammalian cells, plant cells, yeast cells, etc., with mammalian cells being preferred. Examples of mammalian cells include HEK293T cells, COS-7 cells, CHO-K1 cells, and CHO-S cells.

[0117] <Gene introduction into the host>

[0118] According to the conventional method for introducing a gene vector into a host as described in "Targeted Protein Expression Protocols" (Chapter 3 Protein Expression Protocols, ISBN 978-4-7581-0175-2, Yodosha), the gene is introduced into the expression vector of the present invention into the host.

[0119] <Host Cultivation>

[0120] For hosts that have undergone gene introduction, culture is performed according to standard host culture methods. Culture conditions vary depending on the host, but standard methods for each host are generally applicable. For example, if the host is an animal cell, it can be cultured for 1 to 10 days, preferably 3 to 4 days, at a typical CO2 concentration of 5–10%, preferably 5–8%, and at a typical temperature of 36–38°C, preferably 36.5–37.5°C. It should be noted that since the Tim protein of this invention does not contain transmembrane or intracellular regions, it is expressed and secreted in the culture supernatant.

[0121] <Purification of Tim Protein in this Invention>

[0122] Then, the obtained culture medium of the gene-introduced host is centrifuged (usually at 200-400×g for 3-10 minutes, preferably at 300×g for 3-6 minutes), and the culture supernatant is recovered. If necessary, the recovered culture supernatant can be centrifuged for 20-60 minutes at 1000-2000×g, preferably at 1200×g for 20-40 minutes, and / or (ii) filtered using a filter to separate impurities, obtaining a culture supernatant filtrate.

[0123] Furthermore, as needed, the obtained culture supernatant filtrate can be concentrated to a concentration typically 5 to 20 times, preferably 8 to 12 times, using conventional methods such as ultrafiltration, thereby obtaining a concentrated culture supernatant filtrate.

[0124] Then, when the Tim protein of the present invention has an affinity tag, the Tim protein of the present invention (the fusion protein of the Tim protein and the affinity tag) can be purified from the obtained culture supernatant, the obtained culture supernatant filtrate, or a concentrate of the obtained culture supernatant filtrate, according to conventional methods for purifying proteins using affinity tags corresponding to each affinity tag as described in Purpose-Selected Protein Expression Schemes (Chapter 3 Protein Expression Schemes, Section 6 Protein Purification, Purification Using Tags, ISBN 978-4-7581-0175-2, Yodosha), etc.

[0125] In the absence of an affinity tag, the Tim protein of the present invention can be purified according to conventional protein purification methods described in Purpose-Selected Protein Expression Protocols (Purpose-Selected Protein Expression Protocols) (Chapter 3 Protein Expression Protocols, Section 6 Protein Purification, Purification by Chromatography, ISBN 978-4-7581-0175-2, Yodosha), using various chromatographic methods, from the obtained culture supernatant, the obtained culture supernatant filtrate, or a concentrate of the obtained culture supernatant filtrate.

[0126] The above purification methods can be combined appropriately for purification.

[0127] <Specific preparation method of Tim protein of the present invention>

[0128] As a specific method for preparing the Tim protein of the present invention, for example, when using the Fc tag as an affinity tag, the following method can be cited. First, according to conventional methods, the Tim coding sequence of the present invention is combined into a pEF-Fc vector or a commercially available vector to construct the expression vector of the present invention. Then, according to conventional methods, the gene is introduced into the expression vector of the present invention into a host cell, and cultured for 1 to 10 days, preferably 3 to 4 days, under conditions of 5 to 10% CO2, preferably 5 to 8%, and at a temperature of 36°C to 38°C, preferably 36.5°C to 37.5°C. Then, the obtained culture medium of the gene-introduced host is centrifuged (usually at 200–400 × g for 3–10 minutes, preferably at 300 × g for 3–6 minutes), and the culture supernatant is recovered. If necessary, the recovered culture supernatant can be centrifuged for 20–60 minutes at 1000–2000 × g, preferably at 1200 × g for 20–40 minutes, and / or (ii) filtered to separate impurities, obtaining a culture supernatant filtrate. Further, if necessary, the obtained culture supernatant filtrate can be concentrated to 5–20 times, preferably 8–12 times, using conventional methods such as ultrafiltration, to obtain a concentrated culture supernatant filtrate. Then, in the case that 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 concentrate of the obtained culture supernatant filtrate according to the conventional method of purification using the affinity tag corresponding to each affinity tag, thereby obtaining the Tim protein of the present invention.

[0129] <5. The Tim carrier of the present invention>

[0130] The Tim protein-bound carrier of the present invention (hereinafter, sometimes referred to as "the Tim carrier of the present invention") is a carrier formed by binding the Tim protein of the present invention as described above with the carrier of the present invention.

[0131] Specifically, examples of Tim vectors of the present invention include vectors that bind to the Tim1 protein of the present invention (hereinafter sometimes referred to as "Tim1 vectors of the present invention"), vectors that bind to the Tim3 protein of the present invention (hereinafter sometimes referred to as "Tim3 vectors of the present invention"), and vectors that bind to the Tim4 protein of the present invention (hereinafter sometimes referred to as "Tim4 vectors of the present invention"). Furthermore, vectors that bind to two or more proteins selected from the Tim1 protein, the Tim3 protein, and the Tim4 protein of the present invention are also included in the Tim vectors of the present invention.

[0132] As the Tim carrier of the present invention, the Tim4 carrier of the present invention is particularly preferred.

[0133] <Carrier of the Invention>

[0134] As the carrier of this invention, any insoluble carrier used in conventional immunological assays can be used, such as organic materials like polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, glycidyl methacrylate, polypropylene, polyolefins, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, and ethylene-maleic anhydride copolymer; inorganic materials like glass, silica, diatomaceous earth, porous glass, frosted glass, alumina, silica gel, and metal oxides; magnetic materials like iron, cobalt, nickel, magnetite, and chromite; and materials prepared from alloys of the aforementioned magnetic materials. Furthermore, the carrier can be used in various forms such as microplates, tubes, discs, and particles (beads).

[0135] It should be noted that, when used in the acquisition method and removal method of the present invention described later, it is preferred to use them 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 that are usually 10 nm to 100 μm, preferably 100 nm to 10 μm, can be cited.

[0136] Furthermore, when used in 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 application, particles with 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 pores in the microplate, but depending on the purpose and application, microplates with a size of 12 to 1536 pores, preferably 96 to 384 pores, can be cited.

[0137] <Method for binding the Tim protein of the present invention to the carrier of the present invention>

[0138] As a method for binding the Tim protein of the present invention to the carrier of the present invention, it can be carried out according to a known method of binding the protein to the carrier, for example: a method of binding by affinity binding; a method of binding by chemical binding (for example, the method described in Japanese Patent No. 3269554 and WO2012 / 039395); a method of binding by physical adsorption (for example, the method described in Japanese Patent Publication No. 5-41946), etc., but the method of binding by affinity binding and the method of binding by physical adsorption are preferred.

[0139] It should be noted that, when used in the acquisition and removal methods of the present invention described later, a binding method via affinity bonding is preferred. Furthermore, when used in the detection method of the present invention described later, a binding method via affinity bonding or physical adsorption is preferred.

[0140] <The binding mode of the Tim protein of the present invention with the carrier of the present invention>

[0141] As for the binding mode between the Tim protein of the present invention and the carrier of the present invention, there is no limitation on the binding mode 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 to the thiol group (SH group) of the Tim protein of the present invention is preferred. In addition, the Tim protein of the present invention can be directly bound to the carrier of the present invention, or it can be indirectly bound through chemical linkers, affinity substances [e.g., substances with affinity for affinity tags (described later), antibodies against the Tim protein of the present invention, biotinylate (described later), avidin (described later), antibodies, etc.].

[0142] <Methods of binding through affinity>

[0143] As for the above-mentioned method of binding through affinity, any method can be used as long as it utilizes the affinity between substances (affinity). For example, the following (a) to (c) can be cited.

[0144] (a) Binding via affinity binding of biotinylate and avidin.

[0145] For example, by using two or more substances (affinity substances) that are composed of biotinylate (biotin, iminobiotin, desulfobiotin, biocytosine, biotin sulfoxide, etc.) and avidin (avidin, tamavidin, tamavidin 2, streptavidin, etc.) and have mutual affinity, the Tim protein of the present invention can be bound to the carrier of the present invention through the affinity substance.

[0146] It should be noted that any one of the affinity substances can be pre-bound to the Tim protein of the present invention, and the remaining one can be pre-bound to the carrier of the present invention. However, for example, when using biotin and avidin, avidin is usually pre-bound to the carrier of the present invention, and biotin is pre-bound to the Tim protein of the present invention.

[0147] (b) A method of binding by affinity tag to a substance that has an affinity for the affinity tag.

[0148] For example, by using substances that have affinity for the Tim protein of the present invention (such as protein A, protein G, etc.) that have affinity for the affinity tag, the Tim protein of the present invention can be bound to the carrier of the present invention by the affinity substance.

[0149] It should be noted that the affinity substance is usually pre-bonded with the carrier of the present invention.

[0150] (c) A 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.

[0151] For example, by using antibodies against the Tim protein of the present invention (antibodies against affinity tags such as 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, and anti-TIM4 antibody (Anti-TIM4 Antibody (clone RMT4-54) (manufactured by LifeSpan Biosciences) etc.), substances (affinity substances) that have affinity for the Tim protein of the present invention can be used to bind the Tim protein of the present invention to the vector of the present invention.

[0152] It should be noted that the affinity substance is usually pre-bonded with the carrier of the present invention.

[0153] It should be noted that in the above methods, the Tim protein of the present invention or / and the carrier of the present invention can be bound to the affinity substance by means of methods that bind it by physical adsorption methods known per se, methods that bind it by means of chemical binding methods, and the binding can be direct or indirect through linkers, etc.

[0154] <Amount of the Tim protein of the present invention bound to the carrier of the present invention>

[0155] With respect to the amount of the Tim protein of the present invention incorporated into the carrier of the present invention, for example, when the carrier of the present invention is a bead, the amount of the protein is typically 0.1 μg to 50 μg relative to 1 mg of the carrier, preferably 0.5 μg to 30 μg, and more preferably 1.0 μg to 20 μg.

[0156] In addition, when the carrier of the present invention is a microplate, the amount of protein relative to 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.

[0157] <Specific preparation method of the Tim carrier of the present invention>

[0158] The following describes the specific preparation method of the Tim vector of the present invention using the methods described in (a)-(c) as an example.

[0159] <(a) Binding via affinity binding of biotinylate and avidin>

[0160] 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 simply referred to as "biotin labeling" or "biotinylation") to form the Tim protein-biotin complex of the present invention. On the other hand, avidin is bound to the carrier of the present invention to form the carrier-avidin complex of the present invention (hereinafter, sometimes simply referred to as "the carrier of the present invention bound with avidin"). The obtained Tim protein-biotin complex of the present invention is brought into contact with the carrier-avidin complex of the present invention, thereby binding the biotin in the Tim protein-biotin complex of the present invention to the avidin in the carrier-avidin complex, thereby obtaining the Tim carrier of the present invention.

[0161] -The binding of Tim protein to biotin (biotin labeling) in this invention-

[0162] In method (a), the binding of the Tim protein of the present invention to biotin can be performed using a commercially available biotinylated protein labeling kit, or by appropriately adjusting the required reagents and performing the binding using conventional methods for biotinylated proteins. As a method using a commercially available biotinylated protein labeling kit, the method described in the accompanying Biotin Labeling Kit-SH (Dojin Scientific Laboratories Co., Ltd.) or Biotin Labeling Kit-NH2 (Dojin Scientific Laboratories Co., Ltd.) can be followed.

[0163] The amount of biotin bound to 1 μg of the Tim protein of the present invention is typically 10 ng to 1.0 μg, preferably 20 ng to 200 ng, and more preferably 30 ng to 150 ng.

[0164] The thiol group of the Tim protein of the present invention is preferably the site where biotinylate binds to the Tim protein of the present invention.

[0165] -The carrier of the present invention-Avidin complex (the carrier of the present invention incorporating avidin-)-

[0166] In method (a) of the present invention, the carrier-avidin complex (the carrier of the present invention incorporating avidin) can be a commercially available complex, or it can be prepared by adjusting the required reagents and following conventional methods. Examples of carrier-avidin complexes of the present invention include, for example, beads or microplates incorporating avidin, beads or microplates incorporating tamavidin, beads or microplates incorporating tamavidin 2, beads or microplates incorporating streptavidin, etc. Examples of commercially available complexes include Dynabeads M-270 streptavidin C1 beads. 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.

[0167] Regarding the combination of the carrier and avidin of the present invention, for example, when the carrier of the present invention is a bead, the amount of avidin in contact with 1 mg of the carrier is typically 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 avidin in contact with one well is typically 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg.

[0168] - The combination of the carrier-avidin complex of the present invention with the Tim protein-biotin complex of the present invention

[0169] In method (a), in order to obtain the Tim carrier of the present invention (by binding 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, typically 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 mg to 3.0 mg, can be combined with the Tim protein-biotin complex of the present invention, typically 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 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 can be brought into contact with each well of a Tim protein-biotin complex of the present invention, typically 1.0–10 μg, preferably 1.0–5.0 μg, more preferably 1.0–2.0 μg, at a temperature typically 4.0°C–37°C, preferably 11°C–30°C, more preferably 20°C–25°C, for a reaction typically 0.5 hours–24 hours, preferably 0.5 hours–8.0 hours, more preferably 0.5 hours–2.0 hours, to allow the avidin and biotin to bind. Thus, the carrier-avidin complex of the present invention binds to the Tim protein-biotin complex of the present invention, yielding the Tim carrier of the present invention.

[0170] It should be noted that, generally, the contact between the carrier-avidin complex of the present invention and the Tim protein-biotin complex of the present invention can be achieved by contacting a solution containing the Tim protein-biotin complex of the present invention with the carrier-avidin complex of the present invention.

[0171] As a solution containing the Tim protein-biotin complex of the present invention, any solution that dissolves the Tim protein-biotin complex of the present invention in a stable state is acceptable. Examples include purified water, or buffer solutions (e.g., PBS, TBS, HBS, etc.) that have buffering properties at a pH of 7.0–8.0, preferably 7.2–7.6. Furthermore, the concentration of the buffer in the buffer solution is typically selected appropriately from the range of 5.0–50 mM, preferably from the range of 10–30 mM, and the NaCl concentration is typically selected appropriately from the range of 100–200 mM, preferably from the range of 140–160 mM. Additionally, the solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc., as long as the amount of contact between the carrier-avidin complex of the present invention and the solution containing the Tim protein-biotin complex of the present invention does not hinder the binding of the carrier-avidin complex of the present invention to the Tim protein-biotin complex of the present invention. Examples of surfactants include Tween 20, and the surfactant concentration in the solution containing the Tim protein-biotin complex of the present invention is typically 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 abbreviated as "a solution containing the Tim protein-biotin complex of the present invention".

[0172] -Specific preparation method of the Tim carrier of the present invention based on method (a)-

[0173] The specific preparation method of the Tim carrier of the present invention in method (a) can be carried out by, for example, the following methods.

[0174] First, the Tim protein of the present invention is prepared according to the preparation method of the Tim protein of the present invention. Then, biotin is bound to the Tim protein of the present invention according to the schemes included in the Biotin Labeling Kit-SH (Dongjin Scientific Laboratories Co., Ltd.), the Biotin Labeling Kit-NH2 (Dongjin Scientific Laboratories Co., Ltd.), or conventional methods for biotin labeling of proteins, thereby forming the Tim protein-biotin complex of the present invention.

[0175] Then, when the carrier of the present invention is a bead, a solution containing, for example, 1.0 to 5.0 μg of the carrier-avidin complex of the present invention (usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 to 3.0 mg) and 1.0 to 50 μg of the Tim protein-biotin complex of the present invention (usually 50 μL to 1500 μL, preferably 100 μL to 1000 μL, more preferably 200 μL to 500 μL) of the carrier-avidin complex of the present invention (usually 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg relative to 1 mg of the carrier-avidin complex of the present invention) is prepared (e.g., in purified water, or in pH). The solution containing the Tim protein-biotin complex of the present invention is contacted with a buffer solution or the like containing a buffer having a buffering effect under conditions of H7.0 to 8.0, preferably 7.2 to 7.6. The reaction is carried out at a temperature of typically 4.0°C to 37°C, preferably 11°C to 30°C, more preferably 20°C to 25°C, for typically 0.5 hours to 24 hours, preferably 0.5 hours to 8.0 hours, more preferably 0.5 hours to 2.0 hours, so that the avidin in the carrier-avidin complex of the present invention binds to the biotin in the Tim protein-biotin complex of the present invention, thereby obtaining the Tim carrier of the present invention.

[0176] When the carrier of the present invention is a microplate, the carrier-avidin complex of the present invention in each well is mixed with a solution of the Tim protein-biotin complex of the present invention, typically 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 100 μL to 200 μL, containing typically 1.0 to 10 μg, preferably 1.0 to 5.0 μg, more preferably 1.0 to 2.0 μg, in pure water or a buffer solution having a buffering effect at pH 7.0 to 8.0, preferably 7.2 to 7.6. The solution containing the Tim protein-biotin complex of the present invention is contacted and reacted at a temperature typically 4.0°C to 37°C, preferably 11°C to 30°C, more preferably 20°C to 25°C, for typically 0.5 hours to 24 hours, preferably 0.5 hours to 8.0 hours, more preferably 0.5 hours to 2.0 hours, to allow the avidin in the carrier-avidin complex of the present invention to bind with the biotin in the Tim protein-biotin complex of the present invention, thereby obtaining the Tim carrier of the present invention.

[0177] <(b) A method of binding by affinity tag to a substance that has an affinity for the affinity tag>

[0178] (b) In this method, firstly, 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. Then, a substance having affinity for the affinity tag (the affinity substance) is bound to the carrier of the present invention to form a carrier-affinity substance complex of the present invention (hereinafter, sometimes abbreviated as "the carrier of the present invention bound with the affinity substance"). The obtained carrier-affinity substance complex of the present invention is contacted with the Tim protein of the present invention having an affinity tag, so that the affinity substance in the carrier-affinity substance complex of the present invention binds 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.

[0179] -Carrier-affinity complex of the present invention-

[0180] In method (b), the carrier-affinity complex of the present invention can be a commercially available product, or the required reagents can be appropriately adjusted and prepared according to conventional methods. Examples of carrier-affinity complexes for this invention include, for example, beads or microplates bound to G proteins, beads or microplates bound to A proteins, etc. Commercially available products include Dynabeads G protein (manufactured by Thermo Fisher Scientific), Dynabeads A protein (manufactured by Thermo Fisher Scientific), FG beads G protein (manufactured by Tamagawa Sei), and FG beads A protein (manufactured by Tamagawa Sei).

[0181] Regarding the binding of the carrier and the affinity substance of the present invention, the amount of affinity substance in contact with 1 mg of the carrier of the present invention is typically 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 affinity substance in contact with one pore is typically 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg.

[0182] -The binding of the carrier-affinity complex of the present invention to the Tim protein of the present invention having an affinity tag--

[0183] In method (b), to obtain the Tim carrier of the present invention (through the binding of the carrier-affinity complex of the present invention to the Tim protein of the present invention with an affinity tag), the following method may be used, for example: When the carrier of the present invention is a bead, a carrier-affinity complex of the present invention, typically 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 mg to 3.0 mg, is contacted with a Tim protein of the present invention with an affinity tag, typically 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg, relative to 1 mg of the carrier-affinity complex of the present invention; when the carrier of the present invention is a microplate, the carrier-affinity complex of the present invention in each well is contacted with a typically 1.0 to 1 mg of the Tim protein protein of the present invention with an affinity tag. 0 μg, preferably 1.0 to 5.0 μg, more preferably 1.0 to 2.0 μg, of the Tim protein of the present invention with an affinity tag is contacted and reacted at a temperature typically 4.0°C to 37°C, preferably 11°C to 30°C, more preferably 20°C to 25°C, for typically 0.5 hours to 24 hours, preferably 0.5 hours to 8.0 hours, more preferably 0.5 hours to 2.0 hours, to bind the carrier-affinity substance complex of the present invention to the Tim protein of the present invention with an affinity tag, thereby obtaining the Tim carrier of the present invention.

[0184] It should be noted that, typically, in method (b), the contact between the carrier-affinity 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 complex of the present invention with a solution containing the Tim protein of the present invention having an affinity tag.

[0185] As a solution containing the Tim protein of the present invention with an affinity tag, any solution that dissolves the Tim protein of the present invention with an affinity tag in a stable state is acceptable. Examples include purified water, or buffer solutions with buffering capacity at pH 7.0–8.0, preferably 7.2–7.6 (e.g., PBS, TBS, HBS, etc.). Furthermore, the concentration of the buffer in the buffer solution is typically selected appropriately from the range of 5.0–50 mM, preferably from the range of 10–30 mM, and the NaCl concentration is typically selected appropriately from the range of 100–200 mM, preferably from the range of 140–160 mM.

[0186] Furthermore, the solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins, etc., as long as the amount of the solution containing the Tim protein of the present invention with the affinity tag, after contacting it with the carrier-affinity complex of the present invention, does not hinder the binding of the Tim protein of the present invention with the affinity tag to the carrier-affinity complex of the present invention. Examples of surfactants include Tween 20, and the concentration of the surfactant in the solution containing the Tim protein of the present invention with the affinity tag is typically 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 abbreviated as "a solution containing the Tim protein of the present invention with the affinity tag".

[0187] -Specific preparation method of the Tim carrier of the present invention based on method (b)-

[0188] In method (b), the specific preparation method of the Tim carrier of the present invention can be carried out by, for example, the following methods.

[0189] First, the Tim protein of the present invention with an affinity tag is prepared according to the preparation method of the Tim protein of the present invention.

[0190] Then, when the carrier of the present invention is a bead, a solution of the carrier-affinity complex of the present invention, typically 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 mg to 3.0 mg, is prepared with a solution of the Tim protein of the present invention having an affinity tag, typically 50 μL to 1500 μL, preferably 100 μL to 1000 μL, more preferably 200 μL to 500 μL, containing, relative to 1 mg, the carrier-affinity complex of the present invention, typically 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg, relative to 1 mg. For example, a solution containing the Tim protein of the present invention with an affinity tag is contacted in purified water, a buffer solution with pH 7.0 to 8.0, etc., and the reaction is carried out at a temperature of typically 4.0°C to 37°C, preferably 11°C to 30°C, more preferably 20°C to 25°C, for typically 0.5 hours to 24 hours, preferably 0.5 hours to 8.0 hours, more preferably 0.5 hours to 2.0 hours, so that the affinity substance in the carrier-affinity substance complex of the present invention binds to the affinity tag in the Tim protein of the present invention with an affinity tag, thereby obtaining the Tim carrier of the present invention.

[0191] When the carrier of the present invention is a microplate, each well contains the carrier-affinity complex of the present invention and a solution of the Tim protein of the present invention with an affinity tag, typically 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 100 μL to 200 μL, containing 1.0 to 10 μg, preferably 1.0 to 5.0 μg, more preferably 1.0 to 2.0 μg, of the present invention. (e.g., in purified water, or in a buffer solution with buffering capacity at pH 7.0 to 8.0, preferably 7.2 to 7.6.) The solution containing the Tim protein-biotin complex of the present invention is contacted with a liquid, etc., and the reaction is carried out at a temperature of generally 4.0°C to 37°C, preferably 11°C to 30°C, more preferably 20°C to 25°C, for generally 0.5 hours to 24 hours, preferably 0.5 hours to 8.0 hours, more preferably 0.5 hours to 2.0 hours, so that the affinity substance in the carrier-affinity substance complex of the present invention binds 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.

[0192] <(c) A 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>

[0193] 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 abbreviated 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 then contacted with the Tim protein of the present invention, allowing the antibody against the Tim protein in the carrier-anti-Tim antibody complex to bind to the Tim protein of the present invention, thereby obtaining the Tim carrier of the present invention.

[0194] It should be noted that, as described above, when using a Tim protein with an affinity tag (a protein having both the amino acid sequence of the Tim protein and the amino acid sequence of the affinity tag) as the Tim protein of the present invention, either an antibody that recognizes the Tim protein (an antibody that recognizes the protein portion based on the amino acid sequence of the Tim protein) or an antibody that recognizes the affinity tag (an antibody that recognizes the protein portion based on the amino acid sequence of the affinity tag) can be used as the anti-Tim antibody. Furthermore, when using a Tim protein without an affinity tag (a protein consisting only of the amino acid sequence of the Tim protein) as the Tim protein of the present invention, either an antibody that recognizes the Tim protein (an antibody that recognizes the protein portion based on the amino acid sequence of the Tim protein) can be used as the anti-Tim antibody.

[0195] -The carrier of the present invention-Anti-Tim antibody complex (the carrier of the present invention bound with antibodies-

[0196] In method (c), the carrier-anti-Tim antibody complex of the present invention can be a commercially available product, or the required reagents can be appropriately adjusted and prepared according to conventional methods. Examples of the carrier-anti-Tim antibody complex of the present invention include beads or microplates bound to anti-Fc-tagged antibodies, beads or microplates bound to anti-FLAG-tagged antibodies, beads or microplates bound to anti-His-tagged antibodies, beads or microplates bound to anti-GST-tagged antibodies, beads or microplates bound to anti-MBP-tagged antibodies, beads or microplates bound to anti-HA-tagged antibodies, beads or microplates bound to anti-Myc-tagged antibodies, beads or microplates bound to anti-Strep(II)-tagged antibodies, and beads or microplates bound to antibodies against anti-Tim proteins. Commercially available products include magnetic beads containing anti-DYKDDDDK-tagged antibodies (manufactured by Wako Pure Chemical Industries, Ltd.).

[0197] There are no particular limitations on the source of the anti-Tim antibody. It can be a polyclonal antibody or a monoclonal antibody, but monoclonal antibodies are preferred. Furthermore, the anti-Tim antibody can be a commercially available antibody, or the required reagents can be appropriately adjusted, and it can be prepared according to conventional methods. Specific examples of anti-Tim antibodies are described above.

[0198] Regarding the binding of the carrier of the present invention to the anti-Tim antibody, when the carrier of the present invention is a bead, the amount of antibody against the Tim protein of the present invention in contact with 1 mg of the carrier of the present invention is typically 5.0 to 50 μg, preferably 10 to 50 μg, and more preferably 20 to 50 μg, in the case of the acquisition method and removal method of the present invention. When the carrier of the present invention is a microplate, the amount per well is typically 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg.

[0199] -The binding of the carrier of the present invention -the anti-Tim antibody complex -to the Tim protein of the present invention-

[0200] In method (c), as a method for obtaining the Tim carrier of the present invention by binding the carrier-anti-Tim antibody complex of the present invention to the Tim protein of the present invention, examples of such methods can be given. That is, when the carrier of the present invention is a bead, a dose of the carrier-anti-Tim antibody complex of the present invention, typically 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 mg to 3.0 mg, is contacted with a dose of the Tim protein of the present invention, typically 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg, 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, each well of the carrier-anti-Tim antibody complex of the present invention is contacted with a dose of typically 1.0 to 50 μg of the Tim protein of the present invention. 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 is contacted and reacted at a temperature typically 4.0°C to 37°C, preferably 11°C to 30°C, more preferably 20°C to 25°C, for typically 0.5 hours to 24 hours, preferably 0.5 hours to 8.0 hours, more preferably 0.5 hours to 2.0 hours, to allow the anti-Tim antibody in the carrier-anti-Tim antibody complex of the present invention to bind to the Tim protein of the present invention, thereby obtaining the Tim4 carrier of the present invention.

[0201] It should be noted that, typically, the contact between the carrier-anti-Tim antibody complex of the present invention and the Tim protein of the present invention in the method described in (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.

[0202] As a solution containing the Tim protein of the present invention, any solution that dissolves the Tim protein of the present invention in a stable state is acceptable. Examples include purified water and buffer solutions (such as PBS, TBS, HBS, etc.) that have buffering properties at a pH of 7.0 to 8.0, preferably 7.2 to 7.6. Furthermore, the concentration of the buffer in the buffer solution is typically selected appropriately from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is typically selected appropriately from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. Additionally, 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-anti-Tim antibody complex of the present invention upon contact with the solution containing the Tim protein of the present invention. Examples of surfactants include Tween 20, and the concentration of the surfactant in the solution containing the Tim protein of the present invention is typically 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 in the solution is sometimes abbreviated as "a solution containing the Tim protein of the present invention".

[0203] -Specific preparation method of the Tim carrier of the present invention using the method described in (c)-

[0204] The specific preparation method of the Tim carrier of the present invention in the method described in (c) can be carried out by, for example, the following methods.

[0205] 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 containing, for example, purified water, of the present invention is prepared by mixing a carrier-anti-Tim antibody complex of the present invention, typically 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, more preferably 0.5 mg to 3.0 mg, of the carrier-anti-Tim antibody complex of the present invention, typically 100 to 1000 μL, preferably 200 to 500 μL, of the Tim protein of the present invention, typically 1.0 to 50 μg, preferably 1.0 to 30 μg, more preferably 1.0 to 20 μg relative to 1 mg of the carrier-anti-Tim antibody complex of the present invention. The solution containing the Tim protein of the present invention is contacted with a buffer solution containing pH 7.0 to 8.0, and the reaction is carried out at a temperature of typically 4°C to 37°C, preferably 11°C to 30°C, more preferably 20°C to 25°C, for typically 0.5 hours to 24 hours, preferably 0.5 hours to 8.0 hours, more preferably 0.5 hours to 2.0 hours. By binding the antibody against the Tim protein of the present invention in the carrier-anti-Tim antibody complex of the present invention to the Tim protein of the present invention, the Tim carrier of the present invention is obtained. When the carrier of the present invention is a microplate, each well contains the carrier-anti-Tim antibody complex of the present invention and a solution of the Tim protein of the present invention, typically 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 100 μL to 200 μL, containing 1.0 to 10 μg, preferably 1.0 to 5.0 μg, more preferably 1.0 to 2.0 μg, of the present invention (in a buffer solution containing, for example, purified water, or a buffer solution having buffering capacity under conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6). The solution of the Tim protein-biotin complex of the present invention is contacted and reacted at a temperature typically 4.0°C to 37°C, preferably 11°C to 30°C, more preferably 20°C to 25°C, for typically 0.5 hours to 24 hours, preferably 0.5 hours to 8.0 hours, more preferably 0.5 hours 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.

[0206] <Methods of binding via physical adsorption>

[0207] As a method for binding the Tim protein of the present invention to the carrier of the present invention through physical adsorption, the Tim protein of the present invention can be brought into contact with the carrier of the present invention under the condition that the Tim protein of the present invention is bound to the carrier of the present invention, according to a method known by itself.

[0208] The reaction temperature between the Tim protein of the present invention and the carrier of the present invention is typically 2°C to 37°C, preferably 4°C to 11°C.

[0209] The reaction time between the Tim protein of the present invention and the carrier of the present invention is typically 4 hours to 48 hours, preferably 12 hours to 24 hours.

[0210] As the Tim protein of the present invention, when the carrier of the present invention is a bead, the amount of Tim protein of the present invention in contact with 1 mg of the carrier is typically 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 Tim protein of the present invention in contact with one well is typically 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg.

[0211] It should be noted that, generally, the physical adsorption of the Tim protein of the present invention to the carrier of the present invention is carried out by contacting the solution containing the Tim protein of the present invention with the carrier of the present invention.

[0212] As a solution containing the Tim protein of the present invention, any solution that dissolves the Tim protein of the present invention in a stable state is acceptable. Examples include purified water, buffer solutions with buffering capacity at pH 6.0 to 9.5, preferably 7.0 to 8.0 (e.g., zwitterionic buffers such as MOPS (3-(N-morpholino)propanesulfonic acid) (Good's buffer), carbonate buffer, PBS, TBS, HBS, etc.). Furthermore, the concentration of the buffer in the buffer solution is typically selected appropriately from the range of 5 to 100 mM, preferably from the range of 10 to 50 mM, and the concentration of NaCl added is typically selected appropriately from the range of 100 to 200 mM, preferably from the range of 140 to 160 mM. Additionally, 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 carrier of the present invention to extracellular membrane vesicles or viruses after contact with the solution containing the Tim protein of the present invention. It should be noted that the solution obtained by dissolving the Tim protein of the present invention in the solution is sometimes referred to as "a solution containing the Tim protein of the present invention".

[0213] In the method of binding the Tim protein of the present invention to the carrier of the present invention, as a specific example of the method of binding by physical adsorption, the following methods can be cited.

[0214] First, the Tim protein of the present invention is prepared according to the preparation method of the Tim protein of the present invention. Then, when the carrier of the present invention is a bead, 1 mg of the carrier of the present invention is contacted with a solution containing 5.0 to 50 μg, preferably 10 to 50 μg, more preferably 20 to 50 μg of Tim protein (in a solution containing the Tim protein of the present invention in, for example, pure water, or a buffer solution with pH 7.0 to 8.0). Alternatively, when the carrier of the present invention is a microplate, 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 5 ... A solution containing 50 μL to 100 μL of Tim protein (preferably in purified water, a buffer solution containing the Tim protein of the present invention, or the like, at pH 7.0 to 8.0) is contacted with one well, and a reaction is carried out at a temperature typically 2°C to 37°C, preferably 4°C to 11°C, for typically 4 hours to 48 hours, preferably 12 hours to 24 hours, to bind the carrier of the present invention to the Tim protein of the present invention, thereby obtaining the Tim carrier of the present invention.

[0215] <Processing of the Tim Carrier in this Invention>

[0216] The Tim carrier of the present invention obtained in the manner described can be subjected to a sealing process typically performed in the field.

[0217] As needed, the Tim carrier of the present invention obtained in the manner described can be subjected to purification treatments commonly performed in the field. As a purification treatment, any impurities adhering to the surface of the carrier can be removed; for example, a method of washing the Tim carrier of the present invention with a washing solution (hereinafter sometimes abbreviated as "washing operation") can be cited.

[0218] As an example of the present invention, the use of magnetic particles will be used for illustration.

[0219] First, a container containing a solution of the Tim carrier of the present invention obtained in the manner described is placed on a magnetic rack, and the Tim carrier of the present invention is aggregated to the tube wall using magnetism. The solution in the container is then discarded. Next, a washing solution is added to the container, and the mixture is stirred. Then, the container is placed on a magnetic rack in the same manner as described above, and the Tim carrier of the present invention is aggregated to the tube wall using magnetism. The solution in the container is then discarded. This washing operation can be repeated multiple times as needed. The washing solution used in this washing operation can be any solution that will not affect the binding of the Tim protein of the present invention to the carrier of the present invention in the Tim carrier of the present invention, such as purified water, or a buffer solution (e.g., PBS, TBS, HBS, etc.) with buffering capacity at pH 7.0–8.0, preferably 7.2–7.6. Furthermore, the concentration of the buffer in the buffer solution is typically selected from the range of 5–50 mM, preferably from the range of 10–30 mM, and the NaCl concentration is typically selected from the range of 100–200 mM, preferably from the range of 140–160 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.

[0220] Examples of surfactants include Tween 20, and the concentration of the surfactant in the washing solution is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0221] Using the Tim vector of the present invention, extracellular membrane vesicles or viruses can be obtained effectively, and extracellular membrane vesicles or viruses in the sample can be obtained with high purity. Furthermore, extracellular membrane vesicles or viruses in the sample can be removed efficiently. Consequently, extracellular membrane vesicles or viruses in the sample can be detected with high sensitivity.

[0222] <6. Methods for obtaining extracellular membrane vesicles or viruses from the sample>

[0223] The method for obtaining extracellular membrane vesicles or viruses of the present invention (hereinafter, sometimes referred to as "the method of obtaining the present invention") is characterized by comprising the following steps.

[0224] (1) The process 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 abbreviated as "complex formation process").

[0225] (2) The process of separating the composite from the sample (hereinafter sometimes referred to as the "composite separation process")

[0226] (3) The process of separating extracellular membrane vesicles or viruses from the complex to obtain extracellular membrane vesicles or viruses (hereinafter sometimes referred to as the “obtaining process”).

[0227] <6-1. Regarding the composite formation process>

[0228] The complex formation process is a process in which Tim protein and carrier form a complex with extracellular membrane vesicles or viruses in the sample in the presence of calcium ions.

[0229] <Sample of the present invention>

[0230] The sample of this invention can be any sample containing the extracellular membrane vesicles or virus of this invention in a liquid, or any sample that may contain the extracellular membrane vesicles or virus of this invention. The sample of this invention can be any sample derived from a living organism, or any sample prepared by dissolving or suspending the extracellular membrane vesicles or virus of this invention in a solution such as a culture medium or buffer solution. Specifically, examples of samples for this invention include bodily fluids such as blood, saliva, urine, breast milk, amniotic fluid, and ascites; and cell culture supernatants.

[0231] As the solution containing (dissolving or suspending) the extracellular membrane vesicles or viruses of the present invention, any solution that 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 to the complex of the extracellular membrane vesicles or viruses in the sample is acceptable. Examples include water and buffer solutions (e.g., TBS, HBS, etc.) that have buffering properties at pH 7.0 to 8.0, preferably 7.2 to 7.6. It should be noted that phosphate buffers are not preferred because they bind with calcium to form a precipitate. In addition, the concentration of the buffer in the buffer solution 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.

[0232] The solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, and proteins, as long as the amount does not hinder the binding of the Tim protein bound to the carrier to the complex of extracellular membrane vesicles or viruses in the sample. Examples of surfactants include Tween 20, and the concentration of the surfactant in the solution containing the extracellular membrane vesicles or viruses of the present invention is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0233] <Calcium ion concentration / Source of calcium ions>

[0234] In this invention, calcium ions are present when the Tim protein of this invention forms a complex with the carrier and extracellular membrane vesicles or viruses in the sample. More specifically, calcium ions are present when the Tim protein of this invention comes into contact with extracellular membrane vesicles in the sample.

[0235] The calcium ion concentration when the Tim protein of the present invention comes into contact with the extracellular membrane vesicles or viruses in the sample of the present invention is typically 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 described above is required in the solution containing the complex from the formation of the Tim protein of the present invention with the extracellular membrane vesicles or viruses in the sample of the present invention up to the acquisition step, i.e., up to the step of separating the complex.

[0236] In addition, there is no particular limitation on the source of calcium ions, and examples include calcium chloride, calcium hydroxide, calcium bicarbonate, calcium iodide, calcium bromide, and calcium acetate. Calcium chloride, calcium bicarbonate, and calcium iodide are preferred, and calcium chloride and calcium bicarbonate are even more preferred.

[0237] It should be noted that, as a method for ensuring the presence of calcium ions when contacting the Tim protein of the present invention with extracellular membrane vesicles or viruses in a sample, the solution containing the Tim carrier of the present invention, the sample, the solution containing the Tim protein of the present invention, and / or the solution containing the carrier of the present invention may typically contain calcium ions as described above, so that the calcium ion concentration when contacting the Tim protein and the carrier of the present invention with extracellular membrane vesicles or viruses in a sample is within the aforementioned range. Alternatively, a solution containing calcium ions may be used, and this solution may be mixed with a solution containing the Tim carrier of the present invention, the sample, the solution containing the Tim protein of the present invention, and / or the carrier of the present invention, so that the calcium ion concentration when contacting the Tim protein and the carrier of the present invention with extracellular membrane vesicles or viruses in a sample is within the aforementioned range.

[0238] 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 specific examples are also the same.

[0239] <Sample Amount>

[0240] In the complex formation process, the amount of sample that comes into contact with 1 μg of the Tim protein of the present invention is typically 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0 ml.

[0241] <Temperature>

[0242] In the complex formation process, the temperature at which the Tim protein of the present invention comes into contact with the extracellular membrane vesicles or viruses in the sample is typically 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0243] <Time>

[0244] In the complex formation process, the contact time between the Tim protein of the present invention and the sample is typically 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0245] The complex formation process is divided into the following two cases: (1-A) using the pre-prepared Tim carrier of the present invention (i.e., using a carrier incorporating the Tim protein of the present invention), and (1-B) using the Tim protein of the present invention and the carrier of the present invention respectively.

[0246] <About (1-A)>

[0247] (1-A) is a method in which the Tim carrier of the present invention is brought into contact with extracellular membrane vesicles or viruses in a sample in the presence of calcium ions, thereby forming a complex (hereinafter sometimes referred to as "the complex of the present invention") of the Tim protein bound to the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample. In (1-A), calcium ions may be present when the Tim carrier of the present invention is brought into contact with extracellular membrane vesicles or viruses in a sample. Specifically, a solution or / and a sample containing a carrier bound to the Tim protein of the present invention may be used, or a solution containing a carrier bound to the Tim protein of the present invention, a sample, and a solution containing calcium ions may be used.

[0248] -Quantity of the sample in this invention-

[0249] In (1-A), the amount of sample that comes into contact with 1 mg of the Tim carrier of the present invention is typically 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0 ml.

[0250] -Contact Temperature-

[0251] In (1-A), the temperature at which the Tim carrier of the present invention comes into contact with the sample is typically 4.0 to 37°C, preferably 4.0 to 25°C, and more preferably 4.0 to 11°C.

[0252] -Contact Time-

[0253] In (1-A), the contact time between the Tim carrier of the present invention and the sample (extracellular membrane vesicles or virus) is typically 0.5 to 24 hours, preferably 0.5 to 8.0 hours, and more preferably 0.5 to 4.0 hours.

[0254] -Amount of Tim carrier in this invention-

[0255] In (1-A), the amount of Tim carrier of the present invention is typically 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 in which the composite of the present invention is formed.

[0256] -1-Specific example of A-

[0257] (1-A) can be carried out, for example, by the following method. That is, the Tim carrier of the present invention is typically 0.1 to 20 mg, preferably 0.3 to 10 mg, more preferably 0.5 to 6.0 mg, of a solution containing calcium ions in a solution containing the Tim carrier of the present invention, the sample, and the solution containing calcium ions in a solution containing calcium ions in a concentration typically 0.5 to 100 mM, preferably 1.0 to 10 mM, more preferably 2.0 to 5.0 mM, relative to 1 mL of the solution containing the Tim carrier of the present invention, the sample, and the solution containing calcium ions in a concentration typically 0.5 to 100 mM, preferably 1.0 to 10 mM, more preferably 2.0 to 5.0 mM, and then... The sample, typically 0.1 to 100 ml, preferably 0.1 to 10 ml, more preferably 0.1 to 1.0 ml, relative to 1 mg of the Tim carrier of the present invention, is contacted at a temperature typically 4.0 to 37°C, preferably 4.0 to 25°C, more preferably 4.0°C to 11°C, for typically 0.5 to 24 hours, preferably 0.5 to 8.0 hours, more preferably 0.5 to 4.0 hours, to form a complex of the Tim protein bound to the carrier of the present invention with the extracellular membrane vesicles or viruses in the sample.

[0258] <About (1-B)>

[0259] (1-B) describes a method of using the Tim protein of the present invention and the vector of the present invention separately. For example, when binding the Tim protein of the present invention to the vector of the present invention by affinity binding, it can be done as shown in (1-Bi), (1-B-ii), or (1-B-iii) below.

[0260] (1-Bi) In the presence of calcium ions, the Tim protein of the present invention, the carrier of the present invention, and the extracellular membrane vesicles or viruses in the sample are simultaneously brought into contact, thereby forming the complex of the present invention.

[0261] (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 the sample. After the Tim protein of the present invention forms a complex with extracellular membrane vesicles or viruses, the complex is then brought into contact with the carrier of the present invention to form the complex of the present invention.

[0262] (1-B-iii) After contacting the carrier of the present invention with extracellular membrane vesicles or viruses in the sample, it is further contacted with the Tim protein of the present invention in the presence of calcium ions, thereby forming the complex of the present invention.

[0263] -About (1-Bi)-

[0264] (1-Bi) is a method for forming the complex of the present invention, consisting of [extracellular membrane vesicles or viruses - (Tim protein of the present invention) - affinity substance - (carrier of the present invention)], by simultaneously contacting the Tim protein of the present invention with the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions.

[0265] Specifically, for example, when two or more substances with mutual affinity are used as affinity substances, in the presence of calcium ions, the Tim protein of the present invention (affinity substance-bound Tim protein) bound to one of the affinity substances is simultaneously brought into contact with a carrier (affinity substance-bound carrier) bound to the remaining affinity substances and extracellular membrane vesicles or viruses in the sample, so that the Tim protein of the present invention in the affinity substance-bound Tim protein binds to the extracellular membrane vesicles or viruses, and the affinity substances in the affinity substance-bound Tim protein bind to the affinity substances in the affinity substance-bound carrier, thereby forming the complex of the present invention composed of [extracellular membrane vesicles or viruses in the sample - (Tim protein of the present invention) - affinity substances - (carrier of the present invention)].

[0266] In (1-Bi), calcium ions can be present when the Tim protein of the present invention is simultaneously contacted with the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample. Specifically, a solution or sample containing calcium ions in at least one of the following can be used: a solution containing the Tim protein of the present invention, a solution containing the carrier of the present invention, and a sample. Alternatively, the contact can be made by contacting the solution containing the Tim protein of the present invention, the solution containing the carrier of the present invention, the sample, and the solution containing calcium ions.

[0267] -Quantity of the sample in this invention-

[0268] In (1-Bi), the amount of sample that comes into contact with 1 μg of the Tim protein of the present invention is typically 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0 ml.

[0269] -Amount of Tim protein in this invention-

[0270] In (1-Bi), the amount of Tim protein of the present invention is typically 0.01 to 200 μg, preferably 0.15 to 50 μg, and more preferably 0.5 to 24 μg, relative to 1 mL of the solution in which the complex of the present invention is formed.

[0271] -Amount of the carrier in this invention-

[0272] In (1-Bi), the amount of the carrier of the present invention is typically 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 in which the composite of the present invention is formed.

[0273] -Contact Temperature-

[0274] In (1-Bi), the temperature at which the Tim protein of the present invention comes into contact with the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample is typically 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0275] -Contact Time-

[0276] The contact time between the Tim protein of the present invention and the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample is typically 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0277] It should be noted that the contact between the Tim protein of the present invention and the carrier of the present invention, as well as the extracellular membrane vesicles or viruses in the sample, is usually carried out by contacting the 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 the solution containing calcium ions.

[0278] As a solution containing the Tim protein of the present invention, any solution that allows the Tim protein of the present invention to dissolve stably and does not hinder the binding of the Tim protein of the present invention to the carrier, extracellular membrane vesicles, or viruses of the present invention is acceptable. Examples include purified water and buffer solutions (e.g., PBS, TBS, HBS, etc.) with buffering capacity at pH 7.0–8.0, preferably 7.2–7.6. Furthermore, the concentration of the buffer in the buffer solution is typically selected appropriately from the range of 5–50 mM, preferably from the range of 10–30 mM, and the NaCl concentration is typically selected appropriately from the range of 100–200 mM, preferably from the range of 140–160 mM. The solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, and proteins, as long as the amount allows the Tim protein of the present invention to dissolve stably and does not hinder the binding of the Tim protein of the present invention to the carrier, extracellular membrane vesicles, or viruses of the present invention. Examples of surfactants include Tween 20, and the concentration of the surfactant in the solution containing the Tim protein of the present invention is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0279] 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 specific examples are also the same.

[0280] The solution containing calcium ions is a solution in which the concentration of calcium ions in the solution in which the composite of the present invention is formed is typically 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM.

[0281] Specific examples of -(1-Bi)-

[0282] (1-Bi) can be performed, for example, by the following method: A solution containing, typically 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 (a solution prepared by containing, for example, the Tim protein of the present invention in a buffer solution having a buffering effect under conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6) is prepared in a volume of typically 0.1 to 100 ml, preferably 0.1 to 10 ml, more preferably 0.5 μL to 24 μg relative to 1 mL of the solution in which the complex of the present invention is formed (a solution prepared by containing, for example, the Tim protein of the present invention in purified water, for example, a buffer solution having a buffering effect under conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6); the volume of the Tim protein of the present invention is typically 0.1 to 100 ml, preferably 0.1 to 10 ml, more preferably 0.5 μL to 10 μL). The preferred sample is 0.1 to 1 ml; a solution containing calcium ions in a concentration typically 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM, in the solution used to form the composite of the present invention; and the composite of the present invention is formed by contacting 1 mL of the carrier of the present invention (typically 0.1 to 20 mg, preferably 0.3 to 10 mg, more preferably 0.5 to 6 mg) with the solution containing the Tim protein of the present invention, the sample, the solution containing calcium ions, and the carrier of the present invention.

[0283] -About (1-B-ii)-

[0284] (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 of the Tim protein of the present invention with extracellular membrane vesicles or viruses, and then contacting the complex with the carrier of the present invention, thereby forming a complex composed of [extracellular membrane vesicles or viruses - (Tim protein of the present invention) - affinity substance - (carrier of the present invention)].

[0285] Specifically, for example, when two or more substances with mutual affinity are used as affinity substances, in the presence of calcium ions, 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 the sample, causing the Tim protein in the affinity substance-bound Tim protein to bind to the extracellular membrane vesicles, 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 bound to another affinity substance (affinity substance-bound carrier), causing the affinity substance in the [extracellular membrane vesicle or virus - (Tim protein of the present invention - affinity substance)] complex to bind 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.

[0286] Alternatively, for example, when a substance with affinity for the affinity tag, such as an anti-Tim antibody, is used as the affinity substance, the Tim protein of the present invention is brought into contact with extracellular membrane vesicles or viruses in the sample in the presence of calcium ions, causing the Tim protein of the present invention to bind to the extracellular membrane vesicles or viruses, 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 binding carrier) bound with the affinity substance, causing the Tim protein of the present invention in the [extracellular membrane vesicle or virus - Tim protein of the present invention] complex to bind to the affinity substance in the affinity substance binding carrier, thereby forming a [extracellular membrane vesicle or virus - Tim protein of the present invention - (affinity substance - carrier of the present invention)] complex.

[0287] In (1-B-ii), calcium ions may be present when the Tim protein of the present invention is brought into contact with extracellular membrane vesicles or viruses in the sample. Specifically, a solution or sample containing calcium ions may be used, or a solution containing the Tim protein of the present invention, a sample, and a solution containing calcium ions may be used.

[0288] -Quantity of the sample in this invention-

[0289] In (1-B-ii), the amount of sample that comes into contact with 1 μg of the Tim protein of the present invention is typically 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0 ml.

[0290] -Amount of Tim protein in this invention-

[0291] In (1-B-ii), the amount of Tim protein of the present invention is typically 0.01 to 200 μg, preferably 0.15 to 50 μg, and more preferably 0.5 to 24 μg, relative to 1 mL of the solution in which the complex of the present invention is formed.

[0292] -Amount of the carrier in this invention-

[0293] In (1-B-ii), the amount of the carrier of the present invention is typically 0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6 mg, relative to 1 mL of the solution in which the composite of the present invention is formed.

[0294] -Contact Temperature-

[0295] In (1-B-ii), the temperature at which the Tim protein of the present invention comes into contact with extracellular membrane vesicles or viruses in the sample is typically 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0296] In (1-B-ii), the temperature at which the complex of the Tim protein of the present invention with the extracellular membrane vesicle or virus is formed and then brought into contact with the carrier of the present invention is typically 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

[0297] -Contact Time-

[0298] In (1-B-ii), the contact time between 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.

[0299] In (1-B-ii), the time for further contacting the complex of the Tim protein of the present invention with the carrier of the present invention after the complex of the complex of the Tim protein of the present invention with the extracellular membrane vesicle or virus is generally 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0300] It should be noted that, typically, the contact between the Tim protein of the present invention and the extracellular membrane vesicles or viruses in the sample is carried out by contacting the sample with a solution containing the Tim protein of the present invention and a solution containing calcium ions.

[0301] As a solution containing the Tim protein of the present invention, any solution is suitable as long as it allows the Tim protein of the present invention to dissolve stably and does not hinder the binding of the Tim protein of the present invention to the carrier of the present invention and to the extracellular membrane vesicles or viruses of the present invention. Examples include purified water and buffer solutions (e.g., TBS, HBS, etc.) with buffering capacity at pH 7.0 to 8.0, preferably 7.2 to 7.6. Furthermore, the concentration of the buffer in the buffer solution is typically selected appropriately from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is typically selected appropriately 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 allows the Tim protein of the present invention to dissolve stably and does not hinder the binding of the Tim protein of the present invention to the carrier of the present invention and to the extracellular membrane vesicles or viruses. Examples of surfactants include Tween 20, and the concentration of the surfactant in the solution containing the Tim protein of the present invention is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0302] 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 specific examples are also the same.

[0303] The solution containing calcium ions is a solution in which the concentration of calcium ions in the solution in which the composite of the present invention is formed is typically 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM.

[0304] Specific examples of -(1-B-ii)-

[0305] For (1-B-ii), for example, it can be done in the following way. That is, a solution containing the Tim protein of the present invention (typically 0.5 μL to 1 ml, preferably 0.15 μL to 50 μg, more preferably 0.5 μL to 10 μL, in 1 mL of the solution in which the complex of the present invention is formed, typically 0.01 to 200 μg, preferably 0.15 to 50 μg, more preferably 0.5 to 24 μg) of 1 μg of the Tim protein of the present invention (in a buffer solution having buffering effect, for example, pure water, at pH 7.0 to 8.0, preferably 7.2 to 7.6); a sample typically 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, contacted at a temperature typically 4 to 37°C, preferably 4 to 25°C, more preferably 4 to 11°C, typically for 0.5 to 24 hours, preferably 0.5 to 8 hours, more preferably 0.5 to 4 hours, wherein the calcium ion-containing solution... The amount of calcium ions in the solution is such that the concentration of calcium ions in the solution containing the sample and calcium ions and the solution containing the Tim protein of the present invention, and in the solution after further contacting the solution with the carrier of the present invention, is typically 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM. After the Tim protein of the present invention forms a complex with extracellular membrane vesicles or viruses, 1 mL of the carrier of the present invention is added, typically 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 in which the complex of the present invention was formed. The obtained complex of the Tim protein of the present invention with extracellular membrane vesicles or viruses is contacted with the carrier of the present invention for typically 0.5 to 24 hours, preferably 0.5 to 8 hours, more preferably 0.5 to 4 hours, at a temperature typically 4 to 37°C, preferably 4 to 25°C, more preferably 4 to 11°C, to form the complex of the present invention.

[0306] -Regarding (1-B-iii)-

[0307] (1-B-iii) is a method for forming the complex of the present invention, which consists of [extracellular membrane vesicles or viruses - (Tim protein of the present invention) - affinity substance - (carrier of the present invention)], by contacting the carrier of the present invention with extracellular membrane vesicles or viruses in a sample and then contacting them with the Tim protein of the present invention in the presence of calcium ions.

[0308] Specifically, for example, when two or more substances with mutual affinity are used as affinity substances, a carrier (affinity substance-binding carrier) bound to one of the affinity substances is brought into contact with extracellular membrane vesicles or viruses in the sample. Then, in the presence of calcium ions, it is brought into contact simultaneously with a Tim protein (affinity substance-binding Tim protein) bound to another affinity substance. The Tim protein in the affinity substance-binding Tim protein binds to the extracellular membrane vesicles or viruses, and the affinity substance in the affinity substance-binding Tim protein binds to the affinity substance in the affinity substance-binding 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.

[0309] Alternatively, for example, when using a substance with affinity for the affinity tag, such as an anti-Tim antibody, as the affinity substance, a carrier (affinity substance binding carrier) bound to the affinity substance is brought into contact with extracellular membrane vesicles or viruses in the sample. 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 binds to the extracellular membrane vesicles or viruses, and the Tim protein of the present invention binds to the affinity substance in the affinity substance binding carrier, thereby forming a [extracellular membrane vesicle or virus - Tim protein of the present invention - (affinity substance - carrier of the present invention)] complex.

[0310] In (1-B-iii), calcium ions may be present when the Tim protein of the present invention is brought into contact with extracellular membrane vesicles or viruses in the sample. Specifically, a solution or sample containing calcium ions in a solution containing the Tim protein of the present invention, and / or extracellular membrane vesicles or viruses in the sample, may be used. Alternatively, a solution containing the Tim4 protein of the present invention, extracellular membrane vesicles or viruses in the sample, and a solution containing calcium ions may be used.

[0311] -Quantity of the sample in this invention-

[0312] In (1-B-iii), the amount of sample in contact with 1 μg of the Tim protein of the present invention is typically 0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1 ml.

[0313] -Amount of Tim protein in this invention-

[0314] In (1-B-iii), the amount of Tim protein of the present invention is typically 0.01 to 200 μg, preferably 0.15 to 50 μg, and more preferably 0.5 to 24 μg, relative to 1 mL of the solution in which the complex of the present invention is formed.

[0315] -Amount of the carrier in this invention-

[0316] In (1-B-iii), the amount of the carrier of the present invention is typically 0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6 mg, relative to 1 mL of the solution in which the composite of the present invention is formed.

[0317] -Contact Temperature-

[0318] In (1-B-iii), the temperature at which the carrier of the present invention comes into contact with the extracellular membrane vesicles or viruses in the sample is typically 4 to 37°C, preferably 4 to 25°C, and more preferably 4 to 11°C.

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

[0320] -Contact Time-

[0321] In (1-B-iii), the contact time when the carrier of the present invention comes into contact with the sample is typically 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0322] In (1-B-iii), the contact time when the carrier of the present invention comes into contact with the extracellular membrane vesicles or viruses in the sample, and then the Tim protein of the present invention comes into contact with them, is typically 0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

[0323] It should be noted that, typically, after the carrier of the present invention comes into contact with the extracellular membrane vesicles or viruses in the sample, and then with the Tim protein of the present invention, a solution containing the Tim protein of the present invention and a solution containing calcium ions are used.

[0324] As a solution containing the Tim protein of the present invention, any solution that allows the Tim protein of the present invention to dissolve stably and does not hinder the binding of the Tim protein of the present invention to the carrier, extracellular membrane vesicles, or viruses of the present invention is acceptable. Examples include purified water and buffer solutions (e.g., PBS, TBS, HBS, etc.) with buffering capacity at pH 7.0–8.0, preferably 7.2–7.6. Furthermore, the concentration of the buffer in the buffer solution is typically selected appropriately from the range of 5–50 mM, preferably from the range of 10–30 mM, and the NaCl concentration is typically selected appropriately from the range of 100–200 mM, preferably from the range of 140–160 mM. The solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, and proteins, as long as the amount allows the Tim4 protein of the present invention to dissolve stably and does not hinder the binding of the Tim protein of the present invention to the carrier, extracellular membrane vesicles, or viruses of the present invention. Examples of surfactants include Tween 20. The concentration of the surfactant in the solution containing the Tim protein of the present invention is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0325] 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 specific examples are also the same.

[0326] The solution containing calcium ions is a solution in which the concentration of calcium ions in the solution in which the composite of the present invention is formed is typically 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM.

[0327] Specific examples of -(1-B-iii)-

[0328] (1-B-iii) This can be carried out, for example, by the following method: A sample of the present invention, typically 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 contacted with a carrier of the present invention, typically 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 used to form the complex of the present invention, at a temperature typically 4 to 37°C, preferably 4 to 25°C, more preferably 4 to 11°C, for typically 0.5 to 24 hours, preferably 0.5 to 8 hours, more preferably 0.5 to 4 hours. Then, a solution containing calcium ions is added, typically 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 used to form the complex of the present invention, containing typically 0.01 to 200 μg, preferably 0.1 mg. A solution of the Tim protein of the present invention (containing the Tim protein of the present invention in, for example, pure water, or in a buffer solution having buffering effect at pH 7.0-8.0, preferably 7.2-7.6) is contacted at a temperature typically 4-37°C, preferably 4-25°C, more preferably 4-11°C for typically 0.5-24 hours, preferably 0.5-8 hours, more preferably 0.5-4 hours, to form the complex of the present invention. The amount of calcium ions in the solution containing calcium ions is such that the concentration of calcium ions in the solution formed by contacting the sample containing the present invention and the solution containing calcium ions with the carrier of the present invention, and then with the solution containing the Tim protein of the present invention, is typically 0.5-100 mM, preferably 1.0-10 mM, more preferably 2-5 mM.

[0329] <6-2. Complex Separation Process>

[0330] The complex separation step in the method of obtaining the present invention is a step of separating the Tim protein (Tim vector of the present invention) bound to the carrier of the present invention (the complex of the present invention) and the extracellular membrane vesicles or viruses in the sample (the complex of the present invention) after the complex formation step, and obtaining the separated complex of the present invention.

[0331] Regarding the composite separation step in the method of obtaining the present invention, any method can be used as long as the composite of the present invention can be separated from the sample and the composite of the present invention can be obtained. For example, the following methods can be cited.

[0332] (1) When using a magnetic carrier as the carrier of the present invention, the container that has undergone the composite formation process is placed on a magnetic frame as needed, and the composite of the present invention is assembled on the tube wall by magnetic force, the sample with supernatant is removed, and thus they are separated.

[0333] (2) When the carrier of the present invention is bead-shaped, the composite of the present invention is separated by centrifugation of the container that has undergone the composite formation process, so that the composite of the present invention is a precipitate aggregate, and the supernatant sample is removed.

[0334] (3) When the carrier of the present invention uses non-beaded plates or the like, the method of separating them by removing only the samples.

[0335] (4) A method for separating the composite of the present invention from the sample by filtration.

[0336] After separating the composite of the present invention from the sample in this manner, the separated composite of the present invention can be obtained (recovered) by methods known per se.

[0337] -Specific examples of complex separation processes-

[0338] When a magnetic carrier is used as the carrier of the present invention, the container that has undergone the composite formation process is placed on a magnetic frame as needed, and the composite of the present invention is assembled on the tube wall by magnetic force, and the supernatant sample is removed.

[0339] <6-3. Washing Operation>

[0340] 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 (hereinafter sometimes referred to as "washing operation") as needed. The washing operation removes impurities from the sample of the present invention, such as cellular components, that are attached to the surface of the carrier of the present invention. As a washing method, in addition to using the calcium-containing washing solution as described above, washing methods commonly used in this field can also be used. The calcium-containing washing solution used in this washing operation is any solution that contains calcium ions typically 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM, and does not affect the binding of the extracellular membrane vesicles or viruses of the present invention in the complex to the Tim4 protein of the present invention and the carrier of the present invention. For example, a buffer solution (e.g., TBS, HBS) containing calcium ions typically 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM, with buffering capacity at pH 7.0 to 8.0, preferably 7.2 to 7.6, and does not cause calcium precipitation. It should be noted that phosphate buffer is not preferred because it binds to calcium and forms a precipitate. Furthermore, the concentration of the buffer solution is typically selected from the range of 5 to 50 mM, preferably from the range of 10 to 30 mM, and the NaCl concentration is typically 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 extracellular membrane vesicles or viruses of the present invention in the complex to the Tim protein and the carrier of the present invention. Examples of surfactants include Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.), and the concentration of the surfactant in the washing solution is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%.

[0341] For example, the use of magnetic particles as the carrier of the present invention will be described. First, a washing solution containing calcium ions is added to a container containing the composite of the present invention obtained through the composite separation process, and the mixture is stirred. Then, the container is placed on a magnetic rack, and the composite is gathered to the tube wall using magnetic force. The solution in the container is then discarded. The washing operation can be repeated multiple times as needed.

[0342] -Specific examples of washing operations-

[0343] First, a washing solution containing calcium ions (e.g., a buffer solution containing typically 0.5–100 mM, preferably 1–10 mM, more preferably 2–5 mM of calcium ions, which has a buffering effect and does not cause calcium precipitation under conditions of pH 7.0–8.0, preferably 7.2–7.6. However, phosphate buffer is not preferred because it binds with calcium to form a precipitate.) is added to a container containing the composite obtained through the composite separation process, and the mixture is stirred. Then, the container is placed on a magnetic rack, and the composite is collected against the tube wall using magnetism. The solution in the container is then discarded. The washing operation can be repeated multiple times as needed.

[0344] <6-4. Regarding the acquisition process>

[0345] The obtaining process is a process of separating extracellular membrane vesicles or viruses from the complex of 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 process, the complex separation process, and the washing process (washing operation) as needed, thereby obtaining the extracellular membrane vesicles or viruses of the present invention.

[0346] Therefore, it is possible to obtain the extracellular membrane vesicles or viruses of the present invention with high purity.

[0347] As specific examples of the acquisition process, examples such as (2-A) and (2-B) can be given below.

[0348] (2-A) Method using protein modifiers

[0349] (2-B) Methods to reduce calcium ion concentration

[0350] <(2-A): Method using protein modifiers>

[0351] (2-A) is a method for separating extracellular membrane vesicles or viruses from the complex of the present invention by applying a protein modifier to the obtained complex after performing the complex formation process, the complex separation process, and the washing operation as needed. This allows for the high purity of the extracellular membrane vesicles or viruses obtained according to the present invention.

[0352] -Protein Modifier-

[0353] As the protein modifier used in (2-A), any substance can be used as long as it is a compound commonly used in this field for modifying proteins. For example, anionic surfactants such as SDS (sodium dodecyl sulfate) and N-lauroyl sarcosine can be used; zwitterionic surfactants such as CHAPS (3-(3-cholamidopropyl)dimethylamino-1-propanesulfonate) and Zwittergent 3-12 (N-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate); Brij 35 (manufactured by Takara Bio Inc.), n-dodecyl-β-D-maltoside, ethyl phenyl polyethylene glycol (Nonidet P-40), octyl-β-D-glucoside, Triton X-100 (polyoxyethylene (10) octylphenyl ether), and Tween 20 (tween Nonionic surfactants such as polyoxyethylene (20) dehydrated sorbitol monolaurate; liquid ionizers such as urea, formamide, and guanidine; preferably anionic surfactants, especially SDS.

[0354] In (2-A), in order for the protein modifier to act on the composite of the present invention, it is generally done by contacting a solution containing the protein modifier (hereinafter sometimes abbreviated as "solution containing protein modifier") and a complex of Tim protein bound to the carrier of the present invention with an extracellular membrane vesicle or virus in the sample, thereby allowing the solution containing the protein modifier to act on the composite of the present invention.

[0355] It should be noted that the contact between the solution containing the protein modifier and the composite of the present invention can be carried out by, for example, suspending the composite in the solution (when the carrier is a bead, etc.), immersing the composite in the solution (when the carrier is a disc, a tube, etc.), or adding the composite (carrier) to the solution (when the carrier is a microplate, a tube, etc.).

[0356] -To make the solution containing the protein modifier-

[0357] In (2-A), examples of solutions containing protein modifiers include purified water and buffer solutions capable of dissolving the protein modifiers. Examples of such buffer solutions include buffers that have buffering properties under normal pH conditions of 6-9, preferably 7-8 (e.g., Tris, HEPES, etc.). Furthermore, the concentration of the buffer in the buffer solution is typically selected from the range of 5-100 mM, preferably from the range of 10-50 mM.

[0358] -A solution containing protein modifier-

[0359] In (2-A), the pH of the solution containing the protein modifier is typically 6.0 to 9.0, preferably 7.0 to 8.0. The concentration of the protein modifier in the solution varies depending on the type of protein modifier; however, it is acceptable as long as it falls within the concentration range commonly used in this field. For example, in the case of SDS, it is typically 0.1% to 10%, preferably 0.3% to 4%, and more preferably 0.5% to 2%. Furthermore, 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, relative to 1 mg of the Tim carrier of the present invention, 10 μL to 500 μL is typically used, preferably 20 μL to 200 μL, and more preferably 50 μL to 100 μL.

[0360] -Contact Conditions-

[0361] In (2-A), the temperature and time at which the protein modifier acts (contacts) the complex are typically 4.0 to 37°C, preferably 10 to 30°C, more preferably 20 to 30°C, and the time is typically 5.0 to 60 seconds, preferably 10 to 30 seconds, more preferably 10 to 20 seconds.

[0362] -Tim protein-

[0363] When the protein modifier is applied (contacted) to 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. However, the Tim4 protein 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 and the Tim3 protein of the present invention are particularly preferred.

[0364] Specific examples of -(2-A)-

[0365] (2-A) can be done, for example, by the following methods. That is, after performing the complex formation process, the complex separation process, and the washing operation as needed, a solution containing 0.1% to 10%, preferably 0.3% to 4.0%, more preferably 0.5% to 2.0% protein modifier (in purified water or a buffer solution with buffering effect under normal pH 6 to 9, preferably 7 to 8) is added to the obtained complex of the present invention, typically 10 μL to 500 μL, preferably 20 μL to 200 μL, more preferably 50 μL to 100 μL relative to 1 mg of the Tim carrier of the present invention. While stirring with a vortex mixer or the like, a reaction is carried out at a temperature typically 4 to 37°C, preferably 10 to 30°C, more preferably 20 to 30°C, typically 5 to 60 seconds, preferably 10 to 30 seconds, more preferably 10 to 20 seconds, so that the Tim protein of the present invention in the complex of the present invention comes into contact with the protein modifier, thereby allowing the protein modifier to exert its effect and causing extracellular membrane vesicles or viruses to separate from the complex of the present invention.

[0366] <(2-B): Methods to reduce calcium ion concentration>

[0367] (2-B) is a method for separating extracellular membrane vesicles or viruses from the complex of the present invention after performing a complex formation process, a complex separation process, and a washing operation as needed, thereby reducing the concentration of calcium ions bound to the obtained complex of the present invention and the concentration of calcium ions in the solution containing the complex.

[0368] Thus, the extracellular membrane vesicles or viruses of the present invention can be obtained with high purity and in an intact state.

[0369] Furthermore, calcium ions are known to lie between the Tim protein and phosphatidylserine (Immunity 2007 December; 27(6): 941-951). After performing the complex formation step, the complex separation step, and the washing operation as needed, for the complex of the present invention obtained, calcium ions must be pre-existing in order 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. For example, when the complex of the present invention coexists in solution, the solution of the complex of the present invention needs to have more than 0.5 mM of calcium ions. The coexistence of the complex of the present invention in solution also includes a carrier in a particulate state after performing the complex formation step, the complex separation step, and the washing operation as needed.

[0370] It should be noted that even if the composite of the present invention dries after the composite formation process, composite separation process, and washing operation as required, calcium ions will not dissolve from the composite as long as the acquisition process is not performed, thereby maintaining the binding of the carrier of the present invention with extracellular membrane vesicles or viruses.

[0371] (2-B) By making the concentration of calcium ions from the calcium ions bound to the complex of the present invention lower than the concentration (effective concentration) required as described above to maintain the binding of the Tim protein of the present invention with the extracellular membrane vesicles or viruses, the extracellular membrane vesicles or viruses of the present invention can be separated from the complex of the present invention.

[0372] Specifically, the calcium ion concentration in the solution containing the composite of the present invention can be set to typically less than 0.5 mM, preferably less than 0.4 mM, and more preferably less than 0.2 mM.

[0373] As a method to reduce the (effective) concentration of calcium ions, examples include (2-Bi) and (2-B-ii).

[0374] (2-Bi) Method using calcium ion chelating agents

[0375] (2-B-ii) Method using a solution that does not contain calcium ions

[0376] -(2-Bi): Method using calcium ion chelating agents-

[0377] (2-Bi) is a method in which, after performing a complex formation step, a complex separation step, and a washing operation as needed, a calcium ion chelating agent is applied to calcium ions introduced by a solution containing calcium ions bound to the obtained complex of the present invention, and then the (effective) concentration of calcium ions introduced by the solution containing calcium ions bound to the complex of the present invention is reduced (calcium ion chelation), thereby separating extracellular membrane vesicles or viruses from the complex of the present invention.

[0378] Thus, the extracellular membrane vesicles or viruses of the present invention can be obtained with high purity and in an intact state.

[0379] -Calcium ion chelating agent-

[0380] As calcium ion chelating agents, any compound capable of chelating calcium ions can be used. Examples include EDTA (ethylenediaminetetraacetic acid), NTA (nitrotriacetic acid), DTPA (diethylenetriaminepentaacetic acid), GLDA (L-glutamic acid diacetic acid), HEDTA (hydroxyethylethylenediaminetetraacetic acid), GEDTA (ethylene glycol bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid), TTHA (triethylenetetramine-N,N,N',N",N”',N”'-hexaacetic acid), HIDA (2-hydroxyethyliminodiacetic acid), DHEG (N,N-bis(2-hydroxyethyl)glycine), and CyDTA (trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate). (e.g., acid, monohydrate), preferably EDTA, GEDTA, or CyDTA. To enable the calcium ion chelating agent to act on calcium ions bound to the complex of the present invention, it is typically achieved by contacting a solution containing the calcium ion chelating agent (hereinafter sometimes abbreviated as "a solution containing the calcium ion chelating agent") with the particulate complex of the present invention, thereby causing a reaction between the calcium ions bound to the complex of the present invention and the calcium ion chelating agent in the solution containing the calcium ion chelating agent.

[0381] It should be noted that the contact between the solution containing the calcium ion chelating agent and the composite of the present invention can be carried out by, for example, suspending the composite in the solution (when the carrier is a bead, etc.), immersing the composite in the solution (when the carrier is a disc, a tube, etc.), or adding the solution to the composite (carrier) (when the carrier is a microplate, a tube, etc.).

[0382] -Solution containing calcium ion chelating agents-

[0383] In (2-Bi), any solution containing the calcium ion chelating agent can be used as a dissolving agent, such as purified water or a buffer solution. The buffer solution is preferably a buffer that provides buffering action at a pH of 7.0–8.0, preferably 7.2–7.6 (e.g., PBS, TBS, HBS). Furthermore, the concentration of the buffer solution is typically selected from the range of 5–50 mM, preferably from 10–30 mM, and the NaCl concentration is typically selected from the range of 100–200 mM, preferably from 140–160 mM. The solution containing the calcium ion chelating agent may contain, for example, sugars, salts such as NaCl, preservatives, and proteins.

[0384] In (2-Bi), the concentration of the calcium ion chelating agent in the solution containing the calcium ion chelating agent is typically 0.5 to 500 mM, preferably 0.5 to 100 mM, and more preferably 0.5 to 50 mM.

[0385] In (2-Bi), the pH of the solution containing the calcium ion chelating agent is typically 6.0 to 9.0, preferably 7.0 to 8.0, and more preferably 7.2 to 7.6.

[0386] In (2-Bi), the amount of the solution containing the calcium ion chelating agent mixed in the reaction solution is only the amount that makes the calcium ion concentration in the reaction solution less than the effective concentration, thereby separating the extracellular membrane vesicles or viruses from the complex of the present invention.

[0387] -Contact Conditions-

[0388] In (2-Bi), the temperature and time at which the calcium ion chelating agent acts (contacts) with the complex are typically 4.0 to 37°C, preferably 10 to 30°C, more preferably 20 to 30°C, and the time is typically 5 to 60 seconds, preferably 10 to 30 seconds, more preferably 10 to 20 seconds.

[0389] -Tim Vector-

[0390] In (2-Bi), as for the binding mode between the Tim protein of the present invention and the carrier of the present invention, there is no limitation on the binding mode as long as the carrier of the present invention binds to the Tim protein of the present invention. When using a calcium ion chelating agent to dissolve extracellular membrane vesicles or viruses (i.e., in the case of (2-Bi)), from the perspective of obtaining a large number 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.

[0391] Specific examples of -(2-Bi)-

[0392] (2-Bi) can be obtained by, for example, the following methods. That is, after performing the complex formation process, the complex separation process, and the washing operation as needed, a solution containing a calcium ion chelating agent (usually 10-500 μL, preferably 20-200 μL, more preferably 50-100 μL, relative to 1 mg of the Tim carrier of the present invention, typically 0.5-500 mM, preferably 0.5-100 mM, more preferably 0.5-50 mM in purified water or a buffer solution having a buffering effect under conditions of normally pH 7.0-8.0, preferably 7.2-7.6) is added to the complex obtained by the present invention. While stirring with a vortex mixer or the like, a reaction is carried out at a temperature of typically 4.0-37°C, preferably 10-30°C, more preferably 20-30°C, typically for 5-60 seconds, preferably 10-30 seconds, more preferably 10-20 seconds, to separate extracellular membrane vesicles or viruses from the complex of the present invention.

[0393] -(2-B-ii): Method using a solution that does not contain calcium ions-

[0394] (2-B-ii) is a method for separating extracellular membrane vesicles or viruses from the complex of the present invention by contacting the obtained complex of the present invention with a solution free of calcium ions, thereby reducing (diluting) the (effective) concentration of calcium ions bound to the complex of the present invention. This is achieved after performing the complex formation process, the complex separation process, and the washing operation as required.

[0395] That is, by contacting the complex of the present invention with a solution that does not contain calcium ions, the (effective) concentration (dilution) 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.

[0396] It should be noted that the contact between the calcium-free solution and the composite of the present invention can be achieved by, for example, suspending the composite in the solution (when the carrier is a bead, etc.), immersing the composite in the solution (when the carrier is a disc, a tube, etc.), or adding the solution to the composite (carrier) (when the carrier is a microplate, a tube, etc.).

[0397] -A solution without calcium ions-

[0398] In (2-B-ii), the calcium-free solution added to the obtained composite of the present invention can be any solution that will not modify the extracellular membrane vesicles or viruses, such as purified water or buffer solution. As the buffer solution, a buffer solution with buffering capacity (e.g., PBS, TBS, HBS, etc.) under normal pH 7.0–8.0, preferably 7.2–7.6, is preferred. Furthermore, the concentration of the buffer in the buffer solution is typically selected from the range of 5–50 mM, preferably from the range of 10–30 mM, and the NaCl concentration is typically selected from the range of 100–200 mM, preferably from the range of 140–160 mM. The calcium-free solvent may contain, for example, sugars, salts such as NaCl, preservatives, proteins, etc.

[0399] In (2-B-ii), the amount of calcium-free solution added to the obtained composite of the present invention is any amount that makes the concentration of calcium ions less than the effective concentration.

[0400] Specific examples of -(2-B-ii)-

[0401] After performing the complex formation process, the complex separation process, and the washing operation as needed, a calcium-free solution 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.

[0402] It should be noted that after the complex formation process, the complex separation process, and the washing operation as needed, when the obtained complex of the present invention is present in a solution containing calcium ions (e.g., the reaction solution after the complex formation process, or the washing solution containing calcium ions), the solution containing calcium ions can be replaced with a solution without calcium ions, or / and diluted with a solution without calcium ions, so that the final concentration of calcium ions is less than the above-mentioned effective concentration.

[0403] Regarding the amount of volume increase of the solution containing extracellular membrane vesicles obtained by the acquisition process, (2-B-ii) is more than (2-Bi), and therefore (2-Bi) is preferred.

[0404] In the above, the solutions that have come into contact with / acted upon the complex (solutions containing protein modifiers, solutions containing calcium ion chelating agents, and solutions without calcium ions) contain a carrier and extracellular membrane vesicles or viruses that have separated (freed) 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.

[0405] If the Tim vector of the present invention is used, extracellular membrane vesicles or viruses can be efficiently removed from the sample, thereby obtaining a sample with fewer impurities.

[0406] The removal method of the present invention can remove extracellular membrane vesicles or viruses in samples that cannot be completely removed by existing methods such as ultracentrifugation and polymer precipitation with good precision and effectiveness.

[0407] <7. Methods for removing extracellular membrane vesicles or viruses from samples>

[0408] The method for obtaining extracellular membrane vesicles or viruses of the present invention (hereinafter, sometimes referred to as "the removal method of the present invention") is characterized by comprising the following steps.

[0409] (1) The process of forming a complex of Tim protein bound to a carrier with an extracellular membrane vesicle or virus in a sample in the presence of calcium ions (hereinafter, sometimes referred to as the "complex formation process").

[0410] (2) The process of separating the composite from the sample (hereinafter, sometimes referred to as the "composite separation process").

[0411] <7-1. Composite Formation Process>

[0412] The composite formation step in the removal method of the present invention is the same as the composite formation step in the acquisition method of the present invention, and the various preferred conditions are also the same.

[0413] <7-2. Complex Separation Process>

[0414] The complex separation step in the removal method of the present invention is a step after the complex formation step, in which the Tim protein (Tim vector of the present invention) bound to the carrier of the present invention is separated from the complex (complex of the present invention) of the extracellular membrane vesicles or viruses in the sample and the sample, and the separated sample is obtained.

[0415] This allows us to obtain samples from which extracellular membrane vesicles or viruses have been removed.

[0416] For the complex separation step in the removal method of the present invention, any method can be used as long as it can remove the complex of the present invention from the sample; examples include methods identical to the complex separation step in the acquisition method of the present invention. After separating the complex of the present invention from the sample in this way, the separated sample can be obtained (recovered) by methods known per se.

[0417] Therefore, it is possible to remove extracellular membrane vesicles or viruses from samples that cannot be completely removed by existing methods such as ultracentrifugation and polymer precipitation with high precision and effectiveness.

[0418] Furthermore, by repeatedly performing the complex formation and complex separation steps, extracellular membrane vesicles or viruses can be removed from the sample more effectively. When repeatedly performing the complex formation and complex separation steps, new carriers can be used, and used carriers can be reused. For the reuse of the carrier, a solution containing a calcium ion chelating agent or a solution without calcium ions can be used to treat the complex of the present invention removed by the complex separation step in the removal method of the present invention in the same way as the acquisition step ((2-B) method for reducing calcium ion concentration) of the acquisition method of the present invention. Thus, the Tim carrier of the present invention and 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 reused in the removal method of the present invention, by reusing the Tim carrier of the present invention, extracellular membrane vesicles or viruses in the sample can be removed with good precision and effectiveness.

[0419] If the Tim vector of the present invention is used, extracellular membrane vesicles or viruses in a sample can be detected with good accuracy and high sensitivity.

[0420] <8. Methods for detecting extracellular membrane vesicles or viruses in samples>

[0421] The method for detecting extracellular membrane vesicles or viruses of the present invention (hereinafter, sometimes referred to as "the detection method of the present invention") is characterized by comprising the following steps.

[0422] (1) The process of forming a complex of Tim protein bound to a carrier with extracellular membrane vesicles or viruses in a sample in the presence of calcium ions (complex formation process).

[0423] (2) The process of testing the composite (testing process)

[0424] <8-1. Composite Formation Process>

[0425] The complex formation step in the detection method of the present invention is a step in which the Tim carrier of the present invention forms a complex with the extracellular membrane vesicles or viruses in the sample in the presence of calcium ions.

[0426] -Sample of the present invention-

[0427] The sample of the present invention is the same as the sample obtained in the method of the present invention.

[0428] -Calcium ion concentration / Source of calcium ions-

[0429] In the detection method of the present invention, calcium ions are present when the Tim carrier of the present invention forms a complex (the complex of the present invention) with the extracellular membrane vesicles or viruses in the sample.

[0430] In the detection method of the present invention, the calcium ion concentration when the Tim carrier of the present invention comes into contact with the extracellular membrane vesicles or viruses in the sample of the present invention is typically 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM. It should be noted that the calcium ion concentration described above is required in the solution containing the complex formed between the Tim carrier of the present invention and the extracellular membrane vesicles or viruses in the sample of the present invention, up to the point where the detection step is performed.

[0431] Furthermore, there is no particular limitation on the source of calcium ions, and specific examples of the same source of calcium ions as in the method of obtaining calcium ions of the present invention can be given.

[0432] It should be noted that, as a method for ensuring the presence of calcium ions when the Tim carrier of the present invention comes into contact with extracellular membrane vesicles or viruses in a sample, the sample can typically contain calcium ions as described above, thereby ensuring that the calcium ion concentration when the Tim carrier of the present invention comes into contact with extracellular membrane vesicles or viruses in the sample is within the aforementioned range.

[0433] 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 specific examples are also the same.

[0434] - Sample quantity -

[0435] In the composite formation step of the detection method of the present invention, the amount of sample is typically 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, the amount per well is typically 50 μL to 300 μL, preferably 100 μL to 200 μL.

[0436] -temperature-

[0437] In the complex formation step of the detection method of the present invention, the temperature at which the Tim carrier of the present invention comes into contact with the extracellular membrane vesicles or viruses in the sample is typically 2 to 37°C, preferably 2 to 30°C.

[0438] -time-

[0439] 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 typically 0.5 to 24 hours, preferably 1 to 20 hours, and more preferably 1 to 12 hours.

[0440] -Tim protein-

[0441] As the Tim protein of the present invention used in the detection method of the present invention, any protein can be used as long as it is the Tim protein of the present invention described above, but the Tim4 protein of the present invention is particularly preferred.

[0442] -Tim Vector-

[0443] As for the binding mode of the Tim protein of the present invention and the vector of the present invention, there is no limitation on the binding mode 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, the binding mode of the vector of the present invention to the thiol group of the Tim protein of the present invention is preferred, and the binding mode of the vector of the present invention to the thiol group of the Tim4 protein of the present invention is particularly preferred.

[0444] -Specific example of the composite formation process in the detection method of the present invention-

[0445] The composite formation step in the detection method of the present invention can be performed by, for example, the following methods.

[0446] That is, when beads are used as the carrier of the present invention, the amount of the Tim carrier of the present invention (the solution in which the composite of the present invention is formed) relative to 1 mL of the solution containing the Tim carrier of the present invention, the sample, and the solution containing calcium ions is typically 0.001 to 20 mg, preferably 0.005 to 10 mg, more preferably 0.01 to 6.0 mg of the Tim carrier of the present invention; the amount of the solution containing calcium ions is typically 0.1 to 1000 ml, preferably 0.1 to 500 ml, more preferably 0.1 to 1 mg of the Tim carrier of the present invention. A 0.00 ml sample is contacted at a temperature of 2–37°C, preferably 2–30°C, for 0.5–24 hours, preferably 1–20 hours, and more preferably 1–12 hours, to allow the Tim protein bound to the carrier of the present invention to form a complex with the extracellular membrane vesicles or viruses in the sample. The amount of calcium ions in the solution containing calcium ions is such that the calcium ion concentration in the solution at the time of formation of the complex of the present invention is typically 0.5–100 mM, preferably 1.0–10 mM, and more preferably 2.0–5.0 mM.

[0447] When a microplate is used as the carrier of the present invention, a solution containing calcium ions and the sample of the present invention are added to each well of the microplate (the Tim carrier of the present invention) containing immobilized Tim protein, typically 50 μL to 300 μL, preferably 100 μL to 200 μL per well. The reaction is carried out at a temperature typically 2°C to 37°C, preferably 2°C to 30°C, typically for 0.5 hours to 24 hours, preferably for 1 hour to 20 hours, and more preferably for 1 to 12 hours. This causes the Tim carrier of the present invention to form a complex (the complex of the present invention) with the extracellular membrane vesicles or viruses in the sample. The calcium ion concentration in the solution containing calcium ions is such that the calcium ion concentration in the solution at the time of formation of the complex of the present invention is typically 0.5 to 100 mM, preferably 1.0 to 10 mM, and more preferably 2.0 to 5.0 mM.

[0448] <8-2. Detection steps in the detection method of the present invention>

[0449] The detection step in the detection method of the present invention is a step of detecting the complex of the Tim vector of the present invention and the extracellular membrane vesicles or viruses in the sample (the complex of the present invention) after the complex formation step, the complex separation step of the sample as needed, and the washing operation as needed.

[0450] -Complex Separation Process-

[0451] The complex separation step in the detection method of the present invention is a step after the complex formation step, whereby the sample is removed from the complex (the complex of the present invention) of the Tim protein (the Tim vector of the present invention) bound to the carrier of the present invention and the extracellular membrane vesicles or viruses in the sample, as needed.

[0452] Regarding the complex separation step in the detection 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; in other words, as long as so-called B / F separation can be performed. The B / F separation method used in this field can be employed. This method is the same as the complex separation step in the acquisition method of the present invention.

[0453] -Specific examples of complex separation processes-

[0454] 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 that has undergone the complex formation step is placed on a magnetic frame as needed, and the magnetic force is used to gather the complex of the present invention on the tube wall, and the sample in the supernatant is removed.

[0455] When a microplate is used as a carrier of the present invention, for example, a microfluidic tube or a plate washer may be used as needed to remove the sample from the microplate after the composite formation process.

[0456] <8-3. Washing Operation>

[0457] After the composite formation process, a composite separation process can be performed as needed, and then the obtained composite of the present invention can be washed with a washing solution containing calcium ions as needed (hereinafter, sometimes abbreviated as "washing operation"). Through the washing operation, impurities such as cell-derived components adhering to the surface of the carrier of the present invention can be removed from the sample of the present invention. The various conditions of the washing method are the same as those in the washing operation of the method for obtaining the present invention.

[0458] -Specific examples of washing operations-

[0459] When using magnetic beads as the carrier of the present invention and employing a 1.5 mL tube, firstly, in a container containing the composite of the present invention obtained through the composite separation process, add 100 μL to 1500 μL, preferably 200 μL to 1000 μL, of a washing solution containing calcium ions (e.g., a buffer solution containing 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2 to 5 mM, of calcium ions, which has a buffering effect and does not cause calcium precipitation under conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6. However, phosphate buffer is not preferred because it binds with calcium to form a precipitate). Stir the solution. Then, place the container on a magnetic rack, use magnetism to collect the composite against the tube wall, and discard the solution in the container. The washing operation can be repeated multiple times as needed.

[0460] When using a microplate as the carrier of the present invention, firstly, 100 μL to 300 μL, preferably 200 μL to 300 μL, of a washing solution containing calcium ions (e.g., a buffer solution containing 0.5 to 100 mM, preferably 1 to 10 mM, more preferably 2.0 to 5.0 mM calcium ions, which has a buffering effect and does not cause calcium precipitation under conditions of pH 7.0 to 8.0, preferably 7.2 to 7.6. However, phosphate buffer is not preferred because it binds to calcium and precipitates it.) is added, and then the added washing solution is removed. The washing operation can be repeated multiple times as needed.

[0461] <8-4. Inspection Procedure>

[0462] For the detection steps in the detection method of the present invention, any method that can detect the presence and / or quantity of the complex of the present invention can be used, and any method known per se for immunological assays can be used.

[0463] The detection process of this invention is not particularly limited except for using the Tim carrier prepared by the method described above. Examples of such immunological assays include: for instance, reverse passive agglutination reaction methods (Tokyo Chemical Dojin, Continued Biochemistry Experiment Lecture 5, Immunobiochemistry Research Methods, pp. 36-37; Kanehara Publishing Co., Ltd., *Summary of Clinical Examination Methods*, 30th edition, pp. 844-845, etc.); and turbidimetric methods (such as the turbidimetric method) (Kanehara Publishing Co., Ltd., *Summary of Clinical Examination Methods*, 30th edition, pp. 851-853, etc.), immunoassays. Turbidimetric assay (Kinbari Publishing Co., Ltd., *Essentials of Clinical Examination Methods*, 30th edition, pp. 853-854, etc.) and other optical methods for measuring agglutination reactions; radioimmunoassay (RIA) (Tokyo Chemical Dojin, *Continued Lectures on Biochemistry Experiments 5, Immunobiochemistry Research Methods*, pp. 57-61; Kinbari Publishing Co., Ltd., *Essentials of Clinical Examination Methods*, 30th edition, pp. 856-862, etc.), immunoradiometric assay (Immunoradiometric) assay, IRMA (Kinbari Publishing Co., Ltd., *Summary of Clinical Examination Methods*, 30th edition, pp. 856-862, etc.), enzyme-linked immunosorbent assay (EIA) (Tokyo 10 Chemical Workers, Continued Lectures on Biochemistry Experiments 5, Immunobiochemistry Research Methods, pp. 62-65; Kinbari Publishing Co., Ltd., *Summary of Clinical Examination Methods*, 30th edition, pp. 862-865; Japanese Patent Application Publication No. 56-106154; Japanese Patent Application Publication No. 58-23796). Among all known immunological assays, including those published in the journal, enzyme-linked immunosorbent assay (ELISA) (Kinbari Publishing Co., Ltd., *Summary of Clinical Examination Methods*, 30th edition, pp. 1145-1149, etc.), fluorescence / luminescent immunoassay (Kinbari Publishing Co., Ltd., *Summary of Clinical Examination Methods*, 30th edition, pp. 865-867, etc.), and flow cytometry, enzyme-linked immunosorbent assay (ELISA) or flow cytometry is preferred.

[0464] When performing the detection steps of the present invention using enzyme-linked immunosorbent assay (ELISA) or flow cytometry, within the scope of the complex of the present invention being detected, methods known in the art can be followed. Examples include methods using anti-extracellular membrane vesicle antibodies that bind to extracellular membrane vesicles or viruses, methods using labeled primary antibodies formed by labeling primary antibodies with markers such as antiviral antibodies, or methods using the primary antibody and labeled secondary antibodies bound to the primary antibody. As for the labeled primary and labeled secondary antibodies, any antibody can be used as a labeling antibody for ELISA or flow cytometry; examples include fluorescently labeled antibodies labeled with fluorescent substances such as Cy3, Cy5, FITC, rhodamine, and PE; enzyme-labeled antibodies labeled with enzymes such as peroxidase and alkaline phosphatase; magnetic bead-labeled antibodies; and infrared-labeled antibodies. The fluorescence of the labeled antibodies can be measured using methods known in the art corresponding to the labeling method (marker) of the labeled antibody.

[0465] -Dilution solution for labeled antibodies-

[0466] In the detection method of the present invention, the diluent for the labeled primary antibody reacting with the complex of the present invention, or the diluent for the primary antibody and the labeled secondary antibody, may contain, except for calcium ions, any buffer that does not hinder the binding of the Tim protein to the extracellular membrane vesicles or viral complexes in the sample, and the antibody. Examples include water and buffer solutions with buffering capacity at pH 7.0–8.0, preferably 7.2–7.6 (e.g., TBS, HBS). It should be noted that phosphate buffers are not preferred because they bind with calcium to form a precipitate. Furthermore, the concentration of the buffer in the buffer solution is typically selected from the range of 5–50 mM, preferably from the range of 10–30 mM, and the NaCl concentration is typically selected from the range of 100–200 mM, preferably from the range of 140–160 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 carrier of the present invention to the extracellular membrane vesicles or viral complexes in the sample. Examples of surfactants include Tween 20, and the concentration of the surfactant in the solution containing the extracellular membrane vesicles or virus of the present invention is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%. Furthermore, the concentration of calcium ions in the diluent is typically 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM.

[0467] In the detection method of the present invention, the dilution ratio of the labeled primary antibody, or the primary antibody and labeled secondary antibody reacting with the complex of the present invention varies depending on the activity and concentration of the antibody, but is usually 10 to 1,000,000 times, preferably 1,000 to 100,000 times.

[0468] In the detection method of the present invention, the amount of the labeled primary antibody, or the diluted solution of the primary antibody and labeled secondary antibody solution reacting with the complex of the present invention, is typically 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, and more preferably 0.1 mL to 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 per well is typically 50 μL to 300 μL, preferably 50 μL to 200 μL, and more preferably 50 μL to 100 μL.

[0469] -Reaction (Contact) Temperature-

[0470] In the detection method of the present invention, the reaction temperature of the complex of the present invention with the labeled primary antibody, or with the primary antibody and the labeled secondary antibody, is generally 2 to 37°C, preferably 11 to 337°C, and more preferably 20 to 30°C.

[0471] -Reaction (Contact) Time-

[0472] In the detection method of the present invention, the reaction time of the complex of the present invention with the labeled primary antibody, or with the primary antibody and the labeled secondary antibody, is usually 0.5 to 12 hours, preferably 1 to 4 hours, and more preferably 1 to 2 hours.

[0473] In the detection steps of the detection method of the present invention, when using a labeled primary antibody, after reacting the complex of the present invention with the labeled primary antibody, a washing operation can remove unreacted labeled primary antibody. Furthermore, when using a primary antibody and a labeled secondary antibody, after reacting the complex of the present invention with the primary antibody, and after reacting the complex of the present invention with the primary antibody and the labeled secondary antibody, a washing operation can remove unreacted primary antibody and labeled secondary antibody. As a washing method, in addition to using a washing solution containing calcium ions as described above, washing methods commonly practiced in this field can be used. As the washing solution containing calcium ions used in this washing operation, any solution containing calcium ions typically 0.5–100 mM, preferably 1–10 mM, more preferably 2–5 mM, that does not affect the binding of the extracellular membrane vesicles or viruses of the present invention to the Tim protein and the vector of the present invention in the complex is acceptable. Examples include buffer solutions containing calcium ions typically 0.5–100 mM, preferably 1–10 mM, more preferably 2–5 mM, that have buffering properties at pH 7.0–8.0, preferably 7.2–7.6, and that do not cause calcium precipitation (e.g., TBS, HBS). It should be noted that phosphate buffers are not preferred because they bind with calcium to form a precipitate. Furthermore, the concentration of the buffer in the buffer solution is typically selected from the range of 5–50 mM, preferably from the range of 10–30 mM, and the NaCl concentration is typically selected from the range of 100–200 mM, preferably from the range of 140–160 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 extracellular membrane vesicles or viruses of the present invention in the complex to the Tim protein and the carrier of the present invention. Examples of surfactants include Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.), and the concentration of the surfactant in the washing solution is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%. When the carrier of the present invention is beads, the amount of washing solution relative to 1 mg of the carrier of the present invention is typically 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, more preferably 0.1 mL to 100 mL. When the carrier of the present invention is a microplate, the amount of washing solution used in one well is typically 100 μL to 300 μL, preferably 200 μL to 300 μL, and is removed after addition. The washing operation can be repeated multiple times as needed.

[0474] -Detection-

[0475] When the detection step in the detection method of the present invention is colorimetric detection, peroxidase, alkaline phosphatase, etc., can be used as markers for labeling primary or secondary antibodies. These markers can be detected according to methods known to the public for each marker.

[0476] As a colorimetric matrix solution in colorimetric detection, any colorimetric matrix solution commonly used in this field can be used. For example, when using peroxidase as a label, TMB (tetramethylbenzidine) solution or OPD (o-phenylenediamine) solution can be used, with TMB solution being preferred.

[0477] Regarding the amount of the colorimetric matrix used in colorimetric detection, when the carrier of the present invention is beads, the amount 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, more preferably 0.1 mL to 100 mL. When the carrier of the present invention is a microplate, the amount per well is usually 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 50 μL to 100 μL.

[0478] When the detection step in the detection method of the present invention is colorimetric detection, the reaction time with the colorimetric matrix solution is usually 5 minutes to 60 minutes, preferably 10 minutes to 40 minutes.

[0479] When the detection step in the detection method of the present invention is colorimetric detection, the reaction temperature with the colorimetric matrix solution is usually 2℃~37℃, preferably 20℃~30℃.

[0480] In the detection method of the present invention, when the detection step 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, which is equal in volume to the colorimetric matrix solution, is usually added as a reaction stopping solution to stop the colorimetric reaction.

[0481] When the detection step in the detection method of the present invention is fluorescence detection, and the fluorescent substance is used to label a primary antibody or a labeling agent for a secondary antibody, a fluorescence assay solution is added to the complex of the present invention and the complex labeled with the primary antibody, or to the complex of the present invention and the complex labeled with the primary antibody and the secondary antibody, and fluorescence is measured. For the fluorescence assay solution, in addition to using a fluorescence assay solution containing calcium ions, a fluorescence assay solution commonly used in this field (hereinafter sometimes abbreviated as "assay solution") can also be used. As the calcium-containing assay solution used for this fluorescence assay, any solution containing calcium ions, typically 0.5–100 mM, preferably 1–10 mM, and more preferably 2–5 mM, that does not affect the binding of the extracellular membrane vesicles or viruses of the present invention to the Tim protein and the vector of the present invention in the complex, is acceptable. For example, buffer solutions containing calcium ions typically 0.5–100 mM, preferably 1–10 mM, and more preferably 2–5 mM, that have buffering properties at pH 7.0–8.0, preferably 7.2–7.6, and do not cause calcium precipitation (e.g., TBS, HBS) are suitable. It should be noted that phosphate buffers are not preferred because they bind with calcium to form a precipitate. Furthermore, the concentration of the buffer in the buffer solution is typically selected from the range of 5–50 mM, preferably from the range of 10–30 mM, and the NaCl concentration is typically selected from the range of 100–200 mM, preferably from the range of 140–160 mM. The solution may contain, for example, sugars, salts such as NaCl, surfactants, preservatives, proteins such as BSA, etc., as long as the amount does not hinder the binding of the extracellular membrane vesicles or viruses of the present invention in the complex to the Tim protein of the present invention and the carrier of the present invention. Examples of surfactants include Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.), and the concentration of the surfactant in the washing solution is typically 0.00001 to 0.2%, preferably 0.0005 to 0.1%. Regarding the amount of fluorescence assay solution used in the detection method of the present invention, when the detection method of the present invention is ELISA and the carrier of the present invention is a microplate, the amount of assay solution used in one well is typically 50 μL to 300 μL, preferably 50 μL to 200 μL. When the detection method of the present invention is flow cytometry and the carrier of the present invention is beads, the amount of the test 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.

[0482] -Specific examples of the detection steps in the detection method of the present invention-

[0483] For example, when using a microplate as a carrier for the present invention and employing an ELISA method, after the complex formation step, the required complex separation step, and then the required washing operation, for each well of the resulting microplate immobilized with the Tim protein of the present invention, a dilution solution is added, typically 50 μL to 300 μL, preferably 50 μL to 200 μL, more preferably 50 μL to 100 μL, to each well. The reaction is carried out at a temperature typically 2 to 37°C, preferably 11 to 37°C, for typically 0.5 to 12 hours, preferably 1 to 4 hours. Then, a washing buffer containing calcium ions (typically containing...) is used. Each well is washed with a solution containing 0.5–100 mM, preferably 1–10 mM, more preferably 2–5 mM of calcium ions, which will not affect the binding of the extracellular membrane vesicles or viruses of the present invention to the Tim protein of the present invention and the carrier of the present invention. The diluent is prepared by diluting peroxidase-labeled primary antibodies against extracellular membrane vesicles or viruses or unlabeled primary antibodies with a diluent containing calcium ions typically 0.5–100 mM, preferably 1–10 mM, more preferably 2–5 mM, to a concentration typically 10–1,000,000 times, preferably 1,000–100,000 times. When using unlabeled primary antibodies, the peroxidase-labeled secondary antibodies are reacted using the same method as with the primary antibodies. Each well is then washed with a washing buffer containing calcium ions (typically 0.5–100 mM, preferably 1–10 mM, and more preferably 2–5 mM calcium ions, which does not affect the binding of the extracellular membrane vesicles or viruses of the present invention to the Tim protein and the vector of the present invention in the complex). Then, a colorimetric matrix solution, typically 50–300 μL, preferably 50–200 μL, and more preferably 50–100 μL, such as TMB or OPD solution, is added to each well, and the reaction is carried out at a temperature typically 2°C–37°C, preferably 20°C–30°C, for 5 to 60 minutes, preferably 10 to 40 minutes. Then, add an equal volume of 1 mol / L hydrochloric acid or 1 mol / L sulfuric acid reaction stop solution to stop the colorimetric reaction, and measure the absorbance using an ELISA reader.

[0484] For example, when beads are used as the carrier of the present invention and flow cytometry is employed, after the complex formation step, the required complex separation step, and then the required washing operation, for the obtained beads immobilized with the Tim protein of the present invention, a dilution solution is added, typically 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, more preferably 0.1 mL to 100 mL, relative to 1 mg of the carrier of the present invention. The reaction is carried out at a temperature typically 2 to 37°C, preferably 11 to 37°C, for 0.5 to 12 hours, preferably 1 to 4 hours. Then, a washing buffer containing calcium ions (containing...) is used. The beads are washed with a solution containing calcium ions typically 0.5–100 mM, preferably 1–10 mM, and more preferably 2–5 mM, which does not affect the binding of the extracellular membrane vesicles or viruses of the present invention to the Tim protein and the vector of the present invention in the complex. The diluent solution is prepared by diluting the fluorescently labeled primary antibody or unlabeled primary antibody to a concentration typically 10 to 1,000,000 times, preferably 1,000 to 100,000 times, with a diluent containing calcium ions typically 0.5–100 mM, preferably 1–10 mM, and more preferably 2–5 mM. When using unlabeled primary antibodies, the fluorescently labeled secondary antibodies are reacted using the same method as with the primary antibodies. The beads are then washed with a washing buffer containing calcium ions (a solution containing typically 0.5–100 mM, preferably 1–10 mM, and more preferably 2–5 mM calcium ions, which will not affect the binding of the extracellular membrane vesicles or viruses of the present invention to the Tim protein and the vector of the present invention in the complex). Then, the beads are suspended by adding 0.1 mL–1000 mL, preferably 0.1 mL–500 mL, and more preferably 0.1 mL–100 mL, of fluorescence assay solution relative to 1 mg of the vector of the present invention, and the fluorescence intensity is measured by flow cytometry.

[0485] It is generally believed that the affinity of an antibody for an antigen is typically at the Kd level of 10 nm to 100 pm. On the other hand, the binding affinity of Tim4 protein to phosphatidylserine has been reported to be approximately 2 nm (Nature (impact factor: 41.46). 12 / 2007; 450(7168): 435-9. DOI: 10.1038 / nature06307). Although the affinity of an antibody for a surface antigen of an extracellular membrane vesicle or viral envelope is similar to the affinity between phosphatidylserine on the surface of an extracellular membrane vesicle or viral envelope and the Tim protein of the present invention, unexpectedly, the detection method of the present invention using the Tim protein of the present invention for the detection of extracellular membrane vesicles or viruses has higher sensitivity than existing methods that use antibodies for the detection of extracellular membrane vesicles or viruses.

[0486] The extracellular membrane vesicles or viruses obtained by the method of this invention can be used for nucleic acid analysis of proteins and small RNAs (microRNAs) on their particle surface and inside, as well as for basic research such as functional analysis of extracellular membrane vesicles or viruses. Furthermore, they can be used in diagnostic drugs, pharmaceuticals, vaccines, and the like.

[0487] <9. The Extracellular Membrane Vesicle or Virus Capture Kit of the Present Invention>

[0488] 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 carrier of the present invention, or 2. a reagent comprising the Tim carrier of the present invention.

[0489] The kit may further include at least one selected from the calcium-containing washing solution, the protein-modifying solution, and the calcium-chelating agent. Specific examples and preferred embodiments of each component are as described above.

[0490] Furthermore, the reagents contained in the kit may include reagents commonly used in this field, such as buffers, sensitizers, surfactants, preservatives (e.g., sodium azide, salicylic acid, benzoic acid, etc.), stabilizers (e.g., albumin, globulin, water-soluble gelatin, surfactants, sugars, etc.), activators, coexisting substance influence avoidance agents, 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. Moreover, in order to maximize the effects of each reagent class, the concentration range of the reagents, etc., can be appropriately selected from commonly used concentration ranges.

[0491] Furthermore, the kit of the present invention may include the acquisition method, removal method, and detection method of the present invention, as well as related instructions. The "instructions" refer to the user manual, accompanying documents, or handbook (leaflet) of the kit that substantially describes the characteristics / principles / operation steps, judgment steps, etc., of the method through articles or diagrams.

[0492] The present invention will now be described in more detail with examples, embodiments and comparative examples, but the present invention is not limited thereto.

[0493] It should be noted that the Tim protein of the present invention and the protein containing T cell immunoglobulin and mucin domain molecule 2 (Tim2) (hereinafter sometimes referred to as "Tim2 protein") are sometimes referred to together as "Tim family proteins".

[0494] Example

[0495] Example 1. Preparation of Fc-tagged Tim4 protein

[0496] The Fc-tagged Tim4 protein was prepared using the following method.

[0497] <(1) Construction / Cultivation of Vectors>

[0498] First, following 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. This vector was formed by combining cDNA (sequence number 26) encoding the N-terminal amino acid domain of mouse-derived Tim4 protein from amino acid position 1 to 273 into the SalI-EcoRV site of the pEF-Fc vector.

[0499] On the other hand, 293T cells (Riken BRC) were cultured for 1 day in 25 150mm cell culture dishes, each containing 20mL of DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (manufactured by Biowest). Then, for each culture dish, 25mL of DMEM containing 10% FBS was replaced with 25mL of DMEM without FBS.

[0500] Then, following standard procedures, 20 μg of pEF-Tim4-Fc was introduced into 293T cells using polyethylenimine "MAX" (manufactured by Poly Technologies Inc.). After gene introduction, the gene-introduced 293T cells were cultured for 4 days at 37°C and 5% CO2.

[0501] <(2) Purification>

[0502] The culture medium of the gene-introduced 293T cells was centrifuged (800×g, 5 min), and the culture supernatant was collected and summarized. The culture supernatant was filtered using an RF (Rapid Flow) 0.2μm filter unit (Thermo Fisher Scientific Co., Ltd.) to separate impurities and obtain the culture supernatant filtrate.

[0503] Then, 500 μL of the obtained culture supernatant filtrate was added to a Poly-Prep column (manufactured by Bio-Rad Laboratories) containing 70 μL of native protein A agarose gel 4FF (nProtein A Sepharose 4 Fast Flow) (manufactured by GE Healthcare Japan, where protein A has an affinity for the Fc tag) washed with 20 mL of PBS. This allowed the Fc tag fused to Tim4 protein in the culture supernatant filtrate to bind to the native protein A agarose gel 4FF. The native protein A agarose gel 4FF was then washed with 20 mL of PBS. Then, each of the five dissolution fractions was dissolved 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 neutralization solution, resulting in five dissolution fractions of 600 μL each.

[0504] For the five obtained dissolution fractions, the absorbance at 280 nm was measured to determine the presence or absence of protein. The protein-containing fractions were mixed together and concentrated using an Amicon Ultra-0.5 mL 10K centrifugal filter column (trade name and model) (Millipore Corporation). The solvent was then replaced with 40 μL of PBS using the same column. The amount of protein in the PBS-replaced dissolution solution was then quantified using the bicinchoninic acid (BCA) method. The protein concentration was adjusted to 88 μg / mL using PBS to obtain a PBS solution containing Fc-tagged fused mouse Tim4 protein (sometimes abbreviated as "Fc-tagged fused mTim4 protein").

[0505] Example 2. Preparation of FLAG-tagged Tim4 protein

[0506] The following method was used to prepare the FLAG-tagged Tim4 protein.

[0507] <(1) Construction and cultivation of the carrier>

[0508] First, following standard procedures, the FLAG tag-fused mouse Tim4 cDNA (a cDNA encoding an amino acid sequence formed by fusing 1×FLAG to the C-terminus of the N-terminal domain of mouse Tim4 protein, sequence number 33 (with a stop codon (taa) and a base sequence encoding 1×FLAG, but without restriction enzyme sites), manufactured by FASMAC) was combined into the XhoI / BamHI site of the pCAG-Neo vector (manufactured by Wako Pure Chemical Industries, Ltd.) to construct a FLAG tag-fused Tim4 protein expression vector (hereinafter sometimes abbreviated as "pCAG-Tim4-FLAG").

[0509] On the other hand, at 225cm 2In a flask, 50 mL of DMEM (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% FBS (manufactured by Biosera) was used to seed 293T cells (Riken BRC) at 70-90% confluence during gene introduction, and the cells were cultured for 1 day. Then, following standard procedures, 60 μg of pCAG-Tim4-FLAG was introduced into the 1-day-old 293T cells using Lipofectamine 2000 (trade name, manufactured by Thermo Fisher Scientific). After gene introduction, the gene-introduced 293T cells were cultured for 1 day at 37°C and 5% CO2. Then, remove all culture medium, wash the gene-introduced 293T cells twice with 10 mL of PBS, replace the culture medium with 50 mL of Opti-MEM (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.), and culture at 37°C and 5% CO2 for 3 days.

[0510] <(2) Purification>

[0511] The culture medium of gene-introduced 293T cells cultured for 3 days was centrifuged (300×g, 5 min), and the culture supernatant was recovered. The recovered culture supernatant was then centrifuged three times (first time: 300×g, 3 min; second time: 1200×g, 20 min; third time: 10000×g, 20 min) to obtain a supernatant free of impurities. The obtained supernatant was ultrafiltered using Vivaspin (trade name, molecular weight cutoff 30000, manufactured by General Electric Company (GE)) and concentrated 10-fold to obtain a concentrated culture supernatant. Add 500 μL (1 / 10 the volume of the culture supernatant concentrate) of ANTI-FLAG M2 affinity gel (manufactured by Sigma Corporation) washed with PBS to 5 mL of the obtained culture supernatant concentrate. Mix inverted for 3 hours to allow the FLAG tag fusion Tim4 protein in the culture supernatant concentrate to react with the anti-FLAG M2 affinity gel, thus binding the FLAG tag fusion Tim4 protein to the anti-FLAG M2 affinity gel. Then, wash the anti-FLAG M2 affinity gel three times with 5 μL of PBS.

[0512] Then, 250 μL of a 200 μg / mL FLAG peptide solution (a solution prepared by diluting DYKDDDDK peptide (manufactured by Wako Pure Chemical Industries, Ltd.) with PBS) (half the amount of the anti-FLAG M2 affinity gel used) was added to the anti-FLAG M2 affinity gel. The mixture was inverted and mixed at 4°C for 30 minutes, followed by centrifugation (4°C, 8000×g, 1 min). The supernatant (dissolution) was then recovered. To remove the FLAG peptide from the recovered supernatant (dissolution), 20 mL of PBS was added to the supernatant (dissolution) and ultrafiltration was performed to obtain a concentrate. PBS was added to the concentrate to adjust the volume to 500 μL, yielding a PBS solution containing FLAG-tagged fused mouse Tim4 protein (sometimes abbreviated as "FLAG-tagged fused mTim4 protein") (sometimes abbreviated as "PBS solution containing FLAG-tagged fused mTim4 protein") (Experimental Example 2).

[0513] Example 3. Preparation of His-tagged Tim4 protein

[0514] The His-tagged Tim4 protein was prepared using the following method.

[0515] <(1) Construction / Cultivation of Vectors>

[0516] Following conventional methods, the His-tagged mouse Tim4 cDNA (cDNA encoding an amino acid sequence formed by fusing a 6×His tag to the C-terminus of the N-terminal domain of mouse Tim4 protein from amino acids 1 to 273, and sequence number 34 (with a stop codon (tga) at the end and cDNA encoding the 6×His tag)) was combined into the XhoI / BamHI site of the pCAG-Neo vector (manufactured by Wako Pure Chemical Industries, Ltd.) to construct a His-tagged Tim4 protein expression vector (hereinafter sometimes abbreviated as "pCAG-Tim4-His").

[0517] On the other hand, at 225cm 2In a flask, 293T cells (Riken BRC) were cultured for 1 day in 50 mL of DMEM (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% FBS (manufactured by Biosera). Then, following standard procedures, 60 μg of pCAG-Tim4-His gene was introduced into the 293T cells using Lipfectamine 2000 (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.). After gene introduction, the 293T cells were cultured for 1 day at 37°C and 5% CO2. Then, remove all culture medium, wash the gene-introduced 293T cells twice with 10 mL of PBS, replace the culture medium with 50 mL of Opti-MEM (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.), and culture at 37°C and 5% CO2 for 3 days.

[0518] <(2) Purification>

[0519] The culture medium of gene-introduced 293T cells cultured for 3 days was centrifuged (300×g, 5 min), and the culture supernatant was recovered. The recovered culture supernatant was then centrifuged three times (first time: 300×g, 3 min; second time: 1200×g, 20 min; third time: 10000×g, 20 min) to obtain a supernatant free of impurities. The obtained supernatant was ultrafiltered using Vivaspin (trade name, molecular weight cutoff 30000, manufactured by General Electric Company (GE)) and concentrated 10-fold to obtain a concentrated culture supernatant.

[0520] Prepare 2.7 mL of Ni-chelated agarose gel 6FF (manufactured by General Electric Company) according to the protocol attached to Ni Sepharose 6Fast Flow gel, and transfer it to the column. While measuring the OD at 280 nm at a flow rate of approximately 1 mL / min, add binding buffer (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole) to the column until the absorbance at OD 280 nm stabilizes. Add 5 mL of the resulting concentrated culture supernatant to the column, and then add binding buffer until the absorbance stabilizes, followed by washing. Then, add dissolution buffer (50 mM Tris-HCl, 500 mM NaCl, 300 mM imidazole) to the column, and recover 1 mL of each of the 10 dissolution fractions.

[0521] <(3) Electrophoresis / Silver Staining>

[0522] Take 15 μL of each dissolved fraction and mix it with 5 μL of 4× sample buffer (manufactured by Wako Pure Chemical Industries, Ltd.). Heat at 98°C for 5 minutes to obtain 20 μL of each sample for electrophoresis. Add 15 μL of each sample to a SuperSep Ace 5-20% gel (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) and perform electrophoresis at 25 mA for 65 minutes.

[0523] The gel was stained using the Wako Silver Staining II Kit (manufactured by Wako Pure Chemical Industries, Ltd.). Five fractions containing the target protein were selected and pooled together. To remove imidazole from the eluent, 20 mL of PBS was added, and the solution was ultrafiltered using Vivaspin (trade name, molecular weight cutoff 30,000, manufactured by General Electric Company) to obtain a concentrate. PBS was added to the concentrate to adjust the volume to 500 μL, yielding a PBS solution containing His-tagged fusion mouse Tim4 protein (sometimes abbreviated as "His-tagged fusion mTim4 protein") (sometimes abbreviated as "PBS solution containing His-tagged fusion mTim4 protein").

[0524] Examples 1-8. Obtaining extracellular membrane vesicles using the Tim4 vector of the present invention (method of obtaining vesicles of the present invention)

[0525] The Tim4 vector of the present invention was prepared as described below, and the extracellular membrane vesicles of the present invention were obtained using the vector.

[0526] <(1) Preparation of culture supernatant samples containing calcium ions>

[0527] Using 80 mL of X-VIVO 15 medium (trade name, manufactured by Lonza), at 37°C and 5% CO2, 1 × 10⁶ human chronic malignant leukemia cell line K562, which secretes extracellular membrane vesicles, were cultured. 7 After culturing the cells for 3 days, centrifugation (300×g, 5 minutes) was performed to precipitate the cells and remove the supernatant. The precipitated cells were then resuspended in 60 mL of X-VIVO15 medium containing 10 μM monensin sodium (manufactured by MP Biomedicals Co., Ltd.) and cultured at 37°C and 5% CO2 for 24 hours.

[0528] The culture medium was then centrifuged (300×g, 5 min), and the culture supernatant was recovered. The recovered 60 mL of culture supernatant was further centrifuged three times (first time: 300×g, 3 min; second time: 1200×g, 20 min; third time: 10000×g, 20 min) to separate impurities and obtain the supernatant. The obtained supernatant was ultrafiltered using Vivaspin (trade name, molecular weight cutoff 30000, manufactured by General Electric Company) until it was reduced to less than 6 mL, obtaining a concentrated solution. X-VIVO15 medium containing 10 μM monensin sodium (manufactured by MP Biomedicals) was added to the concentrated solution, and the volume was adjusted to 6 mL to obtain a 10-fold concentrated K562 cell culture concentrate sample (hereinafter sometimes abbreviated as "culture supernatant sample").

[0529] In addition, CaCl2 was added to the obtained culture supernatant sample to make the final concentration 2mM, resulting in a K562 cell culture supernatant concentrate sample containing 2mM CaCl2 (hereinafter, sometimes referred to as "culture supernatant sample containing calcium ions").

[0530] <(2) Biotinylation of Fc-tagged fused mouse-derived Tim4 protein>

[0531] For 114 μL of PBS solution containing Fc-tagged fused mTim4 protein (containing 10 μg of Fc-tagged fused mTim4 protein) prepared by the same method as in Example 1, the amino group of Fc-tagged fused mTim4 protein was biotinylated using a biotinylated labeling kit - amino (manufactured by Dojin Chemical Research Institute) according to the protocol included in the kit, resulting in 100 μL of PBS solution containing 3.8 μg of amino-biotinylated Fc-tagged fused mouse Tim4 protein (sometimes abbreviated as "amino-biotinylated Fc-tagged fused mTim4 protein") (sometimes abbreviated as "PBS solution containing amino-biotinylated Fc-tagged fused mTim4 protein").

[0532] Additionally, for 114 μL of PBS solution containing Fc-tagged fused mTim4 protein (containing 10 μg of Fc-tagged fused mouse Tim4 protein) prepared by the same method as in Example 1, the thiol group of the Fc-tagged fused mouse Tim4 protein was biotinylated using a biotinylation kit - thiol (manufactured by Dojin Chemical Research Institute) according to the protocol included in the kit, resulting in 100 μL of PBS solution containing 5.9 μg of thiol-biotinylated Fc-tagged fused mouse Tim4 protein (sometimes abbreviated as "thiol-biotinylated Fc-tagged fused mTim4 protein") (sometimes abbreviated as "PBS solution containing thiol-biotinylated Fc-tagged fused mTim4 protein").

[0533] <(3) Dilution of PBS solution containing Fc-tagged fused mouse-derived Tim4 protein>

[0534] 11.4 μL of PBS solution containing Fc-tagged fused mTim4 protein, prepared using the same method as in Example 1, was mixed with 188.6 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of biotin-unlabeled Fc-tagged fused mTim4 protein.

[0535] 26.3 μL of the PBS solution containing the aminobiotin-labeled Fc tag fused to mTim4 protein (containing 1 μg of aminobiotin-labeled Fc tag fused to 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 tag fused to mTim4 protein.

[0536] In addition, 16.9 μL of the PBS solution containing the Fc tag fused with thiol biotin (containing 1 μg of the Fc tag fused with thiol biotin) prepared above was mixed with 183.1 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of the Fc tag fused with thiol biotin.

[0537] <(4) Washing the beads>

[0538] 20 μL of PBS-T solution (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) containing 30 μg / μL Dynabeads G protein (containing 0.6 mg of Dynabeads G protein) was dispensed into three 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.). Then, 500 μL of PBS was added to each of the 1.5 mL tubes, and after stirring, the 1.5 mL tubes were placed on a magnetic rack to use magnetism to aggregate the Dynabeads G protein onto the tube walls. The solution in the 1.5 mL tubes was then discarded using a pipette (hereinafter sometimes referred to as the "washing operation").

[0539] Additionally, 60 μL of PBS solution containing 10 μg / μL Dynabeads M-270 streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) was dispensed into 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.), and washed with 500 μL of PBS in the same manner as described above.

[0540] <(5) Fc tag fusion mouse-derived Tim4 protein immobilized on beads>

[0541] In one of the three 1.5 mL tubes containing Dynabeads G protein (0.6 mg), 200 μL of the PBS solution containing 1 μg of biotin-unlabeled Fc-tagged fused mTim4 protein prepared above was added. The mixture was reacted at 8 °C for 1 hour to obtain 200 μL of PBS solution containing a vector (mTim4 vector) bound to biotin-unlabeled Fc-tagged fused mTim4 protein.

[0542] In the remaining two 1.5 mL tubes containing Dynabeads G protein (0.6 mg), add 200 μL of the total amount of PBS solution containing 1 μg of aminobiotin-labeled Fc tag fused mTim4 protein prepared above to one of them. React at 8 °C for 1 hour to obtain 200 μL of PBS solution containing the vector (mTim4 vector) bound to aminobiotin-labeled Fc tag fused mTim4 protein.

[0543] In another sample, add 200 μL of the PBS solution containing 1 μg of the biotin-labeled Fc tag fused to mTim4 protein prepared above, and react at 8°C for 1 hour to obtain 200 μL of PBS solution containing the carrier (mTim4 carrier) bound to the biotin-labeled Fc tag fused to mTim4 protein.

[0544] For 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 fused with thiol biotin and prepared using the same method as described above. Incubate the reaction at 8 °C for 1 hour to obtain 200 μL of PBS solution containing a carrier (mTim4 carrier) bound to Fc-tagged mTim4 protein fused with thiol biotin.

[0545] Thus, 200 μL of PBS solutions containing 0.6 mg of each of the four mTim4 carriers shown in Table 1 were obtained.

[0546] Table 1

[0547]

[0548] <(6) Obtaining extracellular membrane vesicles using the method of the present invention>

[0549] The four mTim4 carriers listed in Table 1 were washed three times with 500 μL of PBS. Then, 200 μL of the culture supernatant containing calcium ions prepared in (1) was added to each mTim4 carrier in particulate state, and the mixture was reacted at 8 °C for 3 hours.

[0550] The reacted mTim4 vector was washed three times with 500 μL of TBS-T (Tris Buffer, 0.05% Tween 20, 2 mM CaCl2) containing 2 mM CaCl2. During the third wash, 250 μL of each of the four mTim4 vectors was aliquoted into two 1.5 mL tubes.

[0551] To each of the four mTim4 carriers in particulate form, add 20 μL of 1% SDS aqueous solution or 1 mMEDTA aqueous solution as a dissolution solution. Then, mix using a vortex mixer at room temperature for 10 seconds, and then stop spinning (spin down). Place 1.5 mL tubes on a magnetic rack and use magnetism to collect the mTim4 carriers on the tube wall. Collect the supernatant (dissolution solution).

[0552] It should be noted that the types of mTim4 protein and carrier used in each embodiment, the types of lysis solutions used to obtain extracellular membrane vesicles from the mTim4 carrier, and the lane numbers in the protein immunoblotting described later are shown in Table 2 below.

[0553] Table 2

[0554]

[0555] <(7) Western blotting of proteins>

[0556] To 7.5 μL of each supernatant (dissolution solution) obtained in Examples 1-8, add 2.5 μL of 4× sample buffer (manufactured by Wako Pure Chemical Industries, Ltd.), and heat at 98°C for 5 minutes to obtain each protein immunoblot sample. Add 10 μL of each protein immunoblot sample to a SuperSep Ace 5-20% gel (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), and perform electrophoresis at 25 mA for 65 minutes. Using a semi-dry blot 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... 2 Under the specified conditions, the obtained electrophoretic gel was transferred to a PVDF membrane (Millipore) for 60 minutes.

[0557] Add 3% skim milk diluted with PBS-T (PBS buffer, 0.1% Tween 20) to the PVDF membrane and react for 1 hour at room temperature to block it. Then, add 2 mL of anti-human Lamp-1 mouse monoclonal antibody (manufactured by BD Biosciences Co., Ltd., sometimes abbreviated as "anti-human Lamp-1 antibody") diluted 250 times with PBS-T and react overnight at 8°C.

[0558] Then, the PVDF membrane was washed three times with PBS-T. At room temperature, a secondary antibody (anti-mouse IgG (H+L), rabbit, IgG fraction, and peroxidase-conjugating antibody) diluted 10,000 times with PBS-T (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted for 1 hour. After washing five times with PBS-T, ImmunoStar Zeta (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the luminescence signal was detected using a LAS-4000 (manufactured by General Electric Company). It should be noted that the anti-human Lamp-1 antibody is an antibody targeting Lamp-1, one of the marker proteins of exosomes.

[0559] <Results>

[0560] The results of the obtained protein immunoblotting are shown below. Figure 1 . Figure 1 The lanes are as follows.

[0561] Lane 1: Results of Example 1 (using an mTim4 carrier formed by fusing a biotin-unlabeled Fc tag to a G protein bead, and using a 1% SDS aqueous solution as the dissolution medium);

[0562] Lane 2: Results of Example 2 (using an mTim4 carrier formed by fusing a biotin-unlabeled Fc tag with mTim4 protein bound to a G protein bead, and using 1 mM EDTA solution as the dissolution medium);

[0563] Lane 3: Results of Example 3 (using an mTim4 carrier formed by fusing an aminobiotin-labeled Fc tag with mTim4 protein bound to G protein beads, and using a 1% SDS aqueous solution as the dissolution solution);

[0564] Lane 4: Results of Example 4 (using an mTim4 carrier formed by fusing an aminobiotin-labeled Fc tag with mTim4 protein bound to a G protein bead, and using 1 mM EDTA solution as the dissolution medium);

[0565] Lane 5: Results of Example 5 (using an mTim4 carrier formed by fusing an Fc tag labeled with thiol biotin to an mTim4 protein bound to a G protein bead, and using a 1% SDS aqueous solution as the dissolution solution);

[0566] Lane 6: Results of Example 6 (using an mTim4 carrier formed by fusing an Fc tag labeled with thiol biotin to an mTim4 protein bound to a G protein bead, and using 1 mM EDTA solution as the dissolution medium);

[0567] Lane 7: Results of Example 7 (using an mTim4 carrier formed by fusing an Fc tag labeled with thiol biotin to a streptavidin bead, and using a 1% SDS aqueous solution as the dissolution medium);

[0568] Lane 8: Results of Example 8 (using an mTim4 vector formed by fusing mouse-derived Tim4 protein with a thiol-biotin-tagged Fc tag and binding it to streptavidin beads, with 1 mM EDTA solution used as the dissolution medium).

[0569] according to Figure 1 In any of the cases in Examples 1-8, a band of Lamp-1, which serves as an exosome marker, was obtained near 100 kDa. This demonstrates that the method of the present invention can obtain extracellular membrane vesicles containing exosomes (Examples 1-8: lanes 1-8).

[0570] Specifically, by comparing Examples 2, 4, 6, and 8, it can be seen that when a calcium ion chelating agent is used as a dissolution solution, compared to the case where Tim4 protein binds to the carrier through amino groups (Example 4: lane 4) and the case where it binds through affinity tags (Example 2: lane 2; Example 6: lane 6), more extracellular membrane vesicles can be obtained when Tim4 protein binds to the carrier through the sulfhydryl groups of Tim4 protein (Example 8: lane 8).

[0571] Examples 9-16. Obtaining extracellular membrane vesicles using the Tim4 vector of the present invention (method of obtaining vesicles of the present invention)

[0572] The Tim4 vector of the present invention was prepared as shown below, and the extracellular membrane vesicles of the present invention were obtained using the vector.

[0573] <(1) Preparation of culture supernatant>

[0574] The preparation of culture supernatant samples was carried out using the same method as in Examples 1-8, "(1) Preparation of culture supernatant samples".

[0575] <(2) Biotinylation of Fc-tagged fused mouse-derived Tim4 protein>

[0576] For 114 μL of PBS solution containing Fc-tagged fused mTim4 protein (containing 10 μg of Fc-tagged fused mTim4 protein) prepared by the same method as in Experimental Example 1 "(2) Biotin labeling of Fc-tagged fused mTim4 protein", the thiol group of Fc-tagged fused mTim4 protein was biotin-labeled using the same method as in Example 1, resulting in 100 μL of PBS solution containing 3.9 μg of thiol-biotin-labeled Fc-tagged fused mTim4 protein (hereinafter, sometimes abbreviated as "PBS solution containing thiol-biotin-labeled Fc-tagged fused mTim4 protein").

[0577] <(3) Biotinylation of FLAG-tagged fused mouse-derived Tim4 protein>

[0578] Using 99 μL of PBS solution containing FLAG-tagged fused mTim4 protein (containing 10 μg of FLAG-tagged fused mTim4 protein) prepared by the same method as in Example 2, the thiol group of the FLAG-tagged fused mTim4 protein was biotinylated using a biotinylation kit for thiol (manufactured by Dojin Chemical Research Institute Co., Ltd.) according to the instructions included in the kit, resulting in 100 μL of PBS solution containing 4.6 μg of thiol-biotinylated FLAG-tagged fused mTim4 protein (hereinafter, sometimes abbreviated as "PBS solution containing thiol-biotinylated FLAG-tagged fused mTim4 protein").

[0579] <(4) Biotinylation of His-tagged fused mouse-derived Tim4 protein>

[0580] For 54 μL of PBS solution containing His-tagged fused mTim4 protein (containing 10 μg of His-tagged fused mTim4 protein) prepared by the same method as in Experimental Example 3, the thiol group of His-tagged fused mTim4 protein was biotinylated using a biotinylation kit - thiol (manufactured by Dojin Chemical Research Institute Co., Ltd.) according to the protocol included in the kit, resulting in 100 μL of PBS solution containing 7.2 μg of thiol-biotin-labeled His-tagged fused mTim4 protein (hereinafter, sometimes abbreviated as "PBS solution containing thiol-biotin-labeled His-tagged fused mTim4 protein")

[0581] <(5) Dilution of tag-fusion mouse-derived Tim4 protein>

[0582] 11.4 μL of PBS solution containing Fc-tagged fused mTim4 protein (containing 1 μg of Fc-tagged fused mouse Tim4 protein) prepared by the same method as in Experimental Example 1 was mixed with 188.6 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of biotin-unlabeled Fc-tagged fused mouse Tim4 protein.

[0583] 16.9 μL of the PBS solution containing 1 μg of the thiol-biotin-labeled Fc tag fused mTim4 protein prepared in (2) above was mixed with 183.1 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of the thiol-biotin-labeled Fc tag fused mTim4 protein.

[0584] In addition, 21.6 μL of PBS solution containing 1 μg of mTim4 protein fused with FLAG tag with thiol biotin label prepared in (3) was mixed with 178.4 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of mTim4 protein fused with FLAG tag with thiol biotin label.

[0585] 13.9 μL of the PBS solution containing 1 μg of the biotin-labeled His-tagged fused mTim4 protein prepared in (4) was mixed with 186.1 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of the biotin-labeled His-tagged fused mTim4 protein.

[0586] <(6) Washing the beads>

[0587] 20 μL of PBS-T solution containing 30 μg / μL Dynabeads G protein (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) (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 “(4) washing of beads” in Examples 1-8.

[0588] In addition, 60 μL of PBS solution containing 10 μg / μL of Dynabeads M-270 streptavidin (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) (containing 0.6 mg of Dynabeads M-270 streptavidin) 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 “(4) washing of beads” in Examples 1-8.

[0589] <(7) Tag-fused mouse-derived Tim4 protein immobilized on beads>

[0590] Then, in a 1.5 mL tube containing 0.6 mg of washed Dynabeads G protein in particulate form, 200 μL of PBS solution containing 1 μg of biotin-unlabeled Fc-tagged fused mTim4 protein was added, 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 fused mTim4 protein.

[0591] Further, in one of three 1.5 mL tubes containing 0.6 mg of washed Dynabeads M-270 streptavidin in particulate form, 200 μL of the above-prepared PBS solution containing 1 μg of the Fc-tagged mTim4 protein with thiol biotin label was added, and the reaction was carried out at 8 °C for 1 hour to obtain 200 μL of PBS solution containing the carrier (mTim4 carrier) bound to the Fc-tagged mTim4 protein with thiol biotin label.

[0592] In the remaining two 1.5 mL tubes containing 0.6 mg of washed Dynabeads M-270 streptavidin in particulate form, 200 μL of the PBS solution containing 1 μg of the FLAG tag fused with thiol-biotin-labeled mTim4 protein prepared above was added to one of them. The reaction was carried out at 8 °C for 1 hour to obtain 200 μL of PBS solution containing the carrier (mTim4 carrier) bound to the FLAG tag fused with thiol-biotin-labeled mTim4 protein.

[0593] In another sample, 200 μL of the PBS solution containing 1 μg of the biotin-labeled His-tagged fused mTim4 protein prepared above was added, and the reaction was carried out at 8°C for 1 hour to obtain 200 μL of PBS solution containing the vector (mTim4 vector) bound to the biotin-labeled His-tagged fused mTim4 protein.

[0594] Thus, 200 μL of PBS solutions containing 0.6 mg of each of the four mTim4 carriers shown in Table 3 were obtained.

[0595] Table 3

[0596]

[0597] <(8) Obtaining extracellular membrane vesicles using the method of the present invention>

[0598] The four mTim4 carriers described in Table 3 (0.6 mg) were used instead of the four mTim4 carriers described in Table 1. Otherwise, the same method as in Examples 1-8 (6) Obtaining extracellular membrane vesicles using the method of the present invention was employed to obtain the supernatants (dissolution solutions). It should be noted that the types of mTim4 proteins and carriers used in each example, the types of dissolution solutions used to obtain extracellular membrane vesicles from the mTim4 carriers, and the lane numbers in the protein immunoblotting described later are shown in Table 4 below.

[0599] Table 4

[0600]

[0601] <(7) Western blotting of proteins>

[0602] Use “7.5 μL of each supernatant (dissolution solution) obtained in Examples 9-16 (8)” instead of “7.5 μL of each supernatant (dissolution solution) obtained in Examples 1-8”, otherwise use the same method as “(7) protein immunoblotting” in Examples 1-8.

[0603] <Results>

[0604] The results of the obtained protein immunoblotting are shown below. Figure 2 middle. Figure 2 The lanes are as follows.

[0605] They are shown respectively:

[0606] Lane 1: Results of Example 9 (using an mTim4 carrier formed by fusing a biotin-unlabeled Fc tag to a G protein bead, and using a 1% SDS aqueous solution as the dissolution medium);

[0607] Lane 2: Results of Example 10 (using an mTim4 carrier formed by fusing a biotin-unlabeled Fc tag with mTim4 protein bound to a G protein bead, and using 1 mM EDTA solution as the dissolution medium);

[0608] Lane 3: Results of Example 11 (using an mTim4 carrier formed by fusing an Fc tag labeled with thiol biotin to a streptavidin bead and using a 1% SDS aqueous solution as the dissolution medium);

[0609] Lane 4: Results of Example 12 (using an mTim4 carrier formed by fusing an Fc tag labeled with thiol biotin to a streptavidin bead, and using 1 mM EDTA solution as the dissolution medium);

[0610] Lane 5: Results of Example 13 (using an mTim4 carrier formed by fusing mTim4 protein with streptavidin beads using a FLAG tag labeled with thiol biotin and using 1% SDS aqueous solution as the dissolution solution);

[0611] Lane 6: Results of Example 14 (using an mTim4 carrier formed by fusing mTim4 protein with a FLAG tag labeled with thiol biotin to streptavidin beads, and using 1 mM EDTA solution as the dissolution medium);

[0612] Lane 7: Results of Example 15 (using an mTim4 carrier formed by fusing mTim4 protein with a His tag labeled with thiol biotin to streptavidin beads, and using 1% SDS aqueous solution as the dissolution solution);

[0613] Lane 8: Results of Example 16 (using an mTim4 carrier formed by fusing a His tag labeled with thiol biotin to a streptavidin bead, and using 1 mM EDTA solution as the dissolution medium).

[0614] according to Figure 2 It can be seen that in any of the cases in Examples 9-16, a band of Lamp-1, a marker protein of exosomes, was obtained near 100 kDa. Therefore, the method of the present invention can be used to obtain extracellular membrane vesicles containing exosomes (Examples 9-16: lanes 1-8).

[0615] It is also known that if the Tim4 vector of the present invention is used, extracellular membrane vesicles can be obtained regardless of the type, length, or presence of the tag (Examples 9-16: lanes 1-8).

[0616] Comparing Examples 10, 12, 14, and 16, it can be seen that when EDTA, as a calcium ion chelating agent, is used as the dissolution solution, more extracellular membrane vesicles can be obtained when Tim4 protein binds to the carrier through the sulfhydryl group of Tim4 protein (Example 12: Lane 4; Example 14: Lane 6; Example 16: Lane 8) compared to when Tim4 protein binds to the carrier through an affinity tag (Example 10: Lane 2).

[0617] Examples 17-18. Obtaining extracellular membrane vesicles using the Tim4 vector of the present invention (method of obtaining vesicles of the present invention)

[0618] The Tim4 vector of the present invention was prepared as described below, and the extracellular membrane vesicles of the present invention were obtained using the vector.

[0619] <(1) Preparation of culture supernatant>

[0620] The preparation of culture supernatant samples was carried out using the same method as in Examples 1-8, "(1) Preparation of culture supernatant samples".

[0621] <(2) Dilution of biotin-unlabeled FLAG-tagged fusion mouse-derived Tim4 protein>

[0622] 47 μL of PBS solution containing 5 μg of biotin-unlabeled FLAG-tagged fused mTim4 protein (PBS solution containing 5 μg of biotin-unlabeled FLAG-tagged fused mouse-derived Tim4 protein) prepared by the same method as in Experimental Example 2 was mixed with 153 μL of PBS to obtain 200 μL of PBS solution containing 5 μg of biotin-unlabeled FLAG-tagged fused mTim4 protein.

[0623] <(3) Washing the beads>

[0624] 50 μL of 50% glycerol TBS solution (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10 μg / μL anti-DYKDDDDK tag antibody magnetic beads (containing 0.5 mg of anti-DYKDDDDK tag antibody magnetic beads) was dispensed into 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.) and washed with 500 μL of PBS.

[0625] <(4) FLAG tag fusion of mouse-derived Tim4 protein immobilized on vector>

[0626] Then, 200 μL of PBS solution containing 5 μg of FLAG tag fused mTim4 protein was added to the anti-DYKDDDDK tag-labeled magnetic beads after washing, and the reaction was carried out at 8°C for 1 hour to obtain 200 μL of PBS solution containing mTim4 vector.

[0627] As described above, 200 μL of PBS solution containing 0.5 mg of the mTim4 carrier shown in Table 5 was obtained.

[0628] Table 5

[0629]

[0630] <(5) Obtaining extracellular membrane vesicles using the method of the present invention>

[0631] The “0.5 mg of mTim4 carrier described in Table 5” was used instead of “0.6 mg of the four types of mTim4 carriers described in Table 1”. Otherwise, the same method as “(6) Obtaining extracellular membrane vesicles using the method of the present invention” in Examples 1-8 was used to obtain each supernatant (dissolution).

[0632] It should be noted that the types of mTim4 protein and carrier used in each embodiment, the types of lysis solutions used to obtain extracellular membrane vesicles from the mTim4 carrier, and the lane numbers in the protein immunoblotting described later are shown in Table 6 below.

[0633] Table 6

[0634]

[0635] <(6) Western blotting of proteins>

[0636] Use “7.5 μL of each supernatant (dissolution solution) obtained in Examples 1-8” instead of “7.5 μL of each supernatant (dissolution solution) obtained in Examples 1-8”, otherwise perform the same method as “(7) protein immunoblotting” in Examples 1-8.

[0637] <Results>

[0638] The results of the obtained protein immunoblotting are shown below. Figure 3 . Figure 3 The lanes are as follows.

[0639] Lane 1: Results of Example 17 (using an mTim4 vector formed by fusing a biotin-unlabeled FLAG tag with mTim4 protein bound to anti-DYKDDDDK tag antibody beads, and using 1% SDS aqueous solution as the dissolution medium);

[0640] Lane 2: Results of Example 18 (using an mTim4 vector formed by fusing a biotin-unlabeled FLAG tag with mTim4 protein bound to anti-DYKDDDDK tag antibody beads, and using 1 mM EDTA solution as the dissolution medium).

[0641] according to Figure 3 As can be seen, in any of the cases in Examples 17-18, a band of Lamp-1 as an exosome marker was observed near 100 kDa. Therefore, by means of the present invention, even when using a carrier in which Tim4 protein is immobilized by an anti-tag antibody, extracellular membrane vesicles containing exosomes can be obtained (Examples 17-18: lanes 1-2).

[0642] Examples 19-20 and Comparative Examples 1-3: Comparison of the purity of extracellular membrane vesicles obtained by the method of the present invention and by existing methods.

[0643] The purity of extracellular membrane vesicles obtained by the present invention obtained by dissolution using SDS (Example 19), dissolution using EDTA (Example 20), ultracentrifugation (Comparative Example 1), Exo Quick (Comparative Example 2), and total exosome isolation (Comparative Example 3) were compared below.

[0644] <Obtaining extracellular membrane vesicles using the method of the present invention (Examples 19-20)>

[0645] "500 μL of culture supernatant containing calcium ions" was used instead of "200 μL of culture supernatant containing calcium ions". Furthermore, as the dissolution solution, "50 μL of 1% SDS aqueous solution" was used instead of "20 μL of 1% SDS aqueous solution", and "50 μL of 1 mM EDTA aqueous solution" was used instead of "20 μL of 1 mM EDTA aqueous solution". Otherwise, the same method as in Examples 13-14 was used to react the mTim4 protein with the extracellular membrane vesicles in the culture supernatant containing calcium ions, and the reaction was carried out using 1% SDS aqueous solution and 1 mM EDTA aqueous solution as dissolution solutions, respectively. EDTA aqueous solution was used to dissolve extracellular membrane vesicles, and supernatants (dissolution solutions) were obtained. The mTim4 protein was formed by fusing a thiol-biotin-labeled FLAG tag with the mTim4 protein and binding it to Dynabeads M-270 streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.).

[0646] The obtained supernatant (dissolution solution) was used as sample 1 (dissolution with 1% SDS aqueous solution) and sample 2 (dissolution with 1mM EDTA aqueous solution), respectively.

[0647] It should be noted that the types of mTim4 protein and carrier used in Examples 19-20, as well as the types of lysis solutions used to obtain extracellular membrane vesicles from the mTim4 carrier, are shown in Table 7 below.

[0648] Table 7

[0649]

[0650] <Abstract extracellular membrane vesicles obtained by ultracentrifugation (Comparative Example 1)>

[0651] One mL of the culture supernatant sample prepared using the same method as in Example 1 was centrifuged (20000×g, 30 min) to separate impurities and obtain the supernatant. Then, one mL of the obtained supernatant was ultracentrifuged (110000×g, 70 min) to obtain the precipitate. The precipitate was then resuspended in 1 mL of PBS. The suspension of the precipitate was ultracentrifuged again (110000×g, 70 min), and the resulting precipitate was resuspended in 50 μL of PBS. This suspension was used as Sample 3 (Comparative Example 1).

[0652] <Extracellular membrane vesicles were obtained by centrifugation using commercially available reagents (using ExoQuick to obtain extracellular membrane vesicles) (Comparative Example 2)>

[0653] One mL of the culture supernatant sample prepared using the same method as in Example 1 was centrifuged (20000×g, 30 min) to separate impurities and obtain the supernatant. Then, one mL of the obtained supernatant was mixed with 0.2 mL of ExoQuick-TC Reagent (trade name, System Biosciences Co., Ltd.) and allowed to stand overnight at 8°C. The mixture was then centrifuged (1500×g, 30 min), and the resulting precipitate was resuspended in 100 μL of PBS. This suspension was used as Sample 4 (Comparative Example 2).

[0654] <Extracellular membrane vesicles were obtained by centrifugation using commercially available reagents (comparative example 3) (using total exosome isolation)>

[0655] One mL of K562 cell culture supernatant concentrate prepared using the same method as in Example 1 was centrifuged (20000×g, 30 min) to separate impurities and obtain supernatant. Then, one mL of the obtained supernatant was mixed with 0.5 mL of Total Exosome Isolation Reagent (manufactured by Thermo Fisher Scientific Co., Ltd.) and incubated at 8°C for one day. The precipitate obtained by centrifuging this mixture (10000×g, 1 h) was resuspended in 100 μL of PBS. This suspension was used as Sample 5 (Comparative Example 3).

[0656] It should be noted that the methods used in each embodiment and comparative example, the samples obtained, and the lane numbers in the protein immunoblotting and silver staining described later are shown in Table 8 below.

[0657] Table 8

[0658]

[0659] <Western Immune Blotting of Proteins>

[0660] The protein content in samples 1-5 obtained by each method was determined using the BCA method. Based on the determination results, the protein content was used as the baseline for electrophoresis and Western blotting. Specifically, 37.5 μL of PBS solution containing 0.25 μg of each protein from samples 1-5 was mixed with 12.5 μL of 4× sample buffer (manufactured by Wako Pure Chemical Industries, Ltd.), and then heated at 98°C for 5 minutes to obtain 50 μL of each sample for Western blotting.

[0661] Then, in SuperSep Ace 5-20% gel (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), 20 μL of each protein immunoblot was added to two gels, and electrophoresis was performed at 25 mA for 60 minutes. A semi-dry blot apparatus and discontinuous buffers (anodine buffer 1: 0.3 M Tris / 20% methanol; anodine buffer 2: 0.025 M Tris / 20% methanol; cathode buffer: 0.025 M Tris / 0.04 M aminocaproic acid / 20% methanol) were used, and electrophoresis was performed at 1 mA / cm². 2 Under the specified conditions, one of the two gels was transferred onto a PVDF membrane (Millipore, Inc.) 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 for blocking. Then, 2 mL of anti-human Lamp-1 mouse monoclonal antibody (BD Biosciences, Inc.) diluted 250-fold with PBS-T was added and reacted at room temperature for 1 hour. After washing three times with PBS-T, secondary antibodies {anti-mouse IgG (H+L), rabbit, IgG fraction, and peroxidase-conjugating antibody (Wako Pure Chemical Industries, Ltd.)} diluted 10,000-fold with PBS-T were added and reacted at room temperature for 1 hour. After washing five times with PBS-T, ECL prime (trade name, GE, Inc.) was added, and the luminescence signal was detected using a LAS-4000 (trade name, GE, Inc.).

[0662] In addition, another gel was stained with silver using the Wako Silver Staining II Kit (trade name, manufactured by Wako Pure Chemical Industries, Ltd.).

[0663] <Results>

[0664] The results of the obtained protein immunoblotting are shown in the figure. Figure 4 -A. The results of silver staining are shown in Figure 4 -B. Figure 4 -A and Figure 4 In -B, the lanes are as follows.

[0665] Lane 1: Results of Example 19 (Results when the method of obtaining the present invention was carried out using an mTim4 carrier formed by fusing a FLAG tag labeled with thiol biotin to a streptavidin bead and using a 1% SDS aqueous solution as the dissolution solution);

[0666] Lane 2: Results of Example 20 (Results when the method of obtaining the present invention was carried out using an mTim4 carrier formed by fusing a FLAG tag labeled with thiol biotin to a streptavidin bead and using a 1 mM EDTA solution as the dissolution medium);

[0667] Lane 3: Results of Comparative Example 1 (results when extracellular membrane vesicles were obtained by ultracentrifugation);

[0668] Lane 4: Results compared to Example 2 (results when extracellular membrane vesicles were obtained via ExoQuick);

[0669] Lane 5: Results of Comparative Example 3 (results when extracellular membrane vesicles were obtained by total exosome isolation).

[0670] according to Figure 4 -A, In any of the embodiments of Examples 19-20, which are the methods for obtaining the present invention, a band of Lamp-1, which is an exosome marker, was observed near 100 kDa. It can be seen that the method for obtaining the present invention can obtain extracellular membrane vesicles containing exosomes (Examples 19-20: lanes 1-2).

[0671] On the other hand, according to Figure 4 -A, For any existing method (ultracentrifugation (Comparative Example 1), ExoQuick (Comparative Example 2), and Total Exsome Isolation (Comparative Example 3)), only a faint and trace amount of the exosome marker Lamp-1 was observed near 100 kDa (Comparative Examples 1-3: lanes 3-5).

[0672] In addition, according to Figure 4 -B, The method of obtaining the present invention (Examples 19-20) produces fewer bands from impurities such as proteins compared to any existing method (ultracentrifugation (Comparative Example 1), ExoQuick (Comparative Example 2), and Total Exsome Isolation (Comparative Example 3)). (Examples 19-20: lanes 1-2, Comparative Examples 1-3: lanes 3-5).

[0673] Based on the above results, it can be seen that the method of obtaining the present invention (Examples 19 and 20) can obtain extracellular membrane vesicles (lanes 1-5) with better purity compared with any existing method (Ultracentrifugation (Comparative Example 1), ExoQuick (Comparative Example 2), and Total Exsome Isolation (Comparative Example 3)).

[0674] Example 21. Observation of extracellular membrane vesicles obtained by the method of the present invention using electron microscopy.

[0675] To detect the state of the extracellular membrane vesicles obtained by the method of the present invention, they were observed using an electron microscope.

[0676] <(1) Preparation of mouse macrophage culture supernatant>

[0677] To obtain peritoneal macrophages, 2 mL of 3% mercaptoacetate solution (manufactured by Fruka Reagent Co., Ltd.) was injected into the peritoneal cavity of six 8-week-old female C57BL / 6J mice (purchased from SLC Corporation, Japan). Three days later, the macrophages were recovered from the peritoneal cavity and cultured for 2 days in 80 mL of DMEM (manufactured by Nakara Technologies Co., Ltd.) containing 10% FBS (manufactured by Biowest Corporation). The culture supernatant was then collected to obtain the mouse macrophage culture supernatant sample.

[0678] <(2) Preparation of Tim4 carrier>

[0679] Use 1 mg of Dynabeads MyOne streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) instead of 0.6 mg of Dynabeads. MyOne streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) were used. 100 μL of PBS solution containing 100 μg of mTim4 protein fused with a biotin-labeled Fc tag was used instead of 200 μL of PBS solution containing 1 μg of mTim4 protein fused with a biotin-labeled Fc tag. The reaction time of Dynabeads with the PBS solution containing the biotin-labeled Fc tag fused with mTim4 protein was adjusted to 2 hours instead of 1 hour. Otherwise, the mTim4 carrier formed by binding the biotin-labeled Fc tag fused with mTim4 protein to Dynabeads MyOne streptavidin C1 beads was prepared by the same method as in Examples 11-12.

[0680] <(3) Obtaining extracellular membrane vesicles using the Tim4 vector>

[0681] 32 mL of mouse macrophage culture supernatant was centrifuged (first time: 800 × g, 10 min; second time: 12000 × g, 30 min) to obtain the supernatant. CaCl2 was added to the supernatant to achieve a final concentration of 2 mM, followed by the addition of 1 mg of the prepared mTim4 vector. The mixture was stirred at room temperature for 1 hour. The mTim4 vector, after being stirred at room temperature for 1 hour, was then recovered and washed three times with 5 mL of TBS-T containing 2 mM CaCl2, followed by two more washes with 1 mL of TBS. Finally, the mixture was dissolved three times with 100 μL of TBS containing 1 mM EDTA to obtain 300 μL of the extracellular membrane vesicle fraction.

[0682] Then, to reliably recover extracellular membrane vesicles from the mouse macrophage culture supernatant, the extracellular membrane vesicles were again recovered from the mouse macrophage culture supernatant after three dissolutions with 100 μL of TBS containing 1 mM EDTA using the following method, yielding 300 μL of the extracellular membrane vesicle fraction. Specifically, CaCl2 was added to the mouse macrophage culture supernatant after three dissolutions with 100 μL of TBS containing 1 mM EDTA to achieve a final concentration of 2 mM, followed by the addition of 1 mg of mTim4 carrier after three dissolutions with 100 μL of TBS containing 1 mM EDTA, and the mixture was stirred at room temperature for 1 hour. Then, the mTim4 vector, which had been stirred and mixed at room temperature for 1 hour, was recovered and washed three times with 5 mL of TBS-T containing a final concentration of 2 mM CaCl2, followed by two more washes with 1 mL of TBS, and then dissolved three times with 100 μL of TBS containing 1 mM EDTA to obtain 300 μL of extracellular membrane vesicle fraction.

[0683] Then, in order to more reliably recover the extracellular membrane vesicles from the mouse macrophage culture supernatant, the above operation was performed again to recover the extracellular membrane vesicles from the mouse macrophage culture supernatant after 6 dissolutions with 100 μL of TBS containing 1 mM EDTA, yielding 300 μL of extracellular membrane vesicle fraction.

[0684] Using an Amicon Ultra-0.5mL 10K centrifugal filter column, a total of 900μL of extracellular membrane vesicle components obtained through the above 9 dissolution operations was concentrated to 60μL to obtain a sample for electron microscopy observation.

[0685] <(4) Observation of extracellular membrane vesicles obtained by the method of the present invention using an electron microscope>

[0686] Apply 10 μL of the sample for electron microscopy to the screen, and use filter paper to absorb the remaining sample. Then, wash the screen with water, stain it twice with uranium acetate solution, and observe it using a transmission electron microscope with negative staining.

[0687] It should be noted that the types of mTim4 protein and carrier used in Example 21, as well as the types of lysis solutions used to obtain extracellular membrane vesicles from the mTim4 carrier, are shown in Table 9 below.

[0688] Table 9

[0689]

[0690] <Results>

[0691] The obtained electron microscope images are shown below. Figure 5 .according to Figure 5 The method of the present invention enables the acquisition of extracellular membrane vesicles with a diameter of approximately 50–150 nm while maintaining a spherical shape.

[0692] As can be seen from the above, the method of the present invention enables the acquisition of extracellular membrane vesicles in their intact state.

[0693] Examples 22-25. Obtaining extracellular membrane vesicles using the Tim4 vector of the present invention (method of obtaining vesicles of the present invention)

[0694] Extracellular membrane vesicles of the present invention were obtained using a carrier (hereinafter sometimes referred to as "hTim4 carrier") formed by immobilizing human Tim4 protein on beads and an mTim4 carrier.

[0695] <(1) Preparation of culture supernatant>

[0696] The preparation of culture supernatant samples was carried out using the same method as in Examples 1-8, "(1) Preparation of culture supernatant samples".

[0697] <(2) Dilution of Fc tag-fused Tim4 protein>

[0698] 100 μg of a lyophilized human Tim4 protein (manufactured by Wako Pure Chemical Industries, Ltd., Serial No. 7 (a human Tim4 protein formed by fusing an Fc tag to the N-terminal amino acid domain 25-315 of the human Tim4 protein (Gene Bank: GenBank NP_612388.2)) was dissolved in 1 mL of PBS to prepare a PBS solution containing 100 μg / mL of biotin-unlabeled Fc-tagged hTim4 protein. 10 μL of the PBS solution containing biotin-unlabeled Fc-tagged hTim4 protein was mixed with 190 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of biotin-unlabeled Fc-tagged hTim4 protein.

[0699] In addition, 100 μg of a lyophilized mTim4 protein (made by Wako Pure Chemical Industries, Ltd., Serial No. 6 (amino acid domain at positions 22-279 of the N-terminus of the mouse Tim4 protein (Gene Bank: GenBank NP_848874.3)) fused with an Fc tag was dissolved in 1 mL of PBS to prepare a PBS solution containing 100 μg / mL of biotin-unlabeled Fc-tagged mTim4 protein. 10 μL of the PBS solution containing the biotin-unlabeled Fc-tagged mTim4 protein was mixed with 190 μL of PBS to obtain 200 μL of PBS solution containing 1 μg of biotin-unlabeled Fc-tagged mTim4 protein.

[0700] <(3) Washing the beads>

[0701] 20 μL of PBS-T solution containing 30 μg / μL Dynabeads G protein (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) (containing 0.6 mg of Dynabeads G protein) was dispensed into two 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.) and washed with 500 μL of PBS.

[0702] <(4) Biotin-free Fc tag fusion Tim4 protein immobilized on beads>

[0703] In two 1.5 mL tubes containing Dynabeads G protein (0.6 mg), 200 μL of the above-prepared PBS solution containing 1 μg of biotin-unlabeled Fc-tagged fused hTim4 protein was added to one of the tubes. The reaction was carried out at 8 °C for 1 hour to obtain 200 μL of PBS solution containing a carrier (hTim4 carrier) bound to biotin-unlabeled Fc-tagged fused hTim4 protein.

[0704] In the remaining 1.5 mL tube containing Dynabeads G protein (0.6 mg), add 200 μL of the PBS solution containing 1 μg of biotin-unlabeled Fc-tagged fused mTim4 protein prepared above, and react at 8 °C for 1 hour to obtain 200 μL of PBS solution containing the vector (mTim4 vector) bound to the biotin-unlabeled Fc-tagged fused mTim4 protein.

[0705] <(5) Obtaining extracellular membrane vesicles using the method of the present invention>

[0706] The obtained hTim4 and mTim4 vectors (0.6 mg each) were washed three times with 500 μL of PBS. Then, 200 μL of culture supernatant containing calcium ions was added to each of the particulate hTim4 and mTim4 vectors, and the reactions were carried out at 8°C for 3 hours. Finally, the reacted hTim4 and mTim4 vectors were washed three times with 500 μL of TBS-T containing 2 mM CaCl2.

[0707] During the third wash, 250 μL each of hTim4 and mTim4 carriers (0.3 mg carrier) were dispensed into two 1.5 mL tubes. To each of the 0.3 mg mTim4 and hTim4 carrier particles, 20 μL of either a 1% SDS aqueous solution or a 1 mM EDTA aqueous solution was added as a dissolution solution. The mixture was then vortexed at room temperature for 10 seconds, after which the vortexing was stopped (spin-down). The 1.5 mL tubes were then placed on a magnetic rack, and the hTim4 and mTim4 carriers were collected from the tube walls using magnetism. The supernatant (dissolution solution) was then collected separately.

[0708] It should be noted that the types of Tim4 protein and carrier used in Examples 22-25, the types of lysis solutions used to obtain extracellular membrane vesicles from the Tim4 carrier, and the lane numbers in the protein immunoblotting described later are shown in Table 10 below.

[0709] Table 10

[0710]

[0711] <(6) Western blotting of proteins>

[0712] Use “7.5 μL of each supernatant (dissolution) obtained in Examples 22-25 (5)” instead of “7.5 μL of each supernatant (dissolution) obtained in Examples 1-8”, otherwise perform the same method as “(7) protein immunoblotting” in Examples 1-8.

[0713] <Results>

[0714] The results of the obtained protein immunoblotting are shown below. Figure 6 . Figure 6 The lanes are as follows.

[0715] Lane 1: Results of Example 22 (using an hTim4 carrier formed by fusing a biotin-unlabeled Fc tag with hTim4 protein bound to G protein beads, and using a 1% SDS aqueous solution as the dissolution solution);

[0716] Lane 2: Results of Example 23 (using an hTim4 carrier formed by fusing a biotin-unlabeled Fc tag with hTim4 protein bound to a G protein bead, and using a 1 mM EDTA aqueous solution as the dissolution solution);

[0717] Lane 3: Results of Example 24 (using an mTim4 carrier formed by fusing a biotin-unlabeled Fc tag to an mTim4 protein bound to a G protein bead, and using a 1% SDS aqueous solution as the dissolution medium);

[0718] Lane 4: Results of Example 25 (using an mTim4 carrier formed by fusing a biotin-unlabeled Fc tag to an mTim4 protein bound to a G protein bead, and using a 1 mM EDTA aqueous solution as the dissolution medium).

[0719] Depend on Figure 6 As can be seen, in any of the embodiments in Examples 22-25, a band of Lamp-1, which is an exosome marker, was observed near 100 kDa. Therefore, the method of the present invention can be used to obtain extracellular membrane vesicles containing exosomes (Examples 22-25: lanes 1-4).

[0720] Furthermore, it is known that Tim4 protein, regardless of the biological species from which it originates, can be used in the Tim4 vector of this invention and the method for obtaining it.

[0721] Examples 26-27 / Comparative Examples 4-9. Comparison of the amount of extracellular membrane vesicles obtained using Tim4 vector and vector with immobilized PS protein.

[0722] Extracellular membrane vesicles of the present invention were obtained using Tim4 carrier and carriers in which PS proteins (Annexin V from humans, MFG-E8 from humans, and MFG-E8 from mice) were respectively immobilized on beads.

[0723] <(1) Dilution of PBS solution containing PS protein>

[0724] 20 μg of His-tagged fused human membrane adhesion protein-5 (manufactured by Creative BioMart, sometimes abbreviated as "His-tagged fused h-membrane adhesion protein-5") was dissolved in 100 μL of PBS to prepare a PBS solution containing His-tagged fused h-membrane adhesion protein-5 at a concentration of 200 μg / mL. 5 μL of the PBS solution containing 1 μg of His-tagged fused h-membrane adhesion protein-5 was then mixed with 195 μL of PBS and diluted.

[0725] To prepare a PBS solution containing His-tagged fused hMFG-E8, 50 μg of His-tagged fused human MFG-E8 (manufactured by R&D Systems, sometimes abbreviated as "His-tagged fused hMFG-E8") was dissolved in 500 μL of PBS to achieve a concentration of 100 μg / mL. 10 μL of a PBS solution containing 1 μg of His-tagged fused hMFG-E8 protein was then mixed and diluted with 190 μL of PBS. Similarly, to prepare a PBS solution containing His-tagged fused mMFG-E8, 50 μg of His-tagged fused mouse MFG-E8 (manufactured by R&D Systems, sometimes abbreviated as "His-tagged fused mMFG-E8") was dissolved in 500 μL of PBS to achieve a concentration of 100 μg / mL. 10 μL of a PBS solution containing 1 μg of His-tagged fused mouse MFG-E8 protein was then mixed and diluted with 190 μL of PBS.

[0726] <(2) Preparation of anti-His-tagged antibody immobilized beads>

[0727] 100 μL of a solution containing 30 μg / μL of Dynabeads M-270 carboxylic acid (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) (containing 3 mg of Dynabeads M-270 carboxylic acid) was dispensed into 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.) and washed with reaction buffer (0.1 M MES, pH 5.0).

[0728] Then, 60 μL of a 6×His antibody (manufactured by Wako Pure Chemical Industries, Ltd.) solution diluted with 490 μL of reaction buffer (containing 60 μg of 6×His antibody) was added, and the mixture was inverted and mixed for 30 minutes at room temperature. Next, 50 μL of 6 mg / mL WSC (manufactured by Dojin Chemical Research Institute) was added, and the mixture was inverted and mixed for 4 hours at room temperature. After washing the inverted and mixed Dynabeads M-270 carboxylic acid with TBS-T, the mixture was diluted with 100 μL of PBS to obtain 3 mg of anti-His tag antibody immobilized beads.

[0729] <(3) Preparation of Tim4 vector and vectors immobilized with various PS proteins>

[0730] Then, 100 μL of the obtained PBS solution containing anti-His-tagged antibody immobilized beads (containing 3 mg of anti-His-tagged 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-tagged antibody immobilized beads), and each tube was washed with 500 μL of PBS.

[0731] Then, in one of the four 1.5 mL tubes containing 0.6 mg of anti-His-tagged antibody immobilized beads in their particle state after washing, 200 μL of the PBS solution containing the His-tagged fusion mTim4 protein prepared above (containing 1 μg of His-tagged fusion mTim4 protein) was added, and the reaction was carried out at 8 °C for 1 hour.

[0732] Add 200 μL of the above-prepared PBS solution containing His-tagged fused h-membrane-5 (containing 1 μg of His-tagged fused h-membrane-5) to one of the solutions and react at 8°C for 1 hour.

[0733] Add 200 μL of the PBS solution containing His-tagged fused hMFG-E8 prepared above (containing 1 μg of His-tagged fused hMFG-E8) to one of the solutions, and react at 8°C for 1 hour.

[0734] Add 200 μL of the PBS solution containing His-tagged fused mMFG-E8 prepared above (containing 1 μg of His-tagged fused mMFG-E8) to one of the solutions, and react at 8°C for 1 hour.

[0735] Based on the above, 200 μL of PBS solutions containing 0.6 mg of each of the four carriers shown in Table 11 were obtained.

[0736] Table 11

[0737]

[0738] <(4) Obtaining extracellular membrane vesicles>

[0739] After washing 200 μL of PBS solution containing 0.6 mg of each of the four carriers three times with 500 μL of PBS, 200 μL of culture supernatant containing calcium ions, prepared by the same method as in Examples 1-8, was added to each of the five carriers in particulate state, and the reactions were carried out at 8°C for 3 hours.

[0740] Then, the four carriers used to react with the culture supernatant samples containing calcium ions were washed three times with 500 μL of TBS-T containing 2 mM CaCl2.

[0741] During the third wash, 250 μL of each of the four carriers was dispensed into two 1.5 mL tubes. To each of the four carrier particles (0.3 mg), 20 μL of either a 1% SDS aqueous solution or a 1 mM EDTA aqueous solution was added as a dissolution solution. The mixture was then vortexed at room temperature for 10 seconds, after which the vortexing was stopped (spin-down). The 1.5 mL tubes were then placed on a magnetic rack, and the four carriers were collected against the tube wall using magnetism. The supernatant (dissolution solution) was collected from each tube.

[0742] It should be noted that the types of PS proteins and carriers used in each embodiment, the types of lysis solutions used to obtain extracellular membrane vesicles from the carriers, and the lane numbers in the protein immunoblotting described later are shown in Table 12 below.

[0743] Table 12

[0744]

[0745] <(5) Western blotting of proteins>

[0746] Instead of “7.5 μL of each supernatant (dissolution solution) obtained in Examples 26-27 and Comparative Examples 4-9 (described (4))”, “7.5 μL of each supernatant (dissolution solution) obtained in Examples 1-8”, the same method as “(7) protein immunoblotting” in Examples 1-8 was used.

[0747] <Results>

[0748] The results of the obtained protein immunoblotting are shown below. Figure 7 . Figure 7 The lanes are as follows.

[0749] Lane 1: Results of Example 26 (using a carrier formed by immobilizing His tag-fused mTim4 protein onto anti-His tag antibody beads and using 1% SDS aqueous solution as the dissolution medium);

[0750] Lane 2: Results of Example 27 (using a carrier formed by immobilizing His tag-fused mTim4 protein onto anti-His tag antibody beads and using 1 mM EDTA aqueous solution as the dissolution medium);

[0751] Lane 3: Results of Comparative Example 4 (using a carrier formed by immobilizing His tag-fused h-membrane adhesion protein-5 onto anti-His tag antibody beads and using 1% SDS aqueous solution as the dissolution solution);

[0752] Lane 4: Results of Comparative Example 5 (using a carrier formed by immobilizing His tag-fused h-membrane adhesion protein-5 onto anti-His tag antibody beads, and using 1 mM EDTA aqueous solution as the dissolution solution);

[0753] Lane 5: Results of Comparative Example 6 (using a carrier formed by immobilizing His tag with hMFG-E8 bound to anti-His tag antibody beads and using 1% SDS aqueous solution as the dissolution solution);

[0754] Lane 6: Results of Comparative Example 7 (using a carrier formed by immobilizing His tag with hMFG-E8 bound to anti-His tag antibody beads and using 1 mM EDTA aqueous solution as the dissolution solution);

[0755] Lane 7: Results of Comparative Example 8 (using a carrier formed by immobilizing His tag with mMFG-E8 bound to anti-His tag antibody beads and using 1% SDS aqueous solution as the dissolution solution);

[0756] Lane 8: Results of Comparative Example 9 (using a carrier formed by immobilizing His tag-fused mMFG-E8 with anti-His tag antibody beads and using 1 mM EDTA aqueous solution as the dissolution solution).

[0757] Depend on Figure 7 As can be seen, in any of the embodiments in Examples 26-27, a band of Lamp-1, which serves as an exosome marker, was obtained near 100 kDa. Therefore, the method of the present invention can be used to obtain extracellular membrane vesicles containing exosomes (Examples 26-27: lanes 1-2).

[0758] On the other hand, by Figure 7 It can be seen that in any of the comparative examples 4-9, no band of Lamp-1 was observed near 100kDa. Therefore, for the carrier that has been immobilized with PS proteins other than Tim4 protein (membrane adhesion protein-5, MFG-E8), extracellular membrane vesicles containing exosomes cannot be obtained (Comparative Examples 4-9: lanes 3-8).

[0759] Examples 28-33. Obtaining extracellular membrane vesicles using the method of the present invention (exploration of the complex formation process)

[0760] Extracellular membrane vesicles were obtained using the methods of the present invention as described below.

[0761] For Examples 28-29, after reacting the FLAG-tagged thiol biotin-labeled mouse-derived Tim4 protein prepared by the same method as in Examples 13-14 with the carrier of the present invention, the sample was then added to obtain the extracellular membrane vesicles of the present invention.

[0762] For Examples 30-31, after reacting the mouse-derived Tim4 protein with a FLAG tag fused with a thiol biotin-labeled FLAG tag prepared by the same method as in Examples 13-14, the carrier of the present invention was then added to obtain the extracellular membrane vesicles of the present invention.

[0763] For Examples 32-33, thiol-biotin-labeled FLAG-tagged fused mouse-derived Tim4 protein, prepared by the same method as in Examples 13-14, was added simultaneously, along with the sample and the vector of the present invention, to obtain the extracellular membrane vesicles of the present invention.

[0764] <The Washing of Pearls>

[0765] 60 μL of PBS solution containing 10 μg / μL Dynabeads M-270 streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) (containing 0.6 mg of Dynabeads M-270 streptavidin C1 beads) was dispensed into three 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.), and each tube was washed with 500 μL of PBS.

[0766] Using the same method as in Examples 13-14, 270 μL of PBS solution containing His-tagged fused mTim4 protein (containing 50 μg of His-tagged fused mTim4 protein) was biotinylated to obtain 200 μL of PBS solution containing 39.2 μg of FLAG-tagged fused mTim4 protein with thiol biotin. To the obtained 5.1 μL of PBS solution containing 1 μg of FLAG-tagged fused mTim4 protein with thiol biotin, 194.9 μL of PBS solution was added to obtain 200 μL of PBS solution containing 1 μg of FLAG-tagged fused mTim4 protein with thiol biotin.

[0767] <The reaction of the FLAG tag labeled with thiol biotin with the mTim4 protein and the carrier of the present invention, thereby causing the extracellular membrane vesicles in the sample of the present invention to react with it (Examples 28-29)>

[0768] In a 1.5 mL tube containing 0.6 mg of washed Dynabeads M-270 streptavidin C1 beads, 200 μL of PBS solution containing 1 μg of FLAG-tagged mTim4 protein labeled with thiol biotin was added, allowing the Dynabeads M-270 streptavidin C1 beads to contact the FLAG-tagged mTim4 protein. The reaction was carried out at 8°C for 1 hour to obtain the mTim4 carrier. Then, the obtained mTim4 carrier was washed three times with 500 μL of PBS. In a 1.5 mL tube containing 0.6 mg of the washed mTim4 carrier, 200 μL of culture supernatant containing calcium ions, prepared using the same method as in Example 1, was added, and the reaction was carried out at 8°C for 3 hours. Then, the mTim4 carrier obtained by reacting the calcium-containing culture supernatant was washed three times with 500 μL of TBS-T solution containing a final concentration of 2 mM CaCl2. During the third wash, 250 μL of mTim4 carrier was dispensed into two 1.5 mL tubes. 20 μL of either a 1% SDS aqueous solution or a 1 mM EDTA aqueous solution was added to each 0.3 mg mTim4 carrier in particulate form as a dissolution solution. The mixture was then vortexed at room temperature for 10 seconds, after which the vortexing was stopped (spin-down). The 1.5 mL tubes were placed on a magnetic rack, and the mTim4 carrier was magnetically aggregated against the tube wall. The supernatant (dissolution solution) was collected from each tube.

[0769] <The case of reacting the FLAG tag labeled with thiol biotin with the mTim4 protein and the sample of the present invention, and then reacting the carrier of the present invention with it (Examples 30-31)>

[0770] 200 μL of a calcium-containing culture supernatant sample prepared using the same method as in Example 1 was aliquoted into a 1.5 mL tube. 5.1 μL of PBS solution containing 1 μg of FLAG-tagged mTim4 protein labeled with thiol biotin was added to bring the extracellular membrane vesicles in the calcium-containing culture supernatant sample into contact with the FLAG-tagged mTim4 protein labeled with thiol biotin, and the reaction was carried out at 8°C for 3 hours.

[0771] In a 1.5 mL tube containing 0.6 mg of washed Dynabeads M-270 streptavidin C1 beads, a solution was added to which the mTim4 protein, fused with a biotin-labeled FLAG tag from a calcium-containing culture supernatant sample, was reacted with the solution at 8°C for 3 hours. The reaction was then carried out at 8°C for 1 hour to obtain a complex of extracellular vesicles and the mTim4 carrier. The resulting complex was then washed three times with 500 μL of TBS-T solution containing 2 mM CaCl2. During the third wash, 250 μL of the extracellular vesicle and mTim4 carrier complex were dispensed into two separate 1.5 mL tubes. To each of 0.3 mg of the complex of extracellular membrane vesicles in granular form and the mTim4 carrier, add 20 μL of either a 1% SDS aqueous solution or a 1 mM EDTA aqueous solution as a dissolution solution. Then, mix using a vortex mixer at room temperature for 10 seconds, and then stop spinning (spin down). Place 1.5 mL tubes on a magnetic rack and use magnetism to collect the mTim4 carrier on the tube wall. Collect the supernatant (dissolution solution) from each tube.

[0772] <The simultaneous reaction of a thiol-biotin-labeled FLAG tag with mTim4 protein, the sample of the present invention, and the carrier of the present invention (Examples 32-33)>

[0773] In a 1.5 mL tube containing 0.6 mg of washed Dynabeads M-270 streptavidin C1 beads, 200 μL of a calcium-containing culture supernatant prepared using the same method as in Example 1, and 5.1 μL of PBS solution containing 1 μg of FLAG-tagged mTim4 protein labeled with thiol biotin, were added. The reaction was carried out at 8°C for 4 hours. The reacted Dynabeads M-270 streptavidin C1 beads were washed three times with 500 μL of TBS-T solution containing 2 mM CaCl2. During the third wash, 250 μL of mTim4 carrier was dispensed into two 1.5 mL tubes, and 20 μL of either 1% SDS aqueous solution or 1 mM EDTA aqueous solution was added to each 0.3 mg mTim4 carrier as a dissolution solution. The mixture was then vortexed at room temperature for 10 seconds, after which the vortexing was stopped (spin down). Place 1.5 mL tubes on a magnetic rack and use magnetism to collect Dynabeads M-270 streptavidin C1 beads on the tube wall. Collect the supernatant (dissolution) separately.

[0774] It should be noted that the order in which the carrier, mTim4 protein, and culture supernatant sample containing calcium ions are contacted in each embodiment, as well as the type of lysis solution used to obtain extracellular membrane vesicles from the carrier, are shown in Table 13 below.

[0775] Table 13

[0776]

[0777] <Western Immune Blotting of Proteins>

[0778] Use 7.5 μL of each supernatant (dissolution solution) obtained in Examples 28-33 instead of 7.5 μL of each supernatant (dissolution solution) obtained in Examples 1-8, otherwise perform the same method as "(7) protein immunoblotting" in Examples 1-8.

[0779] <Results>

[0780] The results of the obtained protein immunoblotting are shown below. Figure 8 middle. Figure 8 The lanes are as follows.

[0781] Lane 1: Results of Example 28 (Results obtained by contacting the carrier with mTim4 protein and then with calcium ion cell culture supernatant sample, and using 1% SDS aqueous solution as the dissolution solution);

[0782] Lane 2: Results of Example 29 (Results obtained by contacting the carrier with mTim4 protein and then with calcium ion cell culture supernatant sample, and using 1 mM EDTA aqueous solution as the dissolution solution);

[0783] Lane 3: Results of Example 30 (Results obtained by contacting mTim4 protein with calcium ion cell culture supernatant sample and then contacting it with a carrier to obtain extracellular membrane vesicles, and using 1% SDS aqueous solution as the dissolution solution);

[0784] Lane 4: Results of Example 31 (Results obtained by contacting mTim4 protein with calcium ion cell culture supernatant sample and then contacting it with a carrier to obtain extracellular membrane vesicles, and using 1Mm EDTA aqueous solution as the dissolution solution);

[0785] Lane 5: Results of Example 32 (Results obtained by simultaneously contacting the carrier with calcium-ionized cell culture supernatant and mTim4 protein to obtain extracellular membrane vesicles, and using 1% SDS aqueous solution as the dissolution solution);

[0786] Lane 6: Results of Example 33 (Results obtained by simultaneously contacting the carrier with calcium ion cell culture supernatant and mTim4 protein to obtain extracellular membrane vesicles, and using 1 mM EDTA aqueous solution as the dissolution solution).

[0787] according to Figure 8 In any of the embodiments in Examples 28-33, a band of Lamp-1 as an exosome marker was obtained near 100 kDa (Examples 28-33: lanes 1-6).

[0788] Furthermore, based on the results of Examples 28-33, it is known that for the process of forming a complex of the Tim4 protein bound to the carrier and the extracellular membrane vesicles in the sample (complex formation process), the contact order of the Tim4 protein of the present invention, the carrier of the present invention, and the extracellular membrane vesicles in the sample is irrelevant. As a result, it is sufficient to form a complex of the mTim4 protein bound to the carrier and the extracellular membrane vesicles in the sample.

[0789] Examples 34-35 / Comparative Examples 10-13: Comparison of the method of obtaining the present invention with existing methods

[0790] As described below, extracellular membrane vesicles were obtained by using the method of obtaining the present invention by dissolution with SDS (Example 34), the method of obtaining the present invention by dissolution with EDTA (Example 35), the anti-CD63 antibody immobilization method (Comparative Examples 10-11), and the exosome-human CD63 isolation / detection (Comparative Examples 12-13).

[0791] <Tim4 protein immobilized on a carrier>

[0792] 15 μL of a solution containing 10 μg / μL of Dynabeads M-270 streptavidin (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) was prepared (containing 1×10 7 One Dynabeads M-270 streptavidin was dispensed into a 1.5 mL tube (manufactured by BM Equipment Co., Ltd.) and washed with PBS.

[0793] Then, in a 1.5 mL tube containing washed Dynabeads M-270 streptavidin, 200 μL of a total PBS solution containing 0.25 μg of mouse-derived Tim4 protein fused with a FLAG tag and containing thiol-biotin-labeled protein, prepared by the same method as in Examples 11-12, was added. The reaction was carried out under refrigeration for 1 hour to obtain a PBS solution containing the mTim4 carrier.

[0794] <Preparation of Anti-CD63 Antibody (H5C6) Immobilization Vector>

[0795] 50 μL of a solution containing 30 μg / μL of Dynabeads M-270 carboxylic acid (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) (containing 1×10 8 One Dynabeads M-270 carboxylic acid was dispensed into a 1.5 mL tube (made by BM Equipment Co., Ltd.) and washed with reaction buffer (0.1 M MES, pH 5.0).

[0796] On the other hand, 30g (60μL) of antiCD63 antibody (H5C6) (manufactured by BDPharmingen) was diluted with 490μL of reaction buffer (0.1M MES, pH5.0) to obtain a reaction buffer diluted antiCD63 antibody solution.

[0797] Then, in a 1.5 mL tube containing washed Dynabeads M-270 carboxylic acid, 550 μL of the anti-CD63 antibody solution was diluted with the reaction buffer and mixed inverted for 30 minutes at room temperature. Next, 50 μL of 3 mg / mL WSC (manufactured by Dojin Chemical Research Institute) was added, and the mixture was mixed inverted for 4 hours at room temperature. After washing with TBS-T, the solution was diluted with 50 μL of PBS to obtain a PBS solution containing the anti-CD63 antibody (H5C6) vector.

[0798] Based on the above, two types of carriers are shown in Table 14 below.

[0799] Table 14

[0800]

[0801] <Obtaining extracellular membrane vesicles using the method of the present invention>

[0802] Add 500 μL of PBS to 200 μL of the PBS solution containing the mTim4 carrier obtained above (containing 1×10⁻⁶ mTim4). 7 After washing the mTim4 carrier three times, 50 μL of culture supernatant containing calcium ions, prepared by the same method as in Examples 1-8, was added to the mTim4 carrier in particulate state, and the reaction was carried out at 8°C for 3 hours.

[0803] Then, the reacted mTim4 carrier was washed three times with 500 μL of TBS-T containing 2 mM CaCl2. During the third wash, 250 μL of mTim4 carrier was dispensed into two 1.5 mL tubes. To each 0.3 mg of particulate mTim4 carrier, 20 μL of 1% SDS aqueous solution and 1 mM EDTA aqueous solution were added as dissolution solutions. The mixture was then vortexed at room temperature for 10 seconds, after which the vortexing was stopped (spin-down). The 1.5 mL tubes were placed on a magnetic rack, and the mTim4 carrier was magnetically collected against the tube wall. The supernatant (dissolution solution) was then collected.

[0804] <Other cell membrane vesicles obtained using anti-CD63 antibody immobilization>

[0805] Use 5 μL of PBS solution containing the anti-CD63 antibody (H5C6) vector (containing 1 × 10⁻⁶ vectors). 7 "One)" replaces "200 μL of PBS solution containing mTim4 carrier". Otherwise, the extracellular membrane vesicles in the culture supernatant sample containing calcium ions are obtained by the same method as described in "Obtaining extracellular membrane vesicles using the 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 (dissolution solution) is recovered.

[0806] <Obtaining extracellular membrane vesicles using exosome-human CD63 isolation / detection>

[0807] Using "Exosome-Human CD63 Isolation / Detection" (manufactured by Thermo Fisher Scientific Co., Ltd.), 1 mL (containing 1×10⁶ vectors) 7 "One)" replaces "200 μL of PBS solution containing mTim4 carrier". Otherwise, the extracellular membrane vesicles in the culture supernatant sample containing calcium ions are obtained by the same method as described in "Obtaining extracellular membrane vesicles using the 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 (dissolution solution) is recovered.

[0808] It should be noted that the types of vectors used in each embodiment and comparative example, the types of lysis solutions used to obtain extracellular membrane vesicles from the vectors, and the lane numbers in the protein immunoblotting described later are shown in Table 15 below.

[0809] Table 15

[0810]

[0811] <Western Immune Blotting of Proteins>

[0812] Instead of “7.5 μL of each supernatant (dissolution solution) obtained in Examples 34-35 and Comparative Examples 10-13”, “7.5 μL of each supernatant (dissolution solution) obtained in Examples 1-8”, the same method as “(7) protein immunoblotting” in Examples 1-8 was used.

[0813] <Results>

[0814] The results of the obtained protein immunoblotting are shown below. Figure 9 . Figure 9 The lanes are as follows.

[0815] Lane 1: Results of Example 34 (Results when extracellular membrane vesicles were obtained using the method of the present invention and 1% SDS aqueous solution was used as the dissolution solution);

[0816] Lane 2: Results of Example 35 (Results when extracellular membrane vesicles were obtained using the method of the present invention and 1 mM EDTA aqueous solution was used as the dissolution solution);

[0817] Lane 3: Results of Comparative Example 10 (Results when extracellular membrane vesicles were obtained using the anti-CD63 antibody method and 1% SDS aqueous solution was used as the dissolution solution);

[0818] Lane 4: Results of Comparative Example 11 (Results when extracellular membrane vesicles were obtained using the anti-CD63 antibody method and 1 mM EDTA aqueous solution was used as the dissolution solution);

[0819] Lane 5: Results of Comparative Example 12 (Results when extracellular membrane vesicles were obtained using exosome-human CD63 isolation / detection and 1% SDS aqueous solution was used as the dissolution solution);

[0820] Lane 6: Results of Comparative Example 13 (Results when extracellular membrane vesicles were obtained using exosome-human CD63 isolation / detection and 1 mM EDTA aqueous solution was used as the dissolution solution).

[0821] according to Figure 9 In Examples 34-35, which obtained extracellular membrane vesicles using the method of the present invention, a band of CD63, one of the exosome markers, was obtained in the vicinity of 25-60 kDa (Examples 34-35: lanes 1-2).

[0822] On the other hand, according to Figure 9In Comparative Examples 10-11, which obtained extracellular membrane vesicles using the anti-CD63 antibody method, and Comparative Examples 12-13, which obtained extracellular membrane vesicles using exosome-human CD63 isolation / detection, only faint CD63 bands were obtained in the vicinity of 25-60 kDa (Comparative Examples 10-13: lanes 3-6).

[0823] As can be seen from the above, compared with methods such as anti-CD63 antibody method and exosome-human CD63 isolation / detection, which use antibodies against surface antigen proteins of extracellular membrane vesicles and obtain extracellular membrane vesicles through the affinity between the surface antigen protein and the antibody, the method of the present invention can recover more extracellular membrane vesicles.

[0824] Examples 36-38. Residual extracellular membrane vesicles were obtained from samples after ultracentrifugation using the method of the present invention.

[0825] As described below, the method of obtaining the present invention is implemented for a sample after the extracellular membrane vesicles have been removed and obtained by ultracentrifugation (hereinafter, sometimes referred to as ultracentrifugation process) as a conventional method.

[0826] <(1) Removal of extracellular membrane vesicles using ultracentrifugation>

[0827] 15 mL of FBS (manufactured by Corning Incorporated) was centrifuged (10,000 × g, 20 min) to separate impurities. The supernatant was transferred to a new tube to obtain centrifuged FBS. Then, 5 mL of each of the centrifuged FBS were subjected to ultracentrifugation (110,000 × g, overnight) or ultracentrifugation (450,000 × g, overnight). The supernatant was transferred to a new tube to obtain 5 mL of ultracentrifuged FBS (110,000 × g) and 5 mL of ultracentrifuged FBS (450,000 × g).

[0828] <(2) Washing of beads>

[0829] 180 μL of PBS solution containing 10 μg / μL Dynabeads MyOne streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific) was dispensed into three separate tubes. Then, 1500 μL of PBS was added to each tube, and after stirring, the tubes were placed on a magnetic rack to collect the Dynabeads MyOne streptavidin C1 beads on the tube walls using magnetism. The solution in the tubes was then discarded using a pipette (hereinafter, sometimes referred to as a washing operation).

[0830] <(3) Immobilize the Fc tag-fused mouse-derived Tim4 protein onto beads>

[0831] After washing, 1500 μL of PBS solution containing 3 μg of mouse-derived Tim4 protein fused with an Fc tag and labeled with thiol biotin was added to 1.8 mg of Dynabeads MyOne streptavidin C1 beads in granular state. The mixture was reacted at 8 °C for 10 minutes to obtain 1500 μL of PBS solution containing a vector (mTim4 vector) fused with an Fc tag and labeled with thiol biotin.

[0832] <(4) Obtaining extracellular membrane vesicles using the method of the present invention>

[0833] The obtained mTim4 carrier (1.8 mg) was washed three times with 1500 μL of PBS to obtain particulate mTim4 carrier. Then, 5 mL of each of the centrifuged FBS, ultracentrifuged FBS (110000×g), and ultracentrifuged FBS (450000×g) were aliquoted into three 15 mL centrifuge tubes (manufactured by Corning Incorporated), and 1.8 mg of particulate mTim4 carrier was added. The reaction was carried out at 8°C for 2 hours. After the reaction, the 15 mL centrifuge tubes were placed on a magnetic rack, and the mTim4 carrier was collected from the tube wall using magnetism. The supernatant (each FBS) was then collected into a new 15 mL centrifuge tube. After washing the mTim4 carrier particles remaining in the centrifuge tube with 3 mL of TBS-T (Tris buffer, 0.0005% Tween 20, 2 mM CaCl2) containing 2 mM CaCl2, 1 mL of TBS-T containing 2 mM CaCl2 was added to each of the mTim4 carrier particles to suspend them. After transferring all the suspensions to 1.5 mL tubes, the particles were washed twice.

[0834] To 1.8 mg of mTim4 carrier in particulate form, 50 μL of a TBS solution containing 1 mM EDTA was added as a dissolution solution. The mixture was then vortexed at room temperature for 10 seconds, after which the vortexing was stopped (spin-down). 1.5 mL tubes were placed on a magnetic rack, and the mTim4 carrier was magnetically aggregated against the tube wall to collect the dissolution solution. Another 50 μL of TBS solution containing 1 mM EDTA was added, and the same method was used to collect the dissolution solution using the same mTim4 carrier.

[0835] Then, the beads used in the two recovery of the dissolution solution were added again to the supernatant (each FBS) that had been recovered into the 15 mL centrifuge tube. The operation of recovering the extracellular membrane vesicles from the supernatant (each FBS) was repeated twice using the same method as above to obtain the dissolution solution.

[0836] <(5) Western blotting of proteins>

[0837] The obtained 100 μL of each eluent was ultrafiltered using a VIVASPIN 500 (MVCO 10000) (trade name, manufactured by Sartorius Co., Ltd.) to reduce it to 30 μL. To each 15 μL of ultrafiltered eluent, 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 the protein immunoblotting samples. 20 μL of each protein immunoblotting sample was added to a SuperSep Ace 5-20% gel (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), and electrophoresis was performed at 25 mA for 65 minutes. Using a semi-dry blot apparatus and discontinuous buffers (anodic buffer 1: 0.3M Tris / 20% methanol; anodic buffer 2: 0.025M Tris / 20% methanol; cathode buffer: 0.025M Tris / 0.04M aminocaproic acid / 20% methanol), at 1 mA / cm 2Under the specified conditions, 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) was added to the transferred PVDF membrane, and the mixture was reacted at room temperature for 1 hour. For blocking, 2 mL of anti-bovine CD9 antibody (Novus Biologicals, sometimes abbreviated as "anti-bovine CD9 antibody") diluted 1000-fold with PBS-T was reacted at room temperature for 1 hour. The reacted PVDF membrane was washed three times with PBS-T, and a secondary antibody (anti-mouse IgG (H+L), rabbit, IgG fraction, peroxidase-binding antibody) diluted 10000-fold with PBS-T (Wako Pure Chemical Industries, Ltd.) was reacted at room temperature for 1 hour. After washing five times with PBS-T, ImmunoStar Basic (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) was added to the membrane after reacting with anti-bovine CD9 antibody, and ImmunoStar Zeta (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) was added to the membrane after reacting with anti-flotillin-2 antibody. The luminescence signal was detected using a LAS-4000 (trade name, manufactured by General Electric Company). It should be noted that the anti-bovine CD9 antibody and the anti-flotillin-2 antibody are antibodies that recognize CD9 and Flotillin-2, respectively, as marker proteins of exosomes.

[0838] <(6) Determination of average particle number and average particle size>

[0839] After processing 100 μL of each eluent using a centrifugal filter (0.45 μm, PVDF) (Millipore), the eluent was diluted 5 times with water. Using a NanoSight LM10 (trade name, NanoSight Corporation), the particle count in each diluted eluent was measured three times according to the NanoSight instruction manual to determine the average particle number and average particle size.

[0840] It should be noted that the sample processing methods used in each embodiment, the methods for obtaining residual extracellular membrane vesicles in the sample, etc., are shown in Table 16 below.

[0841] Table 16

[0842]

[0843] <Results>

[0844] The results of the obtained protein immunoblotting are shown below. Figure 10The results of the average number of particles and the average particle size measured using NanoSight are shown in Table 17. Figure 10 The results for each lane are as follows.

[0845] Lane 1: Results of Example 36 (results of centrifugation of the sample and acquisition of extracellular membrane vesicles using the method of the present invention);

[0846] Lane 2: Results of Example 37 (Results of obtaining extracellular membrane vesicles using the method of the present invention after centrifugation of the sample and ultracentrifugation at 110000×g).

[0847] Lane 3: Results of Example 38 (Results of obtaining extracellular membrane vesicles using the method of the present invention after centrifugation of the sample at 450,000 × g).

[0848] according to Figure 10 It is known that when extracellular membrane vesicles are obtained using the method of the present invention after centrifugation of the sample at 110,000×g or 450,000×g (Examples 37-38), the bands of CD9 and fusiformin-2, which are exosome markers, are also confirmed to be present. However, ultracentrifugation cannot completely remove (obtain) the extracellular membrane vesicles contained in the sample, leaving a large number of extracellular membrane vesicles in the sample (Examples 37-38: Lanes 2-3). Furthermore, it is known that the method of the present invention can obtain (remove) extracellular membrane vesicles that cannot be completely removed (obtained) by the ultracentrifugation method and remain in the sample (Examples 37-38: Lanes 2-3).

[0849] According to Table 17, when the sample was centrifuged, subjected to ultracentrifugation at 110,000 × g or 450,000 × g, and then the extracellular membrane vesicles were obtained using the method of the present invention (Examples 37-38), and when the sample was centrifuged and then the extracellular membrane vesicles were obtained using the method of the present invention (Example 36), the size (particle size) of the obtained extracellular membrane vesicles was almost the same. Therefore, the extracellular membrane vesicles remaining in the sample that could not be completely removed by ultracentrifugation did not form fragments but existed as particles.

[0850] Table 17

[0851]

[0852] Examples 39-40. Residual extracellular membrane vesicles were obtained from samples treated with total exosome isolation (polymer precipitation) using the method of the present invention.

[0853] As described below, the method of obtaining the present invention is carried out on a sample after removing (obtaining) extracellular membrane vesicles contained in the sample by total exosome isolation (polymer precipitation) as a conventional method (hereinafter, sometimes referred to as "polymer precipitation treatment").

[0854] <(1) Removal of extracellular membrane vesicles using total exosome isolation (polymer precipitation)>

[0855] 10 mL of FBS (manufactured by Corning) was centrifuged (10000×g, 20 min) to separate impurities, yielding centrifuged FBS. Then, 1 mL of Total Exosome Isolation reagent (derived from serum) (manufactured by Thermo Fisher Scientific) was added to 5 mL of the centrifuged FBS. The mixture was allowed to stand under refrigeration for 30 min, then centrifuged (10000×g, 20 min). The supernatant was collected, yielding 6 mL of FBS after Total Exosome Isolation.

[0856] <(2) Washing of beads>

[0857] 180 μL of PBS solution containing 10 μg / μL Dynabeads MyOne streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific) was dispensed into two separate tubes. Then, 1500 μL of PBS was added to each tube, and the mixture was stirred. The tubes were then placed on a magnetic rack to allow the Dynabeads MyOne streptavidin C1 beads to collect on the tube walls. The solution in the tubes was then discarded using a pipette (hereinafter, sometimes referred to as a washing operation).

[0858] <(3) Fc tag fusion mouse-derived Tim4 protein immobilized on beads>

[0859] 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 in Examples 5-8, was added to 1.8 mg of Dynabeads MyOne streptavidin C1 beads in particulate form after washing. The mixture was reacted at 8°C for 10 minutes to obtain 1500 μL of PBS solution containing a carrier (mTim4 carrier) bound to the thiol-biotin-labeled Fc tag fused mTim4 protein.

[0860] <(4) Obtaining extracellular membrane vesicles using the method of the present invention>

[0861] The obtained mTim4 vector (1.8 mg) was washed three times with 1500 μL of PBS to obtain particulate mTim4 vector. Then, 1.8 mg of particulate mTim4 vector was added to either 5 mL of FBS (prepared by centrifugation) or 6 mL of FBS (prepared by total exosome isolation) in 15 mL centrifuge tubes (manufactured by Corning), and the reaction was carried out at 8°C for 2 hours.

[0862] After the reaction, 15 mL centrifuge tubes were placed on magnetic racks to collect mouse-derived Tim4 vectors against the tube walls using magnetism. The supernatants (each FBS) were then collected into new 15 mL centrifuge tubes. The remaining mTim4 vector particles in the centrifuge tubes were washed with 3 mL of TBS-T (Tris buffer, 0.0005% Tween 20, 2 mM CaCl2) containing 2 mM CaCl2. Then, 1 mL of TBS-T containing 2 mM CaCl2 was added to each mTim4 particle to resuspend it. The suspensions were then transferred to 1.5 mL tubes and washed twice.

[0863] To each 1.8 mg of mTim4 carrier in particulate form, 50 μL of a TBS solution containing 1 mM EDTA was added as a dissolution solution. The mixture was then vortexed at room temperature for 10 seconds, after which the vortexing was stopped (spin-down). 1.5 mL tubes were placed on a magnetic rack, and the mTim4 carrier was magnetically aggregated against the tube wall to collect the dissolution solution. Another 50 μL of TBS solution containing 1 mM EDTA was added, and the same method was used to collect the dissolution solution using the same mTim4 carrier.

[0864] Then, the mTim4 carrier (beads) used in the two recovery of the dissolution solution was added again to each supernatant (each FBS) that had been 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 dissolution solution.

[0865] <(5) Western blotting of proteins>

[0866] The obtained 300 μL of each eluent was ultrafiltered using a VIVASPIN 500 (MVCO 10000) (trade name, manufactured by Sartorius Co., Ltd.) to reduce it to 30 μL. To each 30 μL of ultrafiltered eluent, 10 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 the protein immunoblotting samples. 20 μL of each protein immunoblotting sample was added to two sites in a SuperSep Ace 5-20% gel (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), and electrophoresis was performed at 25 mA for 65 minutes. Using a semi-dry blot apparatus and discontinuous buffers (anodic buffer 1: 0.3M Tris / 20% methanol; anodic buffer 2: 0.025M Tris / 20% methanol; cathode buffer: 0.025M Tris / 0.04M aminocaproic acid / 20% methanol), at 1 mA / cm 2Under the specified conditions, the obtained electrophoresis gel was transferred to a PVDF membrane (Millipore) for 60 minutes. After transfer, 3% skim milk diluted with PBS-T (PBS buffer, 0.1% Tween 20) was added to the PVDF membrane, and the mixture was reacted at room temperature for 1 hour. For blocking, 2 mL of anti-bovine CD9 antibody (Novus Biologicals, sometimes abbreviated as "anti-bovine CD9 antibody") diluted 1000-fold with PBS-T or anti-human nautiloid-2 antibody (BD Biosciences, sometimes abbreviated as "anti-human nautiloid-2 antibody") diluted 250-fold with PBS-T was reacted at room temperature for 1 hour. The PVDF membrane was washed three times with PBS-T after the reaction. Secondary antibodies (anti-mouse IgG (H+L), rabbit, IgG fraction, and peroxidase-binding antibody) diluted 10,000 times with PBS-T (manufactured by Wako Pure Chemical Industries, Ltd.) were then reacted at room temperature for 1 hour. After washing five times with PBS-T, ImmunoStar Basic (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) was added to the membrane reacted with anti-bovine CD9 antibody, and ImmunoStar Zeta (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) was added to the membrane reacted with anti-navone-2 antibody. The luminescence signal was detected using a LAS-4000 (trade name, manufactured by General Electric Company). It should be noted that the anti-bovine CD9 antibody is an antibody against CD9, one of the marker proteins of exosomes, and the anti-navone-2 antibody is an antibody against navone-2, one of the marker proteins of exosomes.

[0867] It should be noted that the sample processing methods used in each embodiment, the methods for obtaining residual extracellular membrane vesicles in the sample, and the lane numbers in the protein immunoblotting are shown in Table 18 below.

[0868] Table 18

[0869]

[0870] <Results>

[0871] The results of the obtained protein immunoblotting are shown below. Figure 11 . Figure 11 In the diagram, each lane represents the following results.

[0872] Lane 1: Results of Example 39 (results of obtaining extracellular membrane vesicles using the method of the present invention after centrifugation of the sample);

[0873] Lane 2: Results of Example 40 (Results of obtaining extracellular membrane vesicles using the method of the present invention after centrifugation and polymer precipitation of the sample).

[0874] according to Figure 11 It is known that when the sample was centrifuged, polymerized, and the extracellular membrane vesicles were obtained using the method of the present invention (Example 40), the bands of CD9 and buoyancy protein-2, which are exosome markers, were also confirmed. Therefore, the extracellular membrane vesicles contained in the sample could not be completely removed (obtained) by the total exosome isolation (polymer precipitation) method, and a large number of extracellular membrane vesicles remained in the sample (Example 40: lane 2).

[0875] Therefore, it can be seen that by using the method of the present invention, extracellular membrane vesicles remaining in the sample that cannot be completely removed by the total exosome isolation (polymer precipitation) method can be obtained (Example 40: lane 2).

[0876] Examples 41-47. Extracellular membrane vesicles were obtained (removed) from the sample using the removal method of the present invention and / or existing methods (ultracentrifugation, total exosome isolation, polymer precipitation).

[0877] As described below, extracellular membrane vesicles are obtained (removed) from a sample using the removal method of the present invention and / or existing methods (ultracentrifugation, total exosome isolation (polymer precipitation)).

[0878] <(1) Removal of extracellular membrane vesicles using ultracentrifugation>

[0879] 28 mL of FBS (manufactured by Corning Incorporated) was centrifuged (10,000 × g, 20 min) to separate impurities, yielding centrifuged FBS (hereinafter sometimes referred to as "sample 1"). Then, 12 mL of the obtained centrifuged FBS ("sample 1") was ultracentrifuged (110,000 × g, overnight) to obtain ultracentrifuged FBS (110,000 × g) (hereinafter sometimes referred to as "sample 2").

[0880] <(2) Removal of extracellular membrane vesicles using total exosome isolation (polymer precipitation)>

[0881] Then, 1.6 mL of Total Exosome Isolation (from serum) reagent (Thermo Fisher Scientific Co., Ltd.) was added to 8 mL of FBS after centrifugation (“sample 1 removed”). The mixture was allowed to stand for 30 minutes under refrigeration and then centrifuged (10000×g, 20 minutes). The supernatant was collected to obtain 9.6 mL of FBS after Total Exosome Isolation (hereinafter, sometimes abbreviated as “sample 3 removed”).

[0882] In addition, 0.8 mL of total exosome isolation reagent was added to 4 mL of FBS (“sample 2 removed”) after ultracentrifugation (110000×g). The mixture was allowed to stand for 30 minutes under refrigeration and then centrifuged (10000×g, 20 minutes). The supernatant was collected to obtain 4.8 mL of FBS (hereinafter sometimes referred to as “sample 4 removed”) after ultracentrifugation (110000×g) / total exosome isolation.

[0883] <(3) Washing the beads>

[0884] Divide 240 μL of PBS solution containing 10 μg / μL Dynabeads MyOne streptavidin C1 beads (trade name, manufactured by Thermo Fisher Scientific Co., Ltd.) into three separate tubes. Then, add 2000 μL of PBS to each tube, stir, and place the tubes on a magnetic rack to collect the Dynabeads MyOne streptavidin C1 beads on the tube wall using magnetism. Discard the solution in the tube using a pipette (hereinafter, sometimes referred to as "washing operation").

[0885] <(4) Fc tag fusion mouse-derived Tim4 protein immobilized on beads>

[0886] After washing, 2000 μL of PBS solution containing 4 μg of mouse-derived Tim4 protein fused with an Fc tag and labeled with thiol biotin was added to 2.4 mg of Dynabeads MyOne streptavidin C1 beads in particulate form. The reaction was carried out at 8 °C for 10 minutes to obtain 2000 μL of PBS solution containing a vector (mTim4 vector) fused with an Fc tag and labeled with thiol biotin.

[0887] <(5) Removal of extracellular membrane vesicles>

[0888] The obtained mTim4 vector 2.4 mg was washed three times with 2000 μL of PBS to obtain particulate mTim4 vector.

[0889] Then, 4 mL of FBS after centrifugation (“sample 1”) or after ultracentrifugation (110000×g) (“sample 2”), and 4.8 mL of FBS after total exosome isolation (“sample 3”) were dispensed into tubes and reacted at 8°C for 1 hour. After the reaction, the tubes were placed on a magnetic rack, and the mTim4 carrier was collected on the tube wall by magnetic force. The supernatant (each FBS) was then collected into a new tube.

[0890] The mTim4 carrier particles remaining in the tube were washed three times with 4 mL of TBS-T (Tris buffer, 0.0005% Tween 20). The supernatant (FBS) was added again after each wash, and this process was repeated five times from reaction to washing. After the fifth reaction, the supernatant (FBS) was transferred to a new tube and centrifuged (10000×g, 20 min). The supernatant was recovered, yielding 4 mL of FBS treated by ultracentrifugation (110000×g) / Tim4 (hereinafter sometimes abbreviated as "sample 5 removed"), 4.8 mL of FBS treated by total exosome isolation / Tim4 (hereinafter sometimes abbreviated as "sample 6 removed"), and 4 mL of FBS treated by Tim4 (hereinafter sometimes abbreviated as "sample 7 removed").

[0891] <(6) Obtaining extracellular membrane vesicles using the method of the present invention>

[0892] Seven portions of 0.6 mg mTim4 vector were prepared again using the same method as described above, and each portion was washed. The amount of sample removed as specified in Table 19 below, added to each 0.6 mg mTim4 vector in particulate form, was the same amount of sample removed added to the mouse-derived Tim4 vector, and the reaction was carried out at 8°C for 3 hours.

[0893] Table 19

[0894]

[0895] After the reaction, 1.5 mL tubes were placed on a magnetic rack, and the mTim4 carrier was aggregated on the tube wall by magnetic force. The supernatant (each FBS) was recovered to obtain ...

Claims

1. A method for obtaining extracellular membrane vesicles from a sample, characterized in that, It includes the following processes: (1) Complex formation process, which is a process of forming a complex of a carrier of the SH group of Tim4 protein and the extracellular membrane vesicles in the sample in the presence of calcium ions. (2) Composite separation process, which is the process of separating the composite from the sample; (3) The obtaining process, which is the process of separating extracellular membrane vesicles from the complex and obtaining extracellular membrane vesicles. The obtaining process is carried out using a calcium ion chelating agent.

2. The method as described in claim 1, wherein, The complex formation process involves contacting the Tim4 carrier with extracellular membrane vesicles in the sample in the presence of calcium ions, thereby forming a complex of Tim4 protein bound to the carrier and extracellular membrane vesicles in the sample.

3. The method as described in claim 1, wherein, The complex formation process involves bringing Tim4 protein, a carrier, and extracellular membrane vesicles in the sample into contact in the presence of calcium ions, thereby forming a complex of Tim4 protein bound to the carrier and extracellular membrane vesicles in the sample.

4. The method according to any one of claims 1 to 3, wherein, The acquisition process is carried out using a protein modifier.

5. The method according to any one of claims 1 to 3, wherein, The Tim4 protein contains an IgV domain.

6. The use of a kit for capturing extracellular membrane vesicles, said kit being formed containing a Tim4 carrier, a solution containing calcium ions, and a solution containing a calcium ion chelating agent, said use being for performing the method of any one of claims 1 to 5.

7. The use of a kit for capturing extracellular membrane vesicles, said kit comprising a reagent containing Tim4 protein, a reagent containing a carrier, a solution containing calcium ions, and a solution containing a calcium ion chelating agent, said use being for performing the method of any one of claims 1 to 5.

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