Method for detecting extracellular vesicle

Abstract

The present invention addresses the problem of providing a method for detecting, with high sensitivity, a target extracellular vesicle in a blood-derived sample. The problem is solved by removing a platelet-derived extracellular vesicle in a blood-derived sample or suppressing the generation of the platelet-derived extracellular vesicle in the blood-derived sample, before detecting a target extracellular vesicle in the blood-derived sample.

Description

Methods for detecting extracellular vesicles

[0001] The present invention relates to a method for detecting target extracellular vesicles in a blood-derived sample.

[0002] Tumor cell-derived proteins are present in bodily fluids such as the blood of cancer patients and in the culture medium of cultured tumor cells. Tumor cell-derived proteins may be localized intracellularly within circulating tumor cells (or dead cell debris), secreted extracellularly by tumor cells, and present as free proteins in the blood, or present as proteins within extracellular vesicles (EVVs). Detection of these proteins can provide information for early tumor detection and predicting post-treatment patient outcomes. In particular, EVVs are colloidal particles surrounded by a lipid bilayer membrane. Their role as a mediator of intercellular communication in vivo and their association with diseases such as cancer and physiological phenomena have recently been reported. Research is underway to elucidate their physiological functions and apply them to disease testing.

[0003] As a disease test using extracellular vesicles, Patent Document 1 discloses a method for detecting proteins present in plasma and localized on the surface of extracellular vesicles released from tumor cells using an enzyme-linked immunosorbent assay (ELISA). This method makes it possible to distinguish between cancer patients and healthy donors.

[0004] Special Publication No. 2011-510309

[0005] An object of the present invention is to provide a method for detecting target extracellular vesicles in a blood-derived sample with high sensitivity.

[0006] The present inventors have found that when detecting target extracellular vesicles in blood-derived samples, platelet-derived extracellular vesicles cause problems such as increased variability in measurement values ​​and increased non-specific signals (background).

[0007] In order to solve the above problems, the present inventors conducted extensive research and discovered that target extracellular vesicles in a blood-derived sample can be detected with high sensitivity by removing platelet-derived extracellular vesicles in the blood-derived sample or by suppressing the generation of platelet-derived extracellular vesicles in the blood-derived sample, thereby completing the present invention.

[0008] That is, the present invention can be exemplified as follows: [1] A method for detecting target extracellular vesicles in a blood-derived sample, comprising: step A1 of removing platelet-derived extracellular vesicles from the blood-derived sample or step A2 of suppressing the generation of platelet-derived extracellular vesicles in the blood-derived sample; and step B of detecting the target extracellular vesicles in the blood-derived sample. [2] A method for improving the detection sensitivity of target extracellular vesicles in a blood-derived sample, comprising step A1 of removing platelet-derived extracellular vesicles from the blood-derived sample or step A2 of suppressing the generation of platelet-derived extracellular vesicles in the blood-derived sample. [3] A method for pretreating a blood-derived sample for detecting target extracellular vesicles in the blood-derived sample, comprising step A1 of removing platelet-derived extracellular vesicles from the blood-derived sample or step A2 of suppressing the generation of platelet-derived extracellular vesicles in the blood-derived sample. [4] The method (specifically, the method described in any of [1] to [3]), wherein the target extracellular vesicles are extracellular vesicles other than platelet-derived extracellular vesicles. [5] The method (specifically described in [4]) wherein the extracellular vesicles other than platelet-derived extracellular vesicles are extracellular vesicles derived from tumor cells. [6] The method (specifically described in [5]) wherein the tumor cell-derived extracellular vesicles are extracellular vesicles derived from prostate cancer cells. [7] The method (specifically described in any of [1] to [3]) wherein the blood-derived sample is plasma or serum. [8] The method (specifically described in any of [1] to [7]) wherein step A1 is performed using a substance that specifically binds to the platelet-derived extracellular vesicles. [9] The method (specifically described in [8]) wherein the substance is an antibody against a marker specifically present on the platelet-derived extracellular vesicles.

[10] The method (specifically described in [9]) wherein the marker is CD41.

[11] The method (specifically described in any of [1] to

[10] ) wherein step A2 is performed by removing platelets from the blood-derived sample.

[12] The method (specifically, the method described in

[11] ) above, wherein the platelets are removed by performing centrifugation two or more times during preparation of the blood-derived sample.

[13] The method (specifically described in any of [1] to

[10] ) wherein step A2 is performed without freezing the blood-derived sample.

[14] The method (specifically described in any of [1] and [4] to

[13] ) wherein step B is performed by immunological assay.

[15] The method (specifically described in any of [1] and [4] to

[14] ) wherein step B includes step B1 of capturing the target extracellular vesicles on a carrier and step B2 of detecting the captured target extracellular vesicles.

[16] The method (specifically described in

[15] ) wherein step B1 is performed using a first substance that binds to the target extracellular vesicles, and step B2 is performed using a second substance that binds to the target extracellular vesicles.

[17] The method (specifically described in

[16] ) wherein the first substance is an antibody against a first marker present on the target extracellular vesicles, and the second substance is an antibody against a second marker present on the target extracellular vesicles.

[18] The method (specifically described in

[17] ) wherein the first marker is a marker specifically present on the target extracellular vesicles.

[19] The method (specifically described in

[17] or

[18] ) wherein the first marker is TM4SF1 or CD81.

[20] The method (specifically described in any of

[17] to

[19] ) wherein the second marker is CD9 or CD81.

[0009] According to the present invention, target extracellular vesicles in blood-derived samples can be detected with high sensitivity.

[0010] A diagram showing the detection results of PC-3-derived extracellular vesicles (Comparative Example 3). A diagram showing the detection results of PC-3-derived extracellular vesicles (Example 6). A diagram showing the detection results of PC-3-derived extracellular vesicles (Reference Example 1). A diagram showing the detection results of PC-3-derived extracellular vesicles (Comparative Example 4). A diagram showing the detection results of PC-3-derived extracellular vesicles (Example 7). A diagram showing the detection results of PC-3-derived extracellular vesicles (Reference Example 2).

[0011] The present invention will be described in detail below. In this specification, the terms "cancer" and "cancer" may be used interchangeably.

[0012] <1> Method of the Present Invention The method of the present invention comprises a step of removing platelet-derived extracellular vesicles from a blood-derived sample or a step of suppressing the generation of platelet-derived extracellular vesicles in a blood-derived sample. The method of the present invention may further comprise a step of detecting target extracellular vesicles in the blood-derived sample.

[0013] The step of removing platelet-derived extracellular vesicles from a blood-derived sample is also referred to as a "removal step." The step of suppressing the occurrence of platelet-derived extracellular vesicles in a blood-derived sample is also referred to as a "suppression step." In the method of the present invention, one or both of the removal step and the suppression step may be performed. The step of detecting target extracellular vesicles in a blood-derived sample is also referred to as a "detection step." The detection step is performed after the removal step or the suppression step. "Target extracellular vesicles" refer to extracellular vesicles to be detected and are to be distinguished from platelet-derived extracellular vesicles. In other words, "target extracellular vesicles" refer to extracellular vesicles other than platelet-derived extracellular vesicles that may be contained in a blood-derived sample. The target extracellular vesicles may be one or more specific types of extracellular vesicles selected from extracellular vesicles other than platelet-derived extracellular vesicles, or may be all extracellular vesicles other than platelet-derived extracellular vesicles. Specific extracellular vesicles include extracellular vesicles derived from tumor cells.

[0014] The type of tumor from which the target extracellular vesicles can be derived is not particularly limited and may include malignant tumors and benign tumors. Malignant tumors may be primary or metastatic cancers. Tumors include hematopoietic cell malignant tumors, head and neck cancer, brain tumors, breast cancer, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, kidney cancer, adrenal cancer, gastrointestinal stromal tumors, mesothelioma, thyroid cancer, lung cancer, osteosarcoma, bone cancer, prostate cancer, testicular tumors, bladder cancer, skin cancer, and anal cancer. Hematopoietic cell malignant tumors include leukemia, lymphoma, and multiple myeloma. Lymphomas include Hodgkin's lymphoma and non-Hodgkin's lymphoma. Head and neck cancers include floor of mouth cancer, gingival cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer, and paranasal sinus cancer.

[0015] "Extracellular vesicles" refer to lipid-covered vesicles with a diameter of 1 nm to 1 μm that are released by cells, whether actively or passively. Examples of extracellular vesicles include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, and apoptotic vesicles (Nature Reviews Immunology, 9, 581-593 (2009)). In general, extracellular vesicles are composed of lipids and proteins with compositions different from those of cell membranes (Bioscience, 65, 783-797 (2015)). However, proteins that reflect the characteristics of the cells from which they were released are localized in the extracellular vesicles. For example, when the source of release is a tumor cell, the presence or absence of extracellular vesicles released from the cell can be used to distinguish between cancer patients and healthy donors (Japanese Patent Publication No. 2011-510309).

[0016] By carrying out the removal step or the suppression step, the detection sensitivity of target extracellular vesicles in a blood-derived sample can be improved. That is, one embodiment of the method of the present invention is a method for improving the detection sensitivity of target extracellular vesicles in a blood-derived sample, which includes a removal step or a suppression step. This method is also referred to as the "method for improving detection sensitivity of the present invention." Specifically, by carrying out the removal step or the suppression step, the detection sensitivity of target extracellular vesicles in a blood-derived sample when carrying out the detection step can be improved. The method for improving detection sensitivity of the present invention may further include a detection step. Examples of improved detection sensitivity include reducing the variability in measured values ​​and reducing non-specific signals (background).

[0017] The removal step or suppression step can be carried out as a pretreatment of a blood-derived sample. That is, one embodiment of the method of the present invention is a method of pretreating a blood-derived sample, which includes a removal step or a suppression step. This method is also referred to as the "pretreatment method of the present invention." The removal step and the suppression step are also collectively referred to as the "pretreatment step." The removal step or suppression step can specifically be carried out as a pretreatment of a blood-derived sample when carrying out a detection step. That is, the pretreatment method of the present invention may specifically be a method of pretreating a blood-derived sample for detecting target extracellular vesicles in the blood-derived sample. The removal step or suppression step can specifically be carried out as a pretreatment for improving the detection sensitivity of target extracellular vesicles in a blood-derived sample. That is, the pretreatment method of the present invention may specifically be a method of pretreating a blood-derived sample for improving the detection sensitivity of target extracellular vesicles in a blood-derived sample.

[0018] By performing the removal or suppression step and the detection step, target extracellular vesicles in a blood-derived sample can be detected. That is, one embodiment of the method of the present invention is a method for detecting target extracellular vesicles in a blood-derived sample, comprising a removal or suppression step and a detection step. This method is also referred to as the "detection method of the present invention." In other words, the detection method of the present invention is also a method for detecting target extracellular vesicles in a blood-derived sample, comprising a step of pretreating the blood-derived sample using the pretreatment method of the present invention and a detection step. By performing the removal or suppression step and the detection step, target extracellular vesicles in a blood-derived sample can be detected with improved detection sensitivity.

[0019] The "detection method of the present invention," "detection sensitivity improving method of the present invention," and "pretreatment method of the present invention" are all generally carried out in vitro.

[0020] The blood-derived sample is not particularly limited as long as it can contain target extracellular vesicles. Examples of blood-derived samples include whole blood, serum, plasma, and fractions thereof. Examples of blood-derived samples include serum and plasma. Blood-derived samples such as serum and plasma may be treated with an anticoagulant such as citric acid, heparin, or EDTA before the removal or suppression step.

[0021] The blood-derived sample may be obtained from a subject. The subject is not particularly limited. The subject may be a human or a non-human animal. Non-human animals include mice, rats, guinea pigs, rabbits, dogs, cats, cows, horses, pigs, monkeys, chimpanzees, and birds. The subject may particularly be a human. The subject may be, for example, a subject suspected of having cancer. The subject may be, for example, a subject to be tested for cancer.

[0022] The embodiment of the removal step is not particularly limited, as long as platelet-derived extracellular vesicles in the blood-derived sample are removed to the desired extent. The removal step may be carried out, for example, using a substance that specifically binds to platelet-derived extracellular vesicles. A substance that specifically binds to platelet-derived extracellular vesicles is also referred to as a "platelet-derived extracellular vesicle-specific binding substance." "Specifically binding to platelet-derived extracellular vesicles" may mean that it binds to platelet-derived extracellular vesicles but does not substantially bind to target extracellular vesicles. Examples of platelet-derived extracellular vesicle-specific binding substances include substances that bind to markers specifically present on the surface of platelet-derived extracellular vesicles. A marker specifically present on the surface of platelet-derived extracellular vesicles is also referred to as a "platelet-derived extracellular vesicle-specific marker." "Specifically present on the surface of platelet-derived extracellular vesicles" may mean that it is present on the surface of platelet-derived extracellular vesicles but is substantially absent on the surface of target extracellular vesicles. Examples of platelet-derived extracellular vesicle-specific markers include proteins specifically present on the surface of platelet-derived extracellular vesicles. Examples of proteins specifically present on the surface of platelet-derived extracellular vesicles include CD41, CD42a, CD42b, CD61, and CD62P. Examples of proteins specifically present on the surface of platelet-derived extracellular vesicles include CD41 in particular. Examples of substances that bind to platelet-derived extracellular vesicle-specific markers include antibodies and aptamers against platelet-derived extracellular vesicle-specific markers. Examples of substances that bind to platelet-derived extracellular vesicle-specific markers include antibodies against platelet-derived extracellular vesicle-specific markers in particular. Examples of antibodies against platelet-derived extracellular vesicle-specific markers include anti-CD41 antibodies, anti-CD42a antibodies, anti-CD42b antibodies, anti-CD61 antibodies, and anti-CD62P antibodies. Examples of antibodies against platelet-derived extracellular vesicle-specific markers include anti-CD41 antibodies. The antibody may be, for example, a whole immunoglobulin or a portion of an immunoglobulin. Antibodies also include Fab, Fab', F(ab')2, Fv, dAb, complementarity determining region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, and diabodies.The antibody may be a monoclonal or polyclonal antibody, and in particular may be a monoclonal antibody.

[0023] Specifically, the removal step may be carried out by binding a substance that specifically binds platelet-derived extracellular vesicles to platelet-derived extracellular vesicles. That is, the removal step may include a step of binding a substance that specifically binds platelet-derived extracellular vesicles to platelet-derived extracellular vesicles. Binding of a substance that specifically binds platelet-derived extracellular vesicles to platelet-derived extracellular vesicles can be carried out by contacting the substance that specifically binds platelet-derived extracellular vesicles with platelet-derived extracellular vesicles. Binding of a substance that specifically binds platelet-derived extracellular vesicles to platelet-derived extracellular vesicles can be carried out by contacting the substance that specifically binds platelet-derived extracellular vesicles with a blood-derived sample (which may contain platelet-derived extracellular vesicles). Contact of the substance that specifically binds platelet-derived extracellular vesicles with platelet-derived extracellular vesicles can be carried out in a liquid medium. Examples of liquid media include aqueous media such as water and aqueous buffer solutions, and organic solvents such as dimethyl sulfoxide. For example, the substance that specifically binds to platelet-derived extracellular vesicles can be brought into contact with platelet-derived extracellular vesicles by mixing the substance that specifically binds to platelet-derived extracellular vesicles with a blood-derived sample in a liquid medium. Alternatively, for example, when the substance that specifically binds to platelet-derived extracellular vesicles is bound to a solid phase and the solid phase is packed in a column, the substance that specifically binds to platelet-derived extracellular vesicles can be brought into contact with platelet-derived extracellular vesicles by passing a blood-derived sample prepared in liquid form through the column.

[0024] The platelet-derived extracellular vesicle-specific binding substance may be bound to a carrier and used in the removal step. By binding the platelet-derived extracellular vesicle-specific binding substance to a carrier, platelet-derived extracellular vesicles can be captured on the carrier via the platelet-derived extracellular vesicle-specific binding substance. The order in which the platelet-derived extracellular vesicle-specific binding substance and the platelet-derived extracellular vesicles are bound to each other is not particularly limited. The platelet-derived extracellular vesicle-specific binding substance and the carrier may typically be bound to each other before the platelet-derived extracellular vesicles are bound to each other. That is, the platelet-derived extracellular vesicle-specific binding substance may typically be bound to a carrier in advance before being used to bind to the platelet-derived extracellular vesicles. The mode of binding between the platelet-derived extracellular vesicle-specific binding substance and the carrier is not particularly limited. The platelet-derived extracellular vesicle-specific binding substance and the carrier may be bound by a covalent bond or a non-covalent bond. Examples of non-covalent bonds include hydrogen bonds and hydrophobic bonds. The bond between the platelet-derived extracellular vesicle-specific binding substance and the carrier may be, in particular, a covalent bond. Examples of the carrier include water-insoluble carriers. The carrier may be hydrophilic or hydrophobic. Specific examples of the carrier include polysaccharides such as agarose, dextran, chitosan, and cellulose; organic synthetic polymers such as polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylonitrile, styrene-divinylbenzene copolymer, polystyrene, acrylic acid ester, methacrylic acid ester, polyethylene, polypropylene, poly4-ethylene fluoride, ethylene-vinyl acetate copolymer, polyamide, polycarbonate, polyvinylidene fluoride, polyvinyl formal, polyarylate, and polyethersulfone; ceramics such as glass, activated carbon, alumina, silica, and hydroxyapatite; and metals such as iron oxide, titanium, and gold. The carrier may be in any shape, for example, particles, fibers, hollow fibers, membranes, or flat plates. The carrier may, in particular, be particles. Examples of particles include magnetic particles. The magnetic particles include particles manufactured by incorporating a magnetic material.

[0025] Platelet-derived extracellular vesicles can be removed from a blood-derived sample by separating the carrier that has captured platelet-derived extracellular vesicles from the blood-derived sample. That is, the removal step may include a step of separating the carrier that has captured platelet-derived extracellular vesicles from the blood-derived sample. Separation of the carrier and the blood-derived sample can be carried out, for example, by a conventional solid-liquid separation means. The solid-liquid separation means can be selected appropriately depending on various conditions, such as the shape of the carrier. For example, when the carrier that has captured platelet-derived extracellular vesicles is dispersed in a liquid containing a blood-derived sample, the carrier can be separated from the blood-derived sample by subjecting the liquid to centrifugation, magnetic separation, or filtration. Specifically, for example, when the carrier is an antibody-immobilized magnetic particle, the blood-derived sample and the antibody-immobilized magnetic particle are mixed to capture the platelet-derived extracellular vesicles on the carrier, and then the antibody-immobilized magnetic particle that has captured the platelet-derived extracellular vesicles is removed using a magnet, thereby separating the carrier (i.e., the antibody-immobilized magnetic particle that has captured platelet-derived extracellular vesicles) from the blood-derived sample. That is, the blood-derived sample can be recovered as a non-adsorbed fraction (i.e., supernatant). Furthermore, for example, when a column is packed with a carrier, the blood-derived sample is passed through the column to capture platelet-derived extracellular vesicles on the carrier, and then the blood-derived sample is discharged from the column, thereby separating the carrier from the blood-derived sample. That is, the blood-derived sample can be recovered from the column as a non-adsorbed fraction.

[0026] The embodiment of the suppression step is not particularly limited, as long as the generation of platelet-derived extracellular vesicles in a blood-derived sample is suppressed to the desired extent. Platelet-derived extracellular vesicles can be generated from platelets contained in a blood-derived sample. Therefore, the suppression step can be achieved by removing platelets from the blood-derived sample. Removal of platelets from a blood-derived sample may be carried out, for example, by performing centrifugation two or more times during preparation of a blood-derived sample (particularly, plasma). The centrifugation conditions are not particularly limited, as long as they allow separation of platelets and target extracellular vesicles. The centrifugation conditions may be, for example, the usual centrifugation conditions used in preparing plasma. Platelet-derived extracellular vesicles can be generated when a blood-derived sample containing platelets is frozen. Therefore, the suppression step may be carried out, for example, by not freezing the blood-derived sample. In other words, "the method of the present invention includes a suppression step, and the suppression step is carried out by not freezing the blood-derived sample" may mean that the method of the present invention does not include a step of freezing the blood-derived sample.

[0027] The embodiment of the detection step is not particularly limited as long as it can detect target extracellular vesicles to the desired extent. Detection of target extracellular vesicles may be qualitative detection (e.g., detection of the presence or absence of target extracellular vesicles) or quantitative detection (e.g., detection of the amount of target extracellular vesicles present). Detection of target extracellular vesicles may particularly be quantitative detection. The method for detecting target extracellular vesicles can be appropriately selected depending on various conditions such as the desired detection sensitivity. "Measurement of target extracellular vesicles" and "detection of target extracellular vesicles" may be used interchangeably. The detection step is performed after the removal step or the suppression step.

[0028] The detection step may be carried out using, for example, a substance that binds to the target extracellular vesicles. A substance that binds to the target extracellular vesicles is also referred to as a "target extracellular vesicle-binding substance." Examples of target extracellular vesicle-specific binding substances include substances that bind to markers present on the surface of the target extracellular vesicles. Markers present on the surface of the target extracellular vesicles are also referred to as "target extracellular vesicle markers." The target extracellular vesicle-binding substance may preferably be a substance that specifically binds to the target extracellular vesicles. A substance that specifically binds to the target extracellular vesicles is also referred to as a "target extracellular vesicle-specific binding substance." "Specifically binding to the target extracellular vesicles" may mean that the substance binds to the target extracellular vesicles but does not substantially bind to the platelet-derived extracellular vesicles. Examples of target extracellular vesicle-specific binding substances include substances that bind to markers specifically present on the surface of the target extracellular vesicles. Markers specifically present on the surface of the target extracellular vesicles are also referred to as "target extracellular vesicle-specific markers." "Specifically present on the surface of target extracellular vesicles" may mean that it is present on the surface of target extracellular vesicles but is substantially absent on the surface of platelet-derived extracellular vesicles.

[0029] Target extracellular vesicle markers include proteins and lipids present on the surface of target extracellular vesicles. Examples of proteins present on the surface of target extracellular vesicles include tetraspanins such as TM4SF1, CD9, CD63, and CD81; antigen presentation-related proteins such as MHC (Major Histocompatibility Complex) I and MHCII; adhesion molecules such as integrins, cadherins, ICAM-1 (Intercellular Adhesion Molecule 1), and EpCAM (Epithelial Cell Adhesion Molecule); cytokines or growth factors such as EGFR (Epidermal Growth Factor Receptor) vIII and TGF (Transforming Growth Factor)-β; receptors for cytokines or growth factors; and enzymes. Lipids present on the surface of target extracellular vesicles include phosphatidylserine (PS) and sphingomyelin. Target extracellular vesicle-specific markers include TM4SF1 and CD81.

[0030] Substances that bind to target extracellular vesicle markers include antibodies and aptamers (peptide aptamers and nucleic acid aptamers) against the target extracellular vesicle marker. The antibody may be, for example, a whole immunoglobulin or a portion of an immunoglobulin. Examples of antibodies include Fab, Fab', F(ab')2, Fv, dAb, complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), single-domain antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, and diabodies. Substances that bind to target extracellular vesicle markers include, in particular, antibodies against the target extracellular vesicle marker. That is, the detection step may be performed, in particular, by immunoassay (i.e., an assay using an antibody). Substances that bind to target extracellular vesicle markers include, more particularly, antibodies against target extracellular vesicle-specific markers. Examples of antibodies against target extracellular vesicle-specific markers include anti-TM4SF1 antibodies and anti-CD81 antibodies. The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may particularly be a monoclonal antibody. Both the antibody and the aptamer may be appropriately modified with a labeling substance. Examples of labeling substances include fluorescent substances such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), and Alexa Fluor (trade name); enzymes such as luciferase, alkaline phosphatase, and horseradish peroxidase; and radioactive substances. The binding mode between the antibody or aptamer and the labeling substance is not particularly limited. The antibody or aptamer may or may not be directly bound to the labeling substance. For example, the antibody may be directly bound to the labeling substance via a chemical bond or the like, or may be indirectly bound to the labeling substance by binding to a secondary antibody (labeled secondary antibody) bound to the labeling substance. Examples of immunoassay methods include EIA (enzyme immunoassay) and Western blotting.Examples of EIA methods include ELISA (Enzyme-Linked Immunosorbent Assay), CLEIA (Chemiluminescent Enzyme Immunoassay), and FEIA (Fluorescence Enzyme Immunoassay). In the detection step, a detection substrate may be used as appropriate. Examples of detection substrates include colorimetric substrates, fluorescent substrates, and luminescent substrates. Examples of detection substrates include luminescent substrates in particular. Examples of luminescent substrates include substrates that generate luminescence through an enzyme reaction, such as horseradish peroxidase. That is, for example, an enzyme corresponding to the luminescent substrate can be used as a labeling substance, and luminescence can be generated by reacting it with the luminescent substrate. Target extracellular vesicles may be detected manually or automatically using a measuring device. Examples of measuring devices include enzyme immunoassay devices such as AIA-900 (Tosoh) and chemiluminescent enzyme immunoassay devices such as AIA-CL2400 (Tosoh).

[0031] The detection step may involve, for example, recovering target extracellular vesicles and detecting the recovered target extracellular vesicles. That is, the detection step may include, for example, a step of recovering target extracellular vesicles on a carrier and a step of detecting the recovered target extracellular vesicles. Methods for recovering target extracellular vesicles include ultracentrifugation, sedimentation, size exclusion chromatography, affinity methods, and polymer precipitation methods. Methods for recovering target extracellular vesicles include, in particular, affinity methods. Specifically, the recovery of target extracellular vesicles may be carried out by capturing the target extracellular vesicles on a carrier. That is, the detection step may include, for example, a step of capturing the target extracellular vesicles on a carrier and a step of detecting the captured target extracellular vesicles. For example, the target extracellular vesicles can be captured on a carrier by an affinity method. Specifically, for example, the target extracellular vesicles can be captured on a carrier via a target extracellular vesicle-binding substance by an affinity method. The capture of target extracellular vesicles onto a carrier can be carried out in the same manner as the capture of platelet-derived extracellular vesicles onto a carrier described above, except that a target extracellular vesicle-binding substance is used instead of a platelet-derived extracellular vesicle-specific binding substance. That is, for example, the carrier is as described above in the description of the capture of platelet-derived extracellular vesicles onto a carrier. Specific examples of the carrier include magnetic particles and microplates. The carrier may be, for example, magnetic particles. The step of capturing target extracellular vesicles onto a carrier may be carried out, for example, using a first substance that binds to the target extracellular vesicles. The step of detecting the captured target extracellular vesicles may be carried out, for example, using a second substance that binds to the target extracellular vesicles. Both the first substance and the second substance are target extracellular vesicle-binding substances. Both the first substance and the second substance may be, for example, an antibody against a target extracellular vesicle marker. That is, the first substance may be an antibody (also referred to as a first antibody) against a first marker present on the target extracellular vesicles. The second substance may be an antibody (also referred to as a second antibody) against a second marker present on the target extracellular vesicles. The first antibody and the second antibody may, for example, be antibodies against different target extracellular vesicle markers.For example, a sandwich EIA method such as a sandwich CLEIA method can be performed using a first antibody and a second antibody. One or both of the first substance and the second substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker). In other words, one or both of the first marker and the second marker may be a target extracellular vesicle-specific marker. For example, at least the first substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker), and the second substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker). In other words, at least the first marker may be a target extracellular vesicle-specific marker, and the second marker may be a target extracellular vesicle-specific marker. Furthermore, for example, at least the second substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker), and further, the first substance may be a target extracellular vesicle-specific binding substance (e.g., an antibody against a target extracellular vesicle-specific marker). In other words, at least the second marker may be a target extracellular vesicle-specific marker, and further, the first marker may be a target extracellular vesicle-specific marker. The first marker may be, for example, a target extracellular vesicle-specific marker such as TM4SF1 or CD81, among others. The second marker may be, for example, CD9 or CD81, among others.

[0032] The use of the detection results of target extracellular vesicles is not particularly limited.

[0033] For example, when the target extracellular vesicles are extracellular vesicles derived from cancer cells, the detection results of the target extracellular vesicles can be used to test for cancer. Specifically, when a blood-derived sample is obtained from a subject and the target extracellular vesicles are extracellular vesicles derived from cancer cells, the detection results of the target extracellular vesicles can be used to test for cancer in the subject. Testing for cancer in a subject includes testing whether the subject has cancer, testing the possibility or severity of cancer in the subject, and testing the progression (e.g., stage) of cancer if the subject has cancer. "Cancer testing" may also be interpreted as "cancer diagnosis."

[0034] <2> Kit of the Present Invention One embodiment of the kit of the present invention is a kit for pretreatment of a blood-derived sample. This kit is also referred to as the "pretreatment kit of the present invention." The pretreatment of the blood-derived sample is as described above in the method of the present invention.

[0035] One embodiment of the kit of the present invention is a kit for detecting target extracellular vesicles in a blood-derived sample. This kit is also referred to as the "detection kit of the present invention." The detection of target extracellular vesicles in a blood-derived sample is as described above in the method of the present invention.

[0036] The kit of the present invention may include, for example, a reagent used in the method of the present invention. The kit of the present invention may include, for example, a laboratory instrument used in the method of the present invention. The kit of the present invention may include, for example, an instruction manual for the kit of the present invention.

[0037] The kit of the present invention may include, for example, a platelet-derived extracellular vesicle-specific binding substance. The kit of the present invention may include, for example, a carrier for binding to the platelet-derived extracellular vesicle-specific binding substance. The kit of the present invention may include, for example, a pre-bound platelet-derived extracellular vesicle-specific binding substance and a carrier.

[0038] The kit of the present invention may include, for example, a target extracellular vesicle-binding substance. The kit of the present invention may include, for example, a first substance and / or a second substance. The kit of the present invention may include, for example, a carrier for binding to the first substance. The kit of the present invention may include, for example, a pre-bound first substance and a carrier. The kit of the present invention may include, for example, a labeling substance for labeling the target extracellular vesicle-binding substance (e.g., a second substance). The kit of the present invention may include, for example, a pre-bound target extracellular vesicle-binding substance (e.g., a second substance) and a labeling substance. The kit of the present invention may include, for example, a detection substrate.

[0039] The kit of the present invention can be used, for example, to carry out the method of the present invention.

[0040] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. Note that the terms "Example," "Comparative Example," and "Reference Example" are relative terms and do not necessarily indicate whether or not the examples fall within the scope of the present invention.

[0041] Example 1 Preparation of antibody-immobilized magnetic particles (1) To 400 μL of a solution of magnetic particles having carboxy groups on the surface (Magnosphere MS300 / Carboxyl, 1.0% (w / v) slurry, manufactured by JSR Life Sciences Corporation), 600 μL of 0.01 M MES (2-morpholinoethanesulfonic acid) buffer solution (pH 6.0) (hereinafter referred to as "Buffer A") was added.

[0042] (2) The magnetic particle suspension obtained in (1) was brought close to a magnet and left for 1 minute, the supernatant was removed, and then 1 mL of buffer A was added to resuspend the magnetic particles, a washing procedure that was carried out twice. The washed magnetic particles were resuspended in 450 μL of buffer A.

[0043] (3) 50 μL of buffer solution A containing 10 mg / mL EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was added to the magnetic particle suspension obtained in (2), and the mixture was shaken at 37° C. for 1 hour to bind EDC to the carboxy groups on the surface of the magnetic particles. This procedure allows the carboxylic acid on the surface of the magnetic particles to be covalently bonded to the amino groups of proteins, etc. via EDC.

[0044] (4) The EDC-bound magnetic particles obtained in (3) were washed once with 1 mL of buffer A and resuspended in 180 μL of buffer A. 20 μL of a solution containing one of the proteins listed below (a) to (c) was added, and the mixture was shaken at 37°C for 3 hours to covalently bond the protein to the magnetic particles via EDC (i.e., immobilize the protein on the magnetic particles). After immobilization, the particles were washed once with 1 mL of buffer A containing 0.4 M sodium chloride. [a] Buffer A containing 1 mg / mL anti-human TM4SF1 antibody (manufactured by R&D Systems), [b] Buffer A containing 0.5 mg / mL anti-human CD41 antibody (manufactured by BioLegend), and [c] Buffer A containing 1 mg / mL mouse IgG (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0045] (5) The protein-immobilized magnetic particles obtained in (4) were washed twice with 1 mL of buffer A containing 0.9% (w / v) Blockmaster CE210 (a low protein-binding polymer, manufactured by JSR Life Sciences Corporation), resuspended in 500 μL of buffer A, and then blocked by shaking overnight (17 to 24 hours) at 37°C.

[0046] (6) After blocking, the particles were washed once with 1 mL of 0.1 M Tris-HCl buffer (pH 8.0) containing 0.2% (w / v) Blockmaster CE210, and then resuspended in 200 μL of buffer A to obtain anti-TM4SF1 antibody-immobilized magnetic particles, anti-CD41 antibody-immobilized magnetic particles, and mouse IgG-immobilized magnetic particles as a control.

[0047] Example 2 Preparation of biotinylated modified antibodies (1) 200 μL of a solution containing one of the antibodies shown below in [d] and [e] was added to a filtration tube (manufactured by Dojin Chemical Research Institute), and the tube was centrifuged at 8,000 × g for 10 minutes at room temperature to immobilize the antibody on the filter. [d] 1 mg / mL anti-human CD9 antibody (manufactured by Frontier Research Institute) [e] 1 mg / mL anti-human CD81 antibody (manufactured by Frontier Research Institute)

[0048] (2) 100 μL of Reaction Buffer (manufactured by Dojin Chemical Laboratory) was added to the filter tube, followed by the addition of NH2-Reactive Biotin (manufactured by Dojin Chemical Laboratory) suspended in 10 μL of dimethyl sulfoxide. After suspension, the filter tube was incubated at 37°C for 10 minutes to biotinylate the antibody.

[0049] (3) 100 μL of WS Buffer (manufactured by Dojin Chemical Laboratory) was added to the filter tube, and the mixture was centrifuged at 8,000×g for 10 minutes at room temperature, and the flow-through was then removed.

[0050] (4) 200 μL of WS Buffer (manufactured by Dojin Chemical Laboratory) was added to the filter tube, and the mixture was centrifuged at 8,000×g for 10 minutes at room temperature, and the flow-through was then removed.

[0051] (5) (4) was repeated.

[0052] (6) 200 μL of WS Buffer (manufactured by Dojin Chemical Laboratory) was added to the filter tube and suspended to obtain a biotin-modified anti-CD9 antibody solution and a biotin-modified anti-CD81 antibody solution.

[0053] Example 3: Detection of background in plasma measurement In the following comparative examples, examples, and reference examples, PC-3-derived extracellular vesicles are used as target extracellular vesicles. TM4SF1 (a member of the transmembrane 4 superfamily (tetraspanin), a protein that is highly expressed in many tumor cells, including PC-3, and in tumor cell-derived extracellular vesicles) was selected as a marker protein for detecting PC-3-derived extracellular vesicles. TM4SF1 is not expressed in blood cell-derived extracellular vesicles contained in plasma. Plasma is used as the specimen sample. In this example, background detection in plasma measurement was performed. Here, "background" refers to the measurement value (signal) of a specimen that does not contain target extracellular vesicles. If a specimen contains a large amount of impurities, the noise signal increases, resulting in a high background and making accurate measurements impossible.

[0054] (1) Preparation of Plasma Samples (1-1) Five mL of blood was collected from one healthy subject who had given informed consent into an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo Corporation). The blood collection tube was centrifuged at 1,500 × g for 20 minutes at 4°C to separate blood cells, and the supernatant was collected. The supernatant was centrifuged at 1,500 × g for 20 minutes at 4°C to separate remaining blood cells, and the supernatant was collected to obtain plasma from a healthy subject.

[0055] (1-2) 250 μL of the obtained plasma from healthy subjects was frozen and stored at −80°C.

[0056] (1-3) Cryopreserved plasma from a healthy subject was thawed at 4°C for approximately 1 hour, diluted with 1 mL of PBS (Phosphate Buffered Saline), and then centrifuged at 10,000 × g for 20 minutes at 4°C to separate blood cells remaining in the plasma sample. The supernatant was then collected to obtain a plasma sample.

[0057] (2) Background Measurement (2-1) 1 mL of PBS containing 3% (w / v) bovine serum albumin (BSA) (hereinafter referred to as "BSA buffer") and 10 μL of the anti-TM4SF1 antibody-immobilized magnetic particles prepared in Example 1 were mixed in a 2 mL tube and stirred by end-over-end for 3 minutes. The 2 mL tube was brought close to a magnet and then left for 1 minute, the solution was removed, and then 1 mL of BSA buffer was added and stirred by end-over-end for 10 minutes or more. The 2 mL tube was brought close to a magnet and then left for 1 minute, the solution was removed, and then 50 μL of BSA buffer was added to prepare an anti-TM4SF1 antibody-immobilized magnetic particle suspension.

[0058] (2-2) 50 μL of the anti-TM4SF1 antibody-immobilized magnetic particle suspension obtained in (2-1) and 100 μL of the plasma sample obtained in (2-1) were mixed in a well of a 96-well plate and stirred for 90 minutes. If the plasma contains target extracellular vesicles (such as cancer cell-derived extracellular vesicles), this procedure allows the target extracellular vesicles to be bound to the magnetic particles via the anti-TM4SF1 antibody.

[0059] (2-3) After stirring, a magnet was brought close to the bottom of the 96-well plate and left there for 1 minute. After removing the solution, 150 μL of PBS containing 0.05% (v / v) Tween 20 (trade name) (hereinafter referred to as "washing buffer") was added and mixed. A magnet was again brought close to the bottom of the 96-well plate and left there for 1 minute, and the solution was removed to wash the magnetic particles. This washing procedure was performed a total of three times.

[0060] (2-4) After removing the washing solution, a mixture of 90 μL of BSA buffer and 10 μL of FcR Blocking Reagent (Miltenyi Biotec) was added as a blocking reagent, and the mixture was stirred for 10 minutes.

[0061] (2-5) After removing the blocking reagent, 100 μL of BSA buffer containing 0.5 μg / mL biotin-modified anti-CD9 antibody was added and stirred for 30 minutes. As in (2-3), the supernatant was removed using a magnet, and the plate was washed three times with the washing buffer.

[0062] (2-6) After removing the washing solution, 100 μL of BSA buffer containing streptavidin polyvalently bound to HRP (horseradish peroxidase) was added and stirred for 20 minutes. As in (2-3), the supernatant was removed using a magnet, and the plate was washed three times with the washing buffer.

[0063] (2-7) After removing the wash solution, 150 μL of a substrate reaction solution containing a luminescent substrate, SuperSignal ELISA Pico Chemiluminescent Substrate (manufactured by ThermoFisher), was added and stirred for 5 minutes. 100 μL of the substrate reaction solution containing a magnet was collected and transferred to a new 96-well plate. The luminescence level was measured using a plate reader (manufactured by Tecan) to detect background light in the plasma measurement.

[0064] Comparative Example 1 Detection of background in plasma measurement (1) Preparation of plasma samples Plasma samples were obtained in the same manner as in Example 3(1-1), except that in Example 3(1-1), the blood collection tubes were each centrifuged at 1,500 × g for 10 minutes at 4°C to separate blood cells, and the supernatant was then collected to obtain plasma from healthy subjects.

[0065] (2) Measurement of background The background in the plasma measurement was detected in the same manner as in Example 3(2), except that the plasma sample obtained in (1) was used instead.

[0066] Comparative Example 2 Detection of background in plasma measurement when pretreatment was performed using mouse IgG-immobilized magnetic particles (1) Preparation of plasma sample Plasma samples were prepared in the same manner as in Comparative Example 1 (1).

[0067] (2) Pretreatment using mouse IgG-immobilized magnetic particles (2-1) 1 mL of BSA buffer and 10 μL of the mouse IgG-immobilized magnetic particles prepared in Example 1 were mixed in a 2 mL tube and stirred by inversion for 3 minutes. The 2 mL tube was brought close to a magnet and then left for 1 minute, the solution was removed, and then 1 mL of BSA buffer was added and stirred by inversion for 10 minutes or more. The 2 mL tube was brought close to a magnet and then left for 1 minute, and the solution was removed.

[0068] (2-2) The mouse IgG-immobilized magnetic particles prepared in (2-1) were suspended in 100 μL of the plasma sample prepared in (1), and the suspension was stirred by inversion for 90 minutes.

[0069] (2-3) After stirring, the plasma sample containing the mouse IgG-immobilized magnetic particles was brought close to a magnet and left for 1 minute, and the supernatant was collected to obtain a pretreated plasma sample.

[0070] (3) Measurement of background The background in plasma measurement was detected in the same manner as in Example 3(2), except that the plasma sample in Example 3(2) was the pretreated plasma sample obtained in (2-3).

[0071] Example 4 Detection of background in plasma measurement when pretreatment was performed using anti-CD41 antibody-immobilized magnetic particles Background in plasma measurement was detected in the same manner as in Comparative Example 2 (2), except that anti-CD41 antibody-immobilized magnetic particles were used.

[0072] The results of Examples 3 and 4 and Comparative Examples 1 and 2 are shown in Table 1. The background luminescence intensity, which was 160,000 in Example 3, increased to 1,000,000 in Comparative Example 1. In Example 3, almost all blood cells were removed from the plasma by two centrifugations, while in Comparative Example 1, a large number of blood cells remained in the plasma due to milder conditions and a single centrifugation. The remaining blood cells are thought to release extracellular vesicles through freezing and thawing. In particular, platelets are known to be activated by physical stimuli such as freezing and thawing, and release extracellular vesicles upon activation. In Comparative Example 1, although the remaining blood cells themselves were removed by centrifugation (10,000 × g) after thawing, it is presumed that the extracellular vesicles derived from the blood cells did not precipitate but remained in the supernatant. In this study, TM4SF1, which was used as a detection marker, is not expressed on blood cells, so it is unlikely that blood cell-derived extracellular vesicles were specifically detected by TM4SF1. However, we decided to examine the possibility that blood cell-derived extracellular vesicles were somehow involved in increasing the background luminescence intensity.

[0073] Therefore, we investigated whether platelet-derived extracellular vesicles, which are particularly abundant among blood cell-derived extracellular vesicles, were removed by pretreatment using magnetic particles immobilized with antibodies against CD41 expressed on platelets to confirm whether background measurement could be reduced. First, to confirm that platelet-derived extracellular vesicles do not nonspecifically adsorb to the antibody-immobilized magnetic particles, a plasma sample was reacted with magnetic particles immobilized with mouse IgG protein as an antibody control, followed by background measurement (Comparative Example 2). The resulting luminescence intensity (1,000,000) was equivalent to that of Comparative Example 1, which did not undergo pretreatment, indicating no reduction in background due to nonspecific adsorption to the antibody-immobilized magnetic particles. Next, a plasma sample was reacted with anti-CD41 antibody-immobilized magnetic particles for pretreatment, followed by background measurement (Example 4). The luminescence intensity was reduced to 380,000 compared to Comparative Examples 1 and 2. This indicates that the background was reduced by specifically binding and removing platelet-derived extracellular vesicles contained in the plasma to the anti-CD41 antibody-immobilized magnetic particles.

[0074] These results demonstrate for the first time that platelet-derived extracellular vesicles are the main cause of increased background luminescence in plasma measurements. Furthermore, we confirmed that pretreatment with anti-CD41 antibody-immobilized magnetic particles removes platelet-derived extracellular vesicles from plasma, thereby reducing background luminescence.

[0075] [Table 1]

[0076] Example 5 Recovery of Extracellular Vesicles Derived from Diseased Cells PC-3 cells, which are human prostate cancer cells, were selected as diseased cells to be used in the following examples.

[0077] (1) 1 x 10 PC-3 cells ５ The cells were suspended in Ham's F-12K (Fujifilm Wako Pure Chemical Industries, Ltd.) medium containing 15% (v / v) FBS (fetal bovine serum) to a concentration of 100 cells / mL, and then seeded in five 15-cm dishes (30 mL each). ２ The cells were cultured at 37°C under ambient conditions.

[0078] (2) To remove bovine extracellular vesicles from the FBS, 15 mL of FBS was added to a centrifugal ultrafiltration filter, Amicon Ultra-15 (100K) (Merck), and centrifuged at 3,500 × g for 10 minutes at room temperature. The solution that passed through the ultrafiltration filter was collected and then passed through a syringe filter (pore size 0.22 μm) to obtain bovine extracellular vesicle-free FBS.

[0079] (3) After culturing the PC-3 cells obtained in (1) for 2 days, the culture supernatant was replaced with Ham's F-12K medium containing 15% (v / v) bovine extracellular vesicle-free FBS, and the cells were cultured.

[0080] (4) The PC-3 cells obtained in (3) were further cultured for 3 days, and the entire culture supernatant (approximately 125 mL) was then collected. The cells were centrifuged at 300 × g for 10 minutes at room temperature to remove floating cells, and 120 mL of the supernatant was collected.

[0081] (5) The supernatant obtained in (4) was diluted to a uniform volume and dispensed into ultracentrifuge tubes. The tubes were centrifuged at 300,000 × g for 70 minutes at 4°C, and 120 mL of the supernatant was removed. The remaining precipitate was suspended in 4 mL of PBS, and the resulting suspension was centrifuged at 259,000 × g for 70 minutes at 4°C, after which 4 mL of the supernatant was removed. 1 mL of the remaining precipitate was used as a PC-3-derived extracellular vesicle solution.

[0082] Comparative Example 3 Detection of PC-3-Derived Extracellular Vesicles in Plasma (1) Preparation of Plasma Samples (1-1) Plasma from healthy subjects was obtained in the same manner as in Example 3 (1-1), except that blood was collected from three healthy subjects, and the blood collection tubes were centrifuged at 1,500 × g for 10 minutes at 4°C to separate blood cells, and the supernatant was collected.

[0083] (1-2) Plasma samples containing PC-3-derived extracellular vesicles at six concentrations shown below in [f] to [k] were prepared by adding 100 μL of a solution prepared by diluting the PC-3-derived extracellular vesicles obtained in Example 5 with PBS to 250 μL of plasma from a healthy subject. These samples were then frozen and stored at −80°C. [f] 2.5 × 10 ７ particles / mL [g]5.0×10 ７ particles / mL [h] 1.0×10 ８particles / mL [i]2.5×10 ８ particles / mL [j]5.0×10 ８ particles / mL [k]1.0×10 ９ particles / mL

[0084] (1-3) To prepare a plasma sample free of PC-3-derived extracellular vesicles, 100 μL of PBS was added to 250 μL of plasma from a healthy subject, and the mixture was frozen and stored at −80°C.

[0085] (1-4) The plasma samples cryopreserved in (1-2) and (1-3) were thawed at 4°C for approximately 1 hour and then diluted with 900 µL of PBS. The diluted plasma samples were centrifuged at 10,000 × g for 20 minutes at 4°C to separate blood cells remaining in the plasma samples, and the supernatant was collected to obtain plasma samples.

[0086] (2) Measurement of PC-3-derived extracellular vesicles PC-3-derived extracellular vesicles were detected in the same manner as in Example 3(2), except that the plasma sample obtained in (1) was used instead.

[0087] Example 6 Detection of PC-3-Derived Extracellular Vesicles in Plasma of Healthy Subjects from Which Platelet-Derived Extracellular Vesicles Have Been Removed (1) Preparation of Plasma Samples Plasma samples were prepared in the same manner as in Comparative Example 3(1).

[0088] (2) Removal of platelet-derived extracellular vesicles A pretreated plasma sample was obtained in the same manner as in Example 4, except that the plasma sample obtained in (1) was used instead.

[0089] (3) Measurement of PC-3-derived extracellular vesicles PC-3-derived extracellular vesicles in plasma were detected in the same manner as in Example 3(2), except that the plasma sample was the pretreated plasma sample obtained in (2).

[0090] Reference Example 1 Detection of PC-3-Derived Extracellular Vesicles in Plasma from Healthy Subjects from Which Platelets Have Been Removed (1) Preparation of Plasma Samples (1-1) Plasma from healthy subjects was obtained in the same manner as in Example 3 (1-1).

[0091] (1-2) In Comparative Example 3 (1-2), the plasma from a healthy subject obtained in (1-1) was used as the plasma from a healthy subject, and a plasma sample was prepared in the same manner as in Comparative Example 3 (1-2), except that the plasma was not frozen and stored at −80° C. and the step (2) was carried out immediately after preparation.

[0092] (1-3) In Example 3 (1-3), the plasma from a healthy subject obtained in (1-1) was used as the plasma from a healthy subject, and a plasma sample not containing PC-3-derived extracellular vesicles was prepared in the same manner as in Comparative Example 3 (1-3), except that the plasma was not frozen and stored at −80° C., and the process was immediately carried out after preparation in step (2).

[0093] (1-4) A plasma sample was obtained in the same manner as in Comparative Example 3 (1-4), except that the thawing procedure was not carried out.

[0094] (2) Measurement of PC-3-derived extracellular vesicles PC-3-derived extracellular vesicles were detected in the same manner as in Example 3(2), except that the plasma sample obtained in (1) was used instead.

[0095] The results of Comparative Example 3, Example 6, and Reference Example 1 are shown in Table 2 and Figures 1 to 3. In Table 2, the "coefficient of variation" is the value obtained by dividing the standard deviation of the luminescence intensity by the average luminescence intensity; the lower this value, the smaller the variation in measured values ​​between samples, and the higher the measurement accuracy. In Table 2, the "threshold value" is the value obtained by adding twice the standard deviation of the luminescence intensity in plasma samples not containing cell-derived extracellular vesicles to the average luminescence intensity. In Table 2, the "lower detection limit concentration" refers to the lowest concentration of added PC-3-derived extracellular vesicles at which the luminescence intensity is higher than the threshold; the lower the luminescence intensity and standard deviation in plasma samples not containing PC-3 cell-derived extracellular vesicles, the lower the lower detection limit concentration, and the higher the detection sensitivity.

[0096] In Comparative Example 3, Example 6, and Reference Example 1, the amount of luminescence increased depending on the concentration of PC-3-derived extracellular vesicles added, confirming that PC-3-derived extracellular vesicles can be detected in plasma.

[0097] However, when focusing on the average luminescence intensity of plasma samples not containing PC-3 cell-derived extracellular vesicles, Comparative Example 3 showed a significantly higher value than Example 6 and Reference Example 1. Similar to the results of Comparative Example 1, this result is thought to be due to an increase in background luminescence intensity caused by platelet-derived extracellular vesicles released by freezing and thawing from platelets remaining in the plasma. Furthermore, in Comparative Example 3, the noise signal amount differed between the samples, resulting in a high coefficient of variation (0.50), and therefore the lower limit of detection concentration was high (1.0 × 10 ９ particles / mL).

[0098] Therefore, when the frozen-thawed plasma was pretreated with anti-CD41 antibody-immobilized magnetic particles (Example 6), the background luminescence intensity was reduced, and the coefficient of variation (0.23) and the lower limit of detection concentration (2.5 × 10 8 Therefore, by removing platelet-derived extracellular vesicles from plasma by pretreatment with anti-CD41 antibody-immobilized magnetic particles, it became possible to detect the target PC-3-derived extracellular vesicles with higher accuracy and sensitivity.

[0099] Furthermore, by subjecting plasma from which almost all platelets had been removed in advance to measurement without freezing and thawing (Reference Example 1), the background luminescence intensity was reduced as in Example 6, and the coefficient of variation (0.36) and the lower limit of detection concentration (1.0 × 10 8 Therefore, it was demonstrated that the detection sensitivity and measurement accuracy of target extracellular vesicles can be improved by suppressing the generation of platelet-derived extracellular vesicles.

[0100] Generally, the concentration of extracellular vesicles derived from diseased cells contained in the plasma of patients with diseases including cancer is 10 ８ Therefore, the lower limit of detection for the measurement method for detecting extracellular vesicles derived from disease cells in plasma is estimated to be 10 ８The concentration must be at least 1000 particles / mL. Therefore, the method for measuring plasma containing platelet-derived extracellular vesicles, as shown in Comparative Example 3, has a lower detection limit that far exceeds the concentration of the disease cell-derived extracellular vesicles, making it difficult to apply to detecting disease cell-derived extracellular vesicles in plasma samples from disease patients. On the other hand, as shown in Example 6 and Reference Example 1, it is suggested that a plasma measurement method that includes a step of removing platelet-derived extracellular vesicles or that suppresses the generation of platelet-derived extracellular vesicles can be applied to plasma samples from disease patients.

[0101] [Table 2]

[0102] Comparative Example 4 Detection of PC-3-Derived Extracellular Vesicles in Plasma PC-3-derived extracellular vesicles in plasma were detected in the same manner as in Comparative Example 3 (2), except that 0.5 μg / mL biotin-modified anti-CD81 antibody was used instead of 0.5 μg / mL biotin-modified anti-CD9 antibody.

[0103] Example 7 Detection of PC-3-derived extracellular vesicles in plasma from healthy subjects from which platelet-derived extracellular vesicles had been removed PC-3-derived extracellular vesicles in plasma were detected in the same manner as in Example 6 (3), except that 0.5 μg / mL biotin-modified anti-CD81 antibody was used instead of 0.5 μg / mL biotin-modified anti-CD9 antibody.

[0104] Reference Example 2 Detection of PC-3-derived extracellular vesicles in platelet-removed plasma of healthy subjects PC-3-derived extracellular vesicles in plasma were detected in the same manner as in Reference Example 1 (2), except that 0.5 μg / mL biotin-modified anti-CD81 antibody was used instead of 0.5 μg / mL biotin-modified anti-CD9 antibody.

[0105] The results of Comparative Example 4, Example 7, and Reference Example 2 are shown in Table 3 and Figures 4 to 6. In Table 3, the "coefficient of variation" is the value obtained by dividing the standard deviation of the luminescence intensity by the average luminescence intensity; the lower this value, the smaller the variation in measured values ​​between samples, and the higher the measurement accuracy. In Table 3, the "threshold value" is the value obtained by adding twice the standard deviation of the luminescence intensity in plasma samples not containing cell-derived extracellular vesicles to the average luminescence intensity. In Table 3, the "lower detection limit concentration" refers to the lowest concentration of added PC-3-derived extracellular vesicles at which the luminescence intensity is higher than the threshold; the lower the lower detection limit concentration, the higher the detection sensitivity. Therefore, the detection sensitivity decreases as the luminescence intensity and standard deviation increase in plasma samples not containing PC-3 cell-derived extracellular vesicles.

[0106] In Comparative Example 4, Example 7, and Reference Example 2, the luminescence intensity increased in a concentration-dependent manner depending on the addition of PC-3-derived extracellular vesicles, confirming that PC-3-derived extracellular vesicles can be detected in plasma. Furthermore, in all results, the average luminescence intensity of plasma samples not containing PC-3 cell-derived extracellular vesicles was equally low. This is presumably because, while CD9, which is also expressed in platelets, was used as a detection marker in Comparative Example 3, Example 6, and Reference Example 1, CD81, which is not expressed in platelets, was used as a detection marker, thereby suppressing noise signal generation.

[0107] Focusing on the lower detection limit concentration, Example 7 showed a lower value compared to Comparative Example 4 and Reference Example 2. This is presumably because, compared to Comparative Example 4 and Reference Example 2, the total amount of extracellular vesicles contained in plasma was reduced in Example 7 by the step of removing platelet-derived extracellular vesicles, and the reactivity between the target extracellular vesicles (PC-3-derived extracellular vesicles) and the anti-TM4SF1 antibody-immobilized magnetic particles was improved, thereby increasing measurement stability and reducing variability in measurement values ​​between samples.

[0108] The above results suggest that even when CD81 is used as a detection marker, the detection sensitivity and measurement accuracy of the target extracellular vesicles can be improved by the step of removing platelet-derived extracellular vesicles, and that this measurement method is applicable to the detection of disease cell-derived extracellular vesicles in plasma samples from disease patients.

[0109] [Table 3]

[0110] As shown in Examples 6 and 7, the detection method of the present invention can detect target extracellular vesicles from plasma with high sensitivity and accuracy, suggesting that the method is also applicable to detecting extracellular vesicles derived from disease cells in plasma samples from disease patients.

Claims

1. A method for detecting target extracellular vesicles in a blood-derived sample, comprising: step A1 of removing platelet-derived extracellular vesicles in the blood-derived sample or step A2 of suppressing the occurrence of platelet-derived extracellular vesicles in the blood-derived sample; and step B of detecting the target extracellular vesicles in the blood-derived sample.

2. A method for improving the detection sensitivity of target extracellular vesicles in a blood-derived sample, comprising step A1 of removing platelet-derived extracellular vesicles in the blood-derived sample or step A2 of suppressing the occurrence of platelet-derived extracellular vesicles in the blood-derived sample.

3. A method for pretreating a blood-derived sample for detecting target extracellular vesicles in the blood-derived sample, the method comprising step A1 of removing platelet-derived extracellular vesicles in the blood-derived sample or step A2 of suppressing the occurrence of platelet-derived extracellular vesicles in the blood-derived sample.

4. The method according to any one of claims 1 to 3, wherein the target extracellular vesicles are extracellular vesicles other than platelet-derived extracellular vesicles.

5. The method of claim 4, wherein the extracellular vesicles other than platelet-derived extracellular vesicles are tumor cell-derived extracellular vesicles.

6. The method of claim 5, wherein the tumor cell-derived extracellular vesicles are prostate cancer-derived extracellular vesicles.

7. The method according to any one of claims 1 to 3, wherein the blood-derived sample is plasma or serum.

8. The method according to any one of claims 1 to 3, wherein step A1 is carried out using a substance that specifically binds to the platelet-derived extracellular vesicles.

9. The method of claim 8, wherein the substance is an antibody against a marker specifically present on the platelet-derived extracellular vesicles.

10. The method of claim 9, wherein the marker is CD41.

11. The method according to any one of claims 1 to 3, wherein step A2 is carried out by removing platelets from the blood-derived sample.

12. The method of claim 11, wherein the removal of platelets is performed by performing two or more centrifugations during preparation of the blood-derived sample.

13. The method according to any one of claims 1 to 3, wherein step A2 is carried out by not freezing the blood-derived sample.

14. The method of claim 1, wherein step B is carried out by an immunoassay.

15. The method of claim 1, wherein step B comprises step B1 of capturing the target extracellular vesicles on a carrier, and step B2 of detecting the captured target extracellular vesicles.

16. The method of claim 15, wherein step B1 is carried out using a first substance that binds to the target extracellular vesicles, and step B2 is carried out using a second substance that binds to the target extracellular vesicles.

17. The method of claim 16, wherein the first substance is an antibody against a first marker present on the target extracellular vesicles, and the second substance is an antibody against a second marker present on the target extracellular vesicles.

18. The method of claim 17, wherein the first marker is a marker that is specifically present in the target extracellular vesicle.

19. The method of claim 17, wherein the first marker is TM4SF1 or CD81.

20. The method of any one of claims 17 to 19, wherein the second marker is CD9 or CD81.