Exosome isolation and detection method, and isolation and detection kit

Abstract

This method for separating and detecting exosomes comprises: a complex formation step for contacting a biological sample with a capture molecule, said capture molecule containing a substance specifically binding to an antigen expressed on cancer cell surface, to form an exosome-capture molecule complex; and a detection step for contacting the complex with a detection molecule, said detection molecule containing a substance specifically binding to an antigen expressed on cancer cell surface and a labeling substance, and then detecting the complex with the use of the detection molecule, wherein, in the capture molecule and / or the detection molecule, the antigen expressed on cancer cell surface is cell surface vimentin.

Description

[Technical field]

[0001] The present invention relates to a method for isolating and detecting exosomes, and a separation and detection kit. [Background technology]

[0002] It is known that cells release exosomes, which are small lipid bilayer vesicles that contain various cellular contents such as proteins, nucleic acids, lipids, and sugars, to the outside of the cell. Exosomes have attracted attention as important clues for understanding the pathology of the body, and research into various diseases including cancer is being actively conducted.

[0003] On the other hand, vimentin is one of the proteins important for cytoplasm formation and is known as a marker for intracellular epithelial-mesenchymal transition (EMT). It has been revealed that vimentin is overexpressed in cancer cells, and that there is a correlation between the amount of expression and the progression of cancer. Under certain circumstances, vimentin migrates to the cell surface. This vimentin that has migrated to the cell surface is called cell surface vimentin (CSV).

[0004] It has been revealed that CSV is involved in autoimmune deficiency, viral infection, and cancer, and is also involved in inflammatory diseases because CSV is expressed in activated macrophages, platelets, and apoptotic T lymphocytes. Recently, a method for detecting circulating tumor cells (CTCs) has been reported by combining an anti-CSV antibody with an antibody against epithelial cell adhesion molecule (EpCAM, CD326), which is known as a diagnostic marker for carcinoma (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Satelli A et al., “Circulating tumor cell enumeration using a combination of EpCAM and Cell-surface vimentin based methods for monitoring breast cancer therapeutic response.”, Clin Chem., Vo. 61, Issue 1, pp. 259-266, 2015. Summary of the Invention [Problem to be solved by the invention]

[0006] However, CTCs exist in blood in extremely small quantities, making it difficult to detect CTC antigen markers for the purpose of cancer diagnosis or companion diagnosis.

[0007] The present invention has been made in consideration of the above circumstances, and provides a method for isolating and detecting exosomes, which can be used for cancer diagnosis or companion diagnosis, and a kit used for said method. [Means for solving the problem]

[0008] That is, the present invention includes the following aspects. (1) a complex formation step of contacting a biological sample with a capture molecule containing a substance that specifically binds to an antigen expressed on the surface of a cancer cell to form a complex between an exosome and the capture molecule; a detection step of contacting the complex with a detection molecule comprising a labeling substance and a substance that specifically binds to an antigen expressed on the surface of a cancer cell, and detecting the complex with the detection molecule; Including, The method for separating and detecting exosomes, wherein an antigen expressed on the surface of the cancer cell in at least one of the capturing molecule and the detecting molecule is CSV. (2) The method according to (1), wherein the substance that specifically binds to CSV is an anti-CSV antibody. (3) The method according to (1) or (2), wherein the capture molecule has a substance that specifically binds to an antigen expressed on the surface of cancer cells, and the detection molecule has a substance that specifically binds to an antigen expressed on the surface of cancer cells, and the detection molecule has a substance that specifically binds to an antigen expressed on the surface of cancer cells, and the substance is an anti-CSV antibody, an anti-EpCAM antibody, or an anti-PD-L1 antibody. (4) The method according to (1) or (2), wherein the detection molecule has a specific binding substance for an antigen expressed on the surface of cancer cells that is an anti-CSV antibody, and the capture molecule has a specific binding substance for an antigen expressed on the surface of cancer cells that is an anti-CSV antibody, an anti-EpCAM antibody, or an anti-PD-L1 antibody. (5) The method according to any one of (1) to (4), wherein the capturing molecule is immobilized on a magnetic particle. (6) After the complex formation step and before the detection step, The method according to any one of (1) to (5), further comprising a washing step of adding a washing solution to remove contaminants other than the complex. (7) The method according to any one of (1) to (6), wherein in the detection step, the complex is contacted with the detection molecule, a washing solution is then added to remove contaminants other than the complex bound to the detection molecule, and the complex is then detected with the detection molecule.

[0009] (8) a capture molecule comprising a substance that specifically binds to an antigen expressed on the surface of a cancer cell; A detection molecule including a substance that specifically binds to an antigen expressed on the surface of a cancer cell and a labeling substance; The exosome separation and detection kit, wherein an antigen expressed on the surface of the cancer cell in at least one of the capture molecule and the detection molecule is CSV. (9) The kit according to (8), wherein the substance that specifically binds to an antigen expressed on the cell surface is an anti-CSV antibody. (10) The kit according to (8) or (9), wherein the capture molecule has a specific binding substance for an antigen expressed on the surface of cancer cells, which is an anti-CSV antibody; and the detection molecule has a specific binding substance for an antigen expressed on the surface of cancer cells, which is an anti-CSV antibody, an anti-EpCAM antibody, or an anti-PD-L1 antibody. (11) The kit according to (8) or (9), wherein the detection molecule has a specific binding substance for an antigen expressed on the surface of cancer cells, which is an anti-CSV antibody; and the capture molecule has a specific binding substance for an antigen expressed on the surface of cancer cells, which is an anti-CSV antibody, an anti-EpCAM antibody, or an anti-PD-L1 antibody. (12) The kit according to any one of (8) to (11), wherein the capturing molecule is immobilized on a magnetic particle. (13) The kit according to any one of (8) to (12), which is used for diagnosing cancer. (14) The kit according to any one of (8) to (13), which is used for diagnosing early stage cancer. (15) The kit according to any one of (8) to (14), which is used for diagnosing lung cancer. (16) The kit according to any one of (8) to (12), which is for companion diagnosis. Effect of the Invention

[0010] According to the exosome separation and detection method and separation and detection kit of the above aspects, exosomes can be separated and detected, and can be used for cancer diagnosis or companion diagnosis. [Brief description of the drawings]

[0011] [Figure 1] 1 is a graph showing the results of measuring CSV-positive exosomes in specimens from lung cancer patients or healthy subjects in Analysis Example 1. In the figure, the numbers in parentheses indicate the number of cases. [Diagram 2] 2 is a graph showing an ROC curve created from the measurement results shown in FIG. 1. [Diagram 3]1 is a graph showing the results of measuring CSV and EpCAM-positive exosomes in samples from lung cancer patients or healthy subjects in Analysis Example 2. In the figure, the numbers in parentheses indicate the number of cases. [Figure 4] 4 is a graph showing an ROC curve created from the measurement results shown in FIG. 3. [Diagram 5] 1 is a graph showing the results of measuring CSV and PD-L1-positive exosomes in samples from lung cancer patients or healthy subjects in Analysis Example 3. In the figure, the numbers in parentheses indicate the number of cases. [Figure 6] 6 is a graph showing an ROC curve created from the measurement results shown in FIG. 5. [Figure 7A] This is a graph classifying the measurement results shown in Figure 1 by type of lung cancer. In the figure, the numbers in parentheses indicate the number of cases. [Figure 7B] This is a graph showing the measurement results shown in Figure 3 categorized by type of lung cancer. In the figure, the numbers in parentheses indicate the number of cases. However, (12000) for the Ad group indicates the signal / noise value. [Figure 7C] This is a graph classifying the measurement results shown in Figure 5 by type of lung cancer. In the figure, the numbers in parentheses indicate the number of cases. [Figure 8A] This is a graph showing the measurement results shown in Figure 1 categorized by pathological stage of lung cancer. In the figure, the numbers in parentheses indicate the number of cases. [Figure 8B] This is a graph showing the measurement results shown in Figure 3 categorized by pathological stage of lung cancer. In the figure, the numbers in parentheses indicate the number of cases. However, (12000) in group III indicates the signal / noise value. [Figure 8C] This is a graph showing the measurement results shown in Figure 5 classified by pathological stage of lung cancer. In the figure, the numbers in parentheses indicate the number of cases. [Figure 9A] This is a graph showing the measurement results shown in Figure 1 classified by pathological stage of adenocarcinoma or squamous cell carcinoma. In the figure, the numbers in parentheses indicate the number of cases. [Figure 9B] This is a graph showing the measurement results shown in Figure 3 categorized by pathological stage of adenocarcinoma or squamous cell carcinoma. In the figure, the numbers in parentheses indicate the number of cases. However, (12000) in the adenocarcinoma III group indicates the signal / noise value. [Figure 9C] This is a graph showing the measurement results shown in Figure 5 classified by pathological stage of adenocarcinoma or squamous cell carcinoma. In the figure, the numbers in parentheses indicate the number of cases. [Figure 10] 1 is a graph showing the measurement results of CSV-positive exosomes, CSV and EpCAM-positive exosomes, and CSV and GPC3-positive exosomes in specimens from lung cancer patients or healthy subjects in Example 4. [Figure 11] 13 is a graph showing the measurement results of CSV- and PD-L1-positive exosomes in the CSV / PD-L1 evaluation system and the PD-L1 / CSV evaluation system in Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in more detail below by showing embodiments, but the present invention is not limited to the following embodiments in any way.

[0013] <Exosome isolation and detection methods> In one embodiment, the present invention provides a method for isolating and detecting exosomes, comprising a complex formation step and a detection step. In the complex formation step, a biological sample is contacted with a capture molecule that contains a substance that specifically binds to an antigen expressed on the surface of cancer cells to form a complex between the exosome and the capture molecule. In the detection step, the complex is contacted with a detection molecule that contains a labeling substance and a substance that specifically binds to an antigen expressed on the surface of a cancer cell, and the complex is detected with the detection molecule. The antigen expressed on the surface of a cancer cell in at least one of the capturing molecule and the detecting molecule is cell surface vimentin (CSV).

[0014] The inventors discovered that CSV is mainly expressed on the surface of exosomes released from cancer cells, and thus completed the present invention. In conventional methods for detecting CTC antigen markers, since 10 mL of blood contains only 1 to 10 CTCs, a large amount of blood sample is required for detection, making it difficult to detect CTC antigen markers for the purpose of cancer diagnosis. In particular, in cancer diagnosis and companion diagnosis, rapid and accurate diagnosis is required, but detection of CTC antigen markers cannot meet this need. In contrast, in the exosome separation and detection method according to one embodiment of the present invention (hereinafter, sometimes simply abbreviated as "the separation and detection method of this embodiment"), exosomes are contained in a sufficient amount for measurement in a blood sample of about several tens of μL, and are therefore suitable as a detection target for cancer diagnosis or companion diagnosis. In addition, by targeting a combination of CSV and a known cancer antigen marker among the antigen markers present in one exosome, it is possible to specifically separate exosomes released from cancer cells. This can improve the accuracy of cancer diagnosis. Furthermore, companion diagnosis can be performed by selecting a cancer antigen that is a target of a therapeutic drug as a known cancer antigen marker to be combined with CSV. Next, each step of the separation and detection method of this embodiment will be described in detail.

[0015] [Complex formation process] In the complex formation step, the biological sample is contacted with a capture molecule containing a substance that specifically binds to an antigen expressed on the surface of cancer cells, and a complex between the exosome and the capture molecule is formed by this contact.

[0016] Examples of biological samples include various liquids such as body fluids, cell culture supernatants, and tissue cell lysates. Of these, body fluids or cell culture supernatants are preferred. Examples of body fluids include whole blood, serum, plasma, various blood cells, blood clots, platelets, and the like, as well as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymphatic fluid, bone marrow fluid, tissue fluid, saliva, gastric juice, synovial fluid, pleural effusion, bile, ascites, and amniotic fluid. Of these, plasma is preferred. The plasma may be treated with an anticoagulant such as citric acid, heparin, or EDTA.

[0017] The species from which the biological sample is derived is not particularly limited, and examples include humans, mice, rats, rabbits, dogs, pigs, sheep, cattle, horses, camels, monkeys, etc. Among these, human-derived samples are preferred. As a combination of species and body fluid, human-derived plasma is preferred.

[0018] The biological sample may be pretreated in advance by adding it to a known buffer, or may be taken from a living body and used as is. That is, according to the separation and detection method of the present embodiment, selective separation of exosomes can be easily performed without pretreatment such as PEG precipitation or isolation using an ultracentrifuge.

[0019] The amount of the biological sample can be adjusted appropriately, and may be, for example, from 1 μL to 50 μL, for example, from 1 μL to 20 μL.

[0020] The capture molecule used in the complex formation step contains a substance that specifically binds to an antigen expressed on the surface of cancer cells. Since exosomes released from cancer cells have an antigen expressed on the surface of cancer cells on their surface, the use of a substance that specifically binds to the antigen makes it possible to capture the exosomes released from cancer cells.

[0021] An antigen expressed on the surface of a cancer cell refers to an antigen known to be more highly expressed in cancer cells than in normal cells, or an antigen known to be specifically expressed only in cancer cells. Examples of such antigens include cell surface vimentin (CSV), epithelial cell adhesion molecule (EpCAM, CD326), programmed cell death ligand-1 (PD-L1), glypican-3 (GPC3), glypican-1 (GPC1), carcinoembryonic antigen (CEA), CD4, CD5, CD16, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD52, CD123, CD171, Cancer Antigen 125, Claudin 18, receptor tyrosine kinase ROR1, sialyl Le x-i (SLX), sialyl Lewis A (CA19-9), immune co-stimulatory protein B7-homolog 6 (B7H6), cell surface glycoprotein (CS-1), receptor tyrosine kinase 3 (FLT3), prostate-specific membrane antigen (PSMA), B-cell maturation antigen (BCMA), Wilms tumor 1 (WT1), NY-ESO-1, Caveolin-1, Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), Galectine3, CD151, Tetraspanin 8, epidermal growth factor receptor (EGFR), HER2, Ribophorin 2 (RPN2), CD44, and TGF-β are included, but are not limited to these.

[0022] Information on the nucleotide and amino acid sequences of antigens expressed on the surface of the above various types of human cancer cells can be obtained from databases such as Genbank. The amino acid sequence of human CSV (as vimentin) is disclosed, for example, under Genbank accession number NP_003371.2. The amino acid sequence of human EpCAM is disclosed, for example, under Genbank accession number NP_002345.2. The amino acid sequence of human PD-L1 is disclosed, for example, under Genbank accession number NP_054862.1. The amino acid sequence of human GPC3 is disclosed, for example, under Genbank accession number NP_001158089.1.

[0023] The capture molecule comprises a specific binding substance for the antigen. Examples of specific binding substances include substances that specifically bind to antigens. Examples of substances that specifically bind to antigens include antibodies and aptamers.

[0024] The antibody may be any antibody that has the ability to bind to an antigen sequence (epitope) on a cancer cell, and specific examples thereof include anti-CSV antibody, anti-EpCAM antibody, anti-PD-L1 antibody, and anti-GPC3 antibody. These antibodies may be monoclonal or polyclonal, but are preferably monoclonal. Furthermore, these antibodies may be bispecific antibodies. Examples of antibody classes include IgG and IgM, with IgG being more preferred. The antibody may also be a chimeric antibody such as a humanized antibody. The antibody may be derived from any animal species, including, for example, mammals such as mice, rabbits, cows, pigs, horses, sheep, and goats, birds such as chickens, and humans, and is not particularly limited. The antibody may be prepared, for example, from serum derived from the animal species by a conventionally known method, or may be prepared from a vector synthesized using known antibody gene sequence information, or a commercially available antibody may be used. An example of a commercially available anti-CSV monoclonal antibody is mouse anti-human CSV monoclonal antibody (isotype: IgG2b, κ) manufactured by Abnova.

[0025] Furthermore, antibody fragments can also be used as antibodies, including, for example, Fv, Fab, Fab', F(ab')2, rIgG, and scFv.

[0026] An aptamer is a substance that has a specific binding ability to a target substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers that have a specific binding ability to an antigen can be selected, for example, by the systematic evolution of ligand by exponential enrichment (SELEX) method. Furthermore, peptide aptamers that have a specific binding ability to an antigen can be selected, for example, by the two-hybrid method using yeast.

[0027] Furthermore, when the antigen is CSV, Nkp46 protein and the like, which are known to have the ability to bind to CSV, can also be used as a specific binding substance.

[0028] The capture molecule is preferably immobilized on a solid support.

[0029] Examples of materials for the solid phase carrier include polymeric compounds such as polystyrenes, polyethylenes, polypropylenes, polyesters, poly(meth)acrylonitriles, styrene-butadiene copolymers, poly(meth)acrylic esters, fluororesins, cross-linked dextran, and polysaccharides; glass; metals; magnetic materials; resin compositions containing magnetic materials; and combinations of these.

[0030] The shape of the solid phase carrier is not particularly limited, and examples thereof include tray-like, spherical, particulate, fibrous, rod-like, disc-like, container-like, and microchannel-like shapes. In this step, it is preferable to use magnetic particles as the solid phase carrier from the viewpoint of ease of solid-liquid separation and washing.

[0031] Examples of the magnetic particles include magnetic particles made of metals such as iron oxide (Fe3O4), iron sesquioxide (γ-Fe2O3), various ferrites, iron, manganese, nickel, cobalt, and chromium, and magnetic particles made of alloys of these metals, which are contained in a resin. Examples of the resin include hydrophobic polymers and hydrophilic polymers. Among them, magnetic particles containing a magnetic material in a resin are preferred, and those having a polymer layer formed on the surface of mother particles containing superparamagnetic fine particles are more preferred. For example, there are magnetic particles described in JP 2008-32411 A (Reference 1), in which a hydrophobic first polymer layer is formed on the surface of mother particles containing superparamagnetic fine particles, a second polymer layer having at least a glycidyl group on the surface is formed on the first polymer layer, and polar groups are introduced by chemically modifying the glycidyl group.

[0032] Here, the superparamagnetic fine particles are typically iron oxide fine particles having a particle diameter of 20 nm or less (preferably 5 nm to 20 nm), and are represented by XFe2O4 (X=Mn, Co, Ni, Mg, Cu, Li0.5 Fe 0.5 Examples of the magnetite include ferrite represented by Fe3O4, magnetite represented by Fe3O4, and γ-Fe2O3. In terms of high saturation magnetization and low residual magnetization, it is preferable to include either γ-Fe2O3 or Fe3O4.

[0033] Moreover, the monomers for forming the hydrophobic first polymer layer are roughly classified into monofunctional monomers and crosslinkable monomers. Examples of the monofunctional monomer include aromatic vinyl monomers such as styrene, α-methylstyrene, and halogenated styrene; and ethylenically unsaturated carboxylic acid alkyl ester monomers such as methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, stearyl acrylate, stearyl methacrylate, cyclohexyl acrylate, cyclohexyl methacrylate, isobornyl acrylate, and isobornyl methacrylate. Examples of the crosslinkable monomer include polyfunctional (meth)acrylates such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, and dipentaerythritol hexamethacrylate; conjugated diolefins such as butadiene and yprene, as well as divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate.

[0034] The monomer for forming the second polymer layer is intended to introduce a functional group onto the particle surface and includes a glycidyl group-containing monomer. The content of the glycidyl group-containing monomer in the monomer for forming the second polymer layer is preferably 20% by mass or more. Examples of the copolymerizable monomer containing a glycidyl group include glycidyl acrylate, glycidyl methacrylate, and allyl glycidyl ether.

[0035] The polar group introduced by chemically modifying the glycidyl group of the second polymer layer is preferably a functional group capable of reacting with a substance that specifically binds to an antigen expressed on the surface of cancer cells, and more preferably contains at least one atom selected from the group consisting of oxygen, nitrogen, and sulfur atoms. Among these, an amino group, an aldehyde group, a carboxyl group, or an active ester group is more preferable. In particular, when the second polymer layer of the magnetic particle has the polar group and a 2,3-dihydroxypropyl group, the binding property with a substance that specifically binds to an antigen expressed on the surface of cancer cells (especially an antibody against an antigen expressed on the surface of cancer cells) is improved.

[0036] The method of binding a specific binding substance for an antigen expressed on the surface of cancer cells to a solid-phase carrier includes a physical adsorption method, a covalent bond method, an ionic bond method, and other chemical binding methods. Examples of the physical adsorption method include a method of directly immobilizing a specific binding substance for an antigen expressed on the surface of cancer cells on a solid-phase carrier, and a method of chemically binding the substance to other proteins such as albumin and then adsorbing and immobilizing the substance. Examples of the chemical binding method include a method of directly binding the substance to the solid-phase carrier using a functional group that is introduced onto the surface of the solid-phase carrier and is capable of reacting with a specific binding substance for an antigen expressed on the surface of cancer cells, a method of chemically introducing a spacer molecule (such as a carbodiimide compound) between the solid-phase carrier and the specific binding substance for the antigen expressed on the surface of cancer cells and then binding the substance, and a method of binding a specific binding substance for an antigen expressed on the surface of cancer cells to other proteins such as albumin and then chemically binding the protein to the solid-phase carrier.

[0037] The complex formation step may be carried out using salts, proteins such as albumin, surfactants, and the like, in addition to the above-mentioned components, if necessary.

[0038] The reaction temperature in the complex formation step is usually within the range of 2° C. to 42° C., and the reaction time is usually 5 minutes to 24 hours. When the reaction is carried out at 2° C. to 8° C., the reaction time is preferably 8 hours to 30 hours, and when the reaction is carried out at room temperature (20° C.) to 42° C., the reaction time is preferably 5 minutes to 4 hours.

[0039] In the complex formation step, the pH in the system is not particularly limited, but is preferably in the range of pH 5 to 10, more preferably pH 6 to 8. In order to maintain the desired pH, a buffer solution is usually used, such as a phosphate buffer, a tris(hydroxymethyl)aminomethane buffer, a HEPES buffer, or an MES buffer.

[0040] [Detection process] In the detection step, the complex is contacted with a detection molecule that contains a labeling substance and a substance that specifically binds to an antigen expressed on the surface of a cancer cell. This contact causes the detection molecule to bind to the exosomes in the complex, and the complex is detected by the detection molecule.

[0041] The detection molecule includes a labeling substance and a substance that specifically binds to an antigen expressed on the surface of a cancer cell. In particular, the detection molecule preferably includes a labeling substance bound to a substance that specifically binds to an antigen expressed on the surface of a cancer cell.

[0042] Examples of specific binding substances for antigens expressed on the surface of cancer cells include those exemplified in the above "capture molecule". The specific binding substance possessed by the capture molecule and the specific binding substance possessed by the detection molecule may be the same or different. When the specific binding substance possessed by the capture molecule and the specific binding substance possessed by the detection molecule are the same, the specific binding substance is a specific binding substance for CSV, and is preferably an anti-CSV antibody.

[0043] Furthermore, when the specific binding substance possessed by the capture molecule or the specific binding substance possessed by the detection molecule is an antibody, a bispecific antibody may be used, or a mixture of multiple antibodies with different antigens may be used.

[0044] It is also preferable that the specific binding substance possessed by the capture molecule is an anti-CSV antibody, and the specific binding substance possessed by the detection molecule is an anti-CSV antibody, an anti-EpCAM antibody, or an anti-PD-L1 antibody. It is also preferable that the specific binding substance possessed by the detection molecule is an anti-CSV antibody, and the specific binding substance possessed by the capture molecule is an anti-CSV antibody, an anti-EpCAM antibody, or an anti-PD-L1 antibody. By using the above combination of a specific binding substance possessed by the capture molecule and a specific binding substance possessed by the detection molecule, exosomes can be detected with high accuracy, as shown in the examples described later.

[0045] Examples of labeling substances include biotin, avidin, streptavidin, neutravidin, glutathione-S-transferase, glutathione, alkaline phosphatase (ALP), fluorescent dyes, polyethylene glycol, charged molecules such as mellitic acid, and the like. When the labeling substance is ALP, exosomes can be detected with high accuracy by adding an ALP luminescent substrate. Examples of ALP luminescent substrates include p-nitrophenyl phosphate (PNP), 4-methylumbelliferyl phosphate (4MUP), 3-(2'-spiroadamantane)4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 3,7 ]decan}-4-yl)phenyl phosphate (CSPD), 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo[3.3.1.1 3,7]decane}-4-yl)-1-phenylphosphate disodium (CDP-Star).

[0046] The detection method in the detection step may be an immunoassay, such as enzyme immunoassay (EIA), ELISA, chemiluminescent enzyme immunoassay (CLEIA), fluorescent immunoassay (FIA), radioimmunoassay (RIA), luminescent immunoassay, immunoblot, or Western blot. When the specific binding substance is an antibody, the CLEIA or ELISA method is preferred because it allows easy and sensitive detection of the antibody. Examples of the ELISA method include the competitive method and sandwich method.

[0047] An example of a separation and detection method using the CLEIA method is shown below. First, an antibody against an antigen expressed on the surface of cancer cells is bound to a solid support to immobilize a capture molecule, and then a biological sample is contacted to form a complex. A detection molecule containing an antibody against an antigen expressed on the surface of cancer cells to which a labeling substance is bound is added to form a further complex. Then, the amount of label in the formed complex can be detected to measure the signal derived from exosomes contained in the biological sample.

[0048] The separation and detection method of this embodiment may include other steps in addition to the above-mentioned complex formation step and detection step. For example, a washing step may be provided after the complex formation step and before the detection step. In the washing step, the complex between the exosome and the capturing molecule formed in the complex formation step is washed, thereby removing impurities such as unreacted components.

[0049] The washing step is usually carried out by different methods depending on the shape of the solid phase carrier, which is a component of the capture molecule. When the solid phase carrier is particulate, such as magnetic particles, for example, the magnetic particles may be dispersed in a washing solution and washed, or the magnetic particles may be immersed in the washing solution while being collected. On the other hand, when the solid phase carrier is in the form of a microplate, the washing method involves repeatedly dispensing and aspirating the washing solution into the plate.

[0050] Furthermore, when the solid phase carrier is a magnetic particle, the washing step preferably includes a magnetic collection step of collecting the magnetic particles by magnetic force and separating the magnetic particles from a liquid phase, and a dispersing step of dispersing the magnetic particles separated in the magnetic collection step in a washing liquid, thereby enabling unreacted substances and impurities in the sample to be washed and separated and removed from the magnetic particle surfaces more efficiently. Specifically, a magnetic field is applied to the reaction vessel, the magnetic particles are caused to adhere to the wall of the reaction vessel and collected, the reaction supernatant is then removed, and if necessary, a washing solution is added, and the same procedure of applying a magnetic field and then removing the supernatant is repeated.

[0051] The washing solution is preferably a washing solution containing a surfactant and a buffer solution as mentioned in the above complex formation step. The pH of the washing solution is preferably in the range of pH 5 to 10, more preferably pH 6 to 8. Specifically, Tris Buffered Saline (TBS) containing 0.1% by mass of Tween 20 relative to the total mass of the solution can be mentioned.

[0052] In the detection step, after the complex is contacted with a detection molecule containing a labeling substance and a substance that specifically binds to an antigen expressed on the surface of a cancer cell, a washing solution can be added to wash the complex to which the detection molecule is bound. This makes it possible to remove unreacted substances and impurities in the sample, and to detect the complex with the detection molecule with higher sensitivity.

[0053] Furthermore, in the separation and detection method of this embodiment, as shown in the examples described later, the detection sensitivity of exosomes can be further improved by carrying out each step in parallel using a combination of two or more types of capture molecules and detection molecules.

[0054] <Exosome isolation and detection kit> An exosome separation and detection kit according to one embodiment of the present invention (hereinafter, may be simply abbreviated as "the separation and detection kit of this embodiment") comprises a capture molecule and a detection molecule. The capture molecule contains a substance that specifically binds to an antigen expressed on the surface of cancer cells. The detection molecule contains a substance that specifically binds to an antigen expressed on the surface of cancer cells and a labeling substance. The antigen expressed on the surface of cancer cells in at least one of the capture molecule and the detection molecule is CSV.

[0055] The capturing molecule and the detecting molecule are the same as those exemplified in the above "Method for separating and detecting exosomes."

[0056] The separation and detection kit of this embodiment may further include a washing solution in addition to the capture molecule and the detection molecule. The washing solution may be the same as those exemplified in the above "Exosome Separation and Detection Method."

[0057] <Usage> The separation and detection method and the separation and detection kit of the present embodiment are preferably used for cancer diagnosis, particularly early stage cancer diagnosis. That is, in one embodiment, the present invention provides a method for determining cancer, comprising a step of comparing an exosome-derived signal detected by the above-mentioned separation and detection method using a biological sample collected from a subject with an exosome-derived background signal in a biological sample collected from a control, and determining that the subject has developed cancer if the exosome-derived signal in the subject sample is stronger than the exosome-derived signal (background) in the control sample. The above separation and detection kit can also be referred to as a cancer diagnostic kit.

[0058] Cancers to be diagnosed include colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer, lung cancer, kidney cancer, and skin cancer, with lung cancer being preferred among these.

[0059] In the determination step, the signal derived from exosomes in the subject sample measured in the detection step is statistically analyzed based on the signal derived from exosomes in the control sample, and compared to determine whether the subject has cancer. For example, if the signal derived from exosomes in the subject sample is stronger than the signal derived from exosomes in the control sample, it is determined that the subject has a high possibility of developing cancer. Note that the control in the determination step is an average of people of the same age and sex as the subject who do not develop cancer, and the signal derived from exosomes in the control may be measured together with the signal derived from exosomes in the subject, or a statistical value obtained from a value measured in advance may be used.

[0060] In addition, the separation and detection method and the separation and detection kit of this embodiment are preferably used for companion diagnosis for selecting a cancer therapeutic drug. That is, in one embodiment, the present invention provides a method for selecting a cancer therapeutic drug, comprising a selection step of comparing a signal derived from exosomes positive for a specific cancer antigen detected by the above-mentioned separation and detection method using a biological sample collected from a subject with a signal derived from exosomes positive for a specific cancer antigen in a biological sample collected from a control, and selecting a cancer therapeutic drug that molecularly targets the specific cancer antigen as a cancer therapeutic drug to be used for the subject when the signal derived from exosomes positive for a specific cancer antigen in the subject is similar to the signal derived from exosomes positive for a specific cancer antigen in the control, or stronger than the signal derived from exosomes positive for a specific cancer antigen in the control. The above separation and detection kit can also be rephrased as a companion diagnostic kit for a cancer therapeutic drug.

[0061] In the selection step, the signal derived from exosomes in the subject sample measured in the detection step is statistically analyzed based on the signal derived from exosomes in the control sample, and a comparison is made to select a cancer therapeutic drug to be used for the subject. For example, when the signal derived from exosomes positive for a specific cancer antigen in the subject sample is comparable to or higher than the signal derived from exosomes positive for the cancer antigen in the control sample, a cancer therapeutic drug that targets the cancer antigen can be selected as a cancer therapeutic drug to be used for the subject. Note that the control in the selection step is a person of the same age and sex as the subject, and is the average of cancer patients who have achieved a therapeutic effect with a candidate cancer therapeutic drug, and the signal derived from exosomes positive for a specific cancer antigen in the control can be a statistical value obtained from the value measured in the control before administration.

[0062] In addition, since exosomes are secreted from various cells, such as immune system cells and various cancer cells, the efficacy of a cancer therapeutic agent in a patient can be evaluated by measuring changes in exosomes in the blood before and after administration of the cancer therapeutic agent (including not only increases or decreases in the amount present but also changes in the amount of membrane proteins). That is, in one embodiment, the present invention provides a method for evaluating the efficacy of a cancer therapeutic agent, comprising an evaluation step of evaluating, using biological samples collected from a subject before and after administration of the cancer therapeutic agent, signals derived from exosomes detected by the above-mentioned separation and detection method, that the cancer therapeutic agent is highly likely to exhibit efficacy when the signals derived from exosomes in the biological samples collected from the subject after administration of the cancer therapeutic agent are lower than the signals derived from exosomes in the biological samples collected from the subject before administration of the cancer therapeutic agent.

[0063] In the evaluation step, the exosome-derived signal detected by the above separation and detection method is compared by performing statistical analysis based on the exosome-derived signal in the biological sample collected from the subject before administration of the cancer therapeutic drug. The analysis method is not particularly limited, and a known method can be used. In addition, the subsequent judgment can be, for example, when the exosome-derived signal in the biological sample collected from the subject after administration of the cancer therapeutic drug is lower than the exosome-derived signal in the biological sample collected from the subject before administration, it can be evaluated that the cancer therapeutic drug is highly likely to have an effect of suppressing cancer. EXAMPLES

[0064] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0065] <Sample preparation> [Preparation of heparin plasma samples] Blood was collected from healthy subjects or lung cancer patients into blood collection tubes containing sodium heparin, centrifuged at room temperature at 1500 to 1800 × g for 15 minutes, and the supernatant was collected in a separate container as a heparin plasma sample. It was stored at -80 ° C until use.

[0066] <Method for measuring exosomes> In the examples and comparative examples described below, the measurement of exosomes was carried out by measuring the luminescence intensity by a chemiluminescence enzyme immunoassay (CLEIA method). The measurement method in each example is described in detail below.

[0067] [Measurement method A] (Emission intensity measurement by CLEIA method in CSV / CSV evaluation system) Each heparin plasma sample was diluted with 50 mM Tris buffer containing 2% (w / v) BSA and 150 mM NaCl as appropriate to prepare a sample solution. 25 μL / well of a 0.05% (w / v) antibody-bound magnetic particle (Magnosphere MS300 / Carboxyl (product number: MSP-S300-CA, JSR Life Sciences) antibody-sensitized by JSR) dispersion solution with anti-CSV antibody (catalog number: H00007431-M08, Abnova) bound thereto was added to a 96-well white microplate (Corning), followed by 25 μL / well of the sample solution. Furthermore, 25 μL / well of a solution containing alkaline phosphatase-labeled anti-CSV antibody (catalog number: H00007431-M08, Abnova labeled by JSR) was added. Thereafter, the 96-well white microplate was stirred at 25°C for 20 minutes, and then the particles after the reaction were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the magnetic particles using a microplate washer (Hydroflex, manufactured by Tecan). Further, 50 μL of TBS was added, and the mixture was stirred for 5 minutes at 25°C. Then, the particles after the reaction were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the magnetic particles using a microplate washer (Hydroflex, manufactured by Tecan). Next, 50 μL of a luminescent substrate (LSI, substrate solution (R5), CDP-Star) was added, and after 5 minutes, the luminescence intensity was measured using a luminometer (Promega, GloMax (registered trademark) Discover System).

[0068] [Measurement method B] (Luminescence intensity measurement by CLEIA method using EpCAM / CSV evaluation system) Each heparin plasma sample was diluted with 50 mM Tris buffer containing 2% (w / v) BSA and 150 mM NaCl as appropriate to prepare a sample solution. 25 μL / well of a dispersion of 0.05% (w / v) antibody-bound magnetic particles (Magnosphere MS300 / Carboxyl (product number: MSP-S300-CA, JSR Life Sciences) with anti-EpCAM antibody (JSR) sensitized by JSR) was added to a 96-well white microplate (Corning), followed by 25 μL / well of the sample solution. Furthermore, 25 μL / well of a solution containing alkaline phosphatase-labeled anti-CSV antibody (catalog number: H00007431-M08, Abnova labeled by JSR) was added. Thereafter, the 96-well white microplate was stirred at 25°C for 20 minutes, and then the particles after the reaction were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the magnetic flux using a microplate washer (Hydroflex, manufactured by Tecan). Further, 50 μL of TBS was added, and the mixture was stirred for 5 minutes at 25°C. Then, the particles after the reaction were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the magnetic flux using a microplate washer (Hydroflex, manufactured by Tecan). Next, 50 μL / well of a luminescent substrate (substrate solution (R5), manufactured by LSI, CDP-Star) was added, and after 5 minutes, the luminescence intensity was measured using a luminometer (GloMax (registered trademark) Discover System, manufactured by Promega).

[0069] [Measurement method C] (Luminescence intensity measurement by CLEIA method using CSV / PD-L1 evaluation system) Each heparin plasma sample was diluted with 50 mM Tris buffer containing 2% (w / v) BSA and 150 mM NaCl as appropriate to prepare a sample solution. 25 μL of 0.05% (w / v) antibody-bound magnetic particles (Magnosphere MS300 / Carboxyl (product number: MSP-S300-CA, JSR Life Sciences) sensitized with antibody by JSR) dispersion with anti-CSV antibody (catalog number: H00007431-M08, Abnova) was added to a 96-well white microplate (Corning), followed by 25 μL of sample solution. In addition, 25 μL / well of a solution containing alkaline phosphatase-labeled anti-PD-L1 antibody (MBL antibody labeled by JSR) was added. Thereafter, the 96-well white microplate was stirred at 25°C for 20 minutes, and then the particles after the reaction were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the magnetic flux using a microplate washer (Hydroflex, manufactured by Tecan). Further, 50 μL of TBS was added, and the mixture was stirred for 5 minutes at 25°C. Then, the particles after the reaction were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the magnetic flux using a microplate washer (Hydroflex, manufactured by Tecan). Next, 50 μL / well of a luminescent substrate (substrate solution (R5), manufactured by LSI, CDP-Star) was added, and after 5 minutes, the luminescence intensity was measured using a luminometer (GloMax (registered trademark) Discover System, manufactured by Promega).

[0070] <Measurement of CSV-positive exosomes in lung cancer patient samples> [Example 1] Heparin plasma samples from lung cancer patients (47 cases), including patients with early stage lung cancer at stage 1 or 2, were used as sample solutions, and each was measured according to the above-mentioned measurement method A. The measured value of each sample was divided by the blank value to calculate the signal / noise ratio (S / N).

[0071] [Comparative Example 1] Heparin plasma samples from healthy subjects (8 cases) were used as sample solutions, and each was measured according to the above-mentioned Measurement Method A. The measured value of each sample was divided by the blank value to calculate the signal-to-noise ratio (S / N).

[0072] [Analysis example 1] The results of Example 1 and Comparative Example 1 are shown in Figure 1. In Figure 1, "Healthy" refers to heparin plasma samples from healthy subjects, and "Cancer" refers to heparin plasma samples from lung cancer patients. The same applies to the following figures. In addition, the ROC curve created from the measurement results shown in Figure 1 is shown in Figure 2. The S / N of healthy individuals obtained in Comparative Example 1 was used as the control group, and the S / N of lung cancer patients obtained in Example 1 was used as the discrimination group.When the cutoff value was set at 29.47, the sensitivity (specificity) was 0.98, suggesting that lung cancer patients can be distinguished from healthy individuals.

[0073] [Example 2] Heparin plasma samples from lung cancer patients (47 cases), including patients with early stage lung cancer at stage 1 or 2, were used as sample solutions, and each was measured according to the above-mentioned measurement method B. The measured value of each sample was divided by the blank value to calculate the signal-to-noise ratio (S / N).

[0074] [Comparative Example 2] Heparin plasma samples from healthy subjects (8 cases) were used as sample solutions, and each was measured according to the above-mentioned measurement method B. The measured value of each sample was divided by the blank value to calculate the signal-to-noise ratio (S / N).

[0075] [Analysis example 2] The results of Example 2 and Comparative Example 2 are shown in Fig. 3. In addition, the ROC curve created from the measurement results shown in Fig. 3 is shown in Fig. 4. The S / N of healthy individuals obtained in Comparative Example 2 was used as the control group, and the S / N of lung cancer patients obtained in Example 2 was used as the discrimination group.When the cutoff value was set at 4.00, the sensitivity (specificity) was 1.00, suggesting that lung cancer patients can be distinguished from healthy individuals.

[0076] [Example 3] Heparin plasma samples from lung cancer patients (47 cases), including patients with early stage lung cancer at stage 1 or 2, were used as sample solutions, and each was measured according to the above-mentioned measurement method C. The measured value of each sample was divided by the blank value to calculate the signal-to-noise ratio (S / N).

[0077] [Comparative Example 3] Heparin plasma samples from healthy subjects (8 cases) were used as sample solutions, and each was measured according to the above-mentioned measurement method C. The measured value of each sample was divided by the blank value to calculate the signal-to-noise ratio (S / N).

[0078] [Analysis example 3] The results of Example 3 and Comparative Example 3 are shown in Fig. 5. In addition, the ROC curve created from the measurement results shown in Fig. 5 is shown in Fig. 6. The S / N of healthy individuals obtained in Comparative Example 3 was used as the control group, and the S / N of lung cancer patients obtained in Example 3 was used as the discrimination group.When the cutoff value was set to 2.91, the sensitivity (specificity) was 0.94, suggesting that lung cancer patients can be distinguished from healthy individuals.

[0079] [Analysis example 4] The measurement results of Example 1 and Comparative Example 1, Example 2 and Comparative Example 2, and Example 3 and Comparative Example 3 were classified according to the type of lung cancer. The results are shown in FIG. 7A (Example 1 and Comparative Example 1: CSV / CSV evaluation system), FIG. 7B (Example 2 and Comparative Example 2: EpCAM / CSV evaluation system), and FIG. 7C (Example 3 and Comparative Example 3: CSV / PD-L1 evaluation system). In FIG. 7A to FIG. 7C, "Ad" is an abbreviation for Adenocarcinoma, which is adenocarcinoma. "Sq" is an abbreviation for Squamous, which is squamous cell carcinoma. "Large" is large cell carcinoma. "Small" is small cell lung cancer. In Figs. 7A and 7B, the S / N ratios of lung cancer patients were higher than those of healthy subjects for all types of cancer.

[0080] [Analysis example 5] The measurement results of Example 1 and Comparative Example 1, Example 2 and Comparative Example 2, and Example 3 and Comparative Example 3 were classified according to the pathological stage. The results are shown in Figure 8A (Example 1 and Comparative Example 1: CSV / CSV evaluation system), Figure 8B (Example 2 and Comparative Example 2: EpCAM / CSV evaluation system), and Figure 8C (Example 3 and Comparative Example 3: CSV / PD-L1 evaluation system). In Figures 8A to 8C, the pathological stages are as follows. The same applies to the subsequent figures.

[0081] (Pathological stage) The TNM clinical staging classification was followed, as outlined in the 8th edition of the Lung Cancer Guidelines compiled by the Japan Lung Cancer Society.

[0082] 8A to 8C, the S / N ratio of lung cancer patients was higher than that of healthy subjects from the early pathological stage, and lung cancer patients could be distinguished from healthy subjects regardless of pathological stage from I to IV.

[0083] [Analysis example 6] The measurement results of the adenocarcinoma patients and squamous cell carcinoma patients in Example 1 and Comparative Example 1, Example 2 and Comparative Example 2, and Example 3 and Comparative Example 3 were classified according to the pathological stage. The results are shown in Figure 9A (Example 1 and Comparative Example 1: CSV / CSV evaluation system), Figure 9B (Example 2 and Comparative Example 2: EpCAM / CSV evaluation system), and Figure 9C (Example 3 and Comparative Example 3: CSV / PD-L1 evaluation system).

[0084] 9A to 9C, the S / N ratios of adenocarcinoma and squamous cell carcinoma patients tended to be higher from the early stage of the pathological stage. This suggests that an evaluation system combining an antibody against an antigen expressed on the surface of cancer cells with an anti-CSV antibody can distinguish adenocarcinoma and squamous cell carcinoma patients from healthy individuals.

[0085] In addition, the existing lung cancer markers, carcinoembryonic antigen (CEA), squamous cell carcinoma-associated antigen (SCC), and sialyl Le were detected in heparin plasma samples from lung cancer patients. xThe concentrations of SLX, NSE, Pro-GRP, and Cytokeratin 19 fragment (Cyfra) were measured. Next, the CSV / CSV evaluation system of Example 1 and Comparative Example 1, the EpCAM / CSV evaluation system of Example 2 and Comparative Example 2, and the CSV / PD-L1 evaluation system of Example 3 and Comparative Example 3 were examined for correlation with the above-mentioned existing lung cancer markers, but no correlation was observed. Furthermore, the correlation between the CSV / CSV evaluation system of Example 1 and Comparative Example 1, the EpCAM / CSV evaluation system of Example 2 and Comparative Example 2, and the CSV / PD-L1 evaluation system of Example 3 and Comparative Example 3 was examined, but no correlation was found. In addition, in the above correlation study, a sample with an S / N of 12,000 in the EpCAM / CSV evaluation system was excluded as an abnormal value. For these reasons, the accuracy of cancer diagnosis can be improved by combining existing markers with this evaluation system, or by combining multiple versions of this evaluation system.

[0086] [Example 4] Heparin plasma samples from healthy subjects and heparin pool plasma samples from lung cancer patients were diluted with 50 mM Tris buffer containing 2% (w / v) BSA and 150 mM NaCl as appropriate to prepare sample solutions. 25 μL / well of 0.05% (w / v) antibody-bound magnetic particles (Magnosphere MS300 / Carboxyl (product number: MSP-S300-CA, JSR Life Sciences) sensitized by JSR) dispersions (3 types) bound to either anti-CSV antibody (catalog number: H00007431-M08, Abnova), anti-EpCAM antibody (JSR), or anti-glypican-3 antibody (anti-GPC3 antibody) (MBL) were added to different wells of a 96-well white microplate (Corning), and then 25 μL / well of the sample solution was added. Further, 25 μL / well of a solution containing an alkaline phosphatase-labeled anti-CSV antibody (catalog number: H00007431-M08, manufactured by Abnova and labeled by JSR) was added. Thereafter, the 96-well white microplate was stirred at 25° C. for 20 minutes, and then the particles after the reaction were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the magnetic flux using a microplate washer (Hydroflex manufactured by Tecan). Further, 50 μL of TBS was added, and the mixture was stirred at 25° C. for 5 minutes, and then the particles after the reaction were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the magnetic flux using a microplate washer (Hydroflex manufactured by Tecan). Next, 50 μL / well of a luminescent substrate (manufactured by LSI, substrate solution (R5), CDP-Star) was added, and 5 minutes later, the luminescence intensity was measured using a luminometer (GloMax (registered trademark) Discover System manufactured by Promega). The measured value of each sample was divided by the blank value to calculate the signal-to-noise ratio (S / N). The results are shown in Figure 10. In Figure 10, "Capture" refers to the capture molecule, and "Detector" refers to the detection molecule.

[0087] As shown in Figure 10, an evaluation system could be constructed using the coexpression of GPC3, a cancer antigen other than EpCAM, and CSV as a marker, suggesting that an evaluation system combining antibodies against antigens expressed on the surface of cancer cells with anti-CSV antibodies can distinguish lung cancer patients from healthy individuals.

[0088] [Example 5] A heparinized plasma sample from a healthy individual was diluted with 50 mM Tris buffer containing 2% (w / v) BSA and 150 mM NaCl to prepare a sample solution. A 96-well white microplate (Corning) was added with 0.05% (w / v) antibody-bound magnetic particles (Magnosphere MS300 / Carboxyl (product number: MSP-S300-CA, JSR Life Sciences) with anti-PD-L1 antibody (MBL) bound thereto, sensitized by JSR) at 25 μL / well, followed by the addition of 25 μL / well of the sample solution. In addition, 25 μL / well of alkaline phosphatase-labeled anti-CSV antibody (catalog number: H00007431-M08, Abnova labeled by JSR) solution was added. Thereafter, the 96-well white microplate was stirred at 25°C for 20 minutes, and then the reacted particles were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the particles using a microplate washer (Hydroflex, manufactured by Tecan). Further, 50 μL of TBS was added, and the mixture was stirred at 25°C for 5 minutes. Then, the reacted particles were washed five times with a washing buffer (TBS containing 0.1% by mass of Tween 20) while collecting the particles using a microplate washer (Hydroflex, manufactured by Tecan). Next, 50 μL / well of a luminescent substrate (substrate solution (R5), CDP-Star, manufactured by LSI) was added, and after 5 minutes, the luminescence intensity was measured using a luminometer (GloMax (registered trademark) Discover System, manufactured by Promega) (hereinafter, this evaluation system may be referred to as the "CSV / PD-L1 evaluation system"). Separately, instead of 0.05% (w / v) antibody-bound magnetic particles (Magnosphere MS300 / Carboxyl (product number: MSP-S300-CA, JSR Life Sciences) sensitized with the antibody by JSR) dispersion solution containing anti-PD-L1 antibody (manufactured by MBL), 0.05% (w / v) antibody-bound magnetic particles (Magnosphere MS300 / Carboxyl (product number: MSP-S300-CA, JSR Life Sciences) sensitized with the antibody by JSR) containing anti-CSV antibody (catalog number: H00007431-M08, Abnova) were used. The luminescence intensity was measured using the same method as in the "CSV / PD-L1 evaluation system" above, except that an alkaline phosphatase-labeled anti-PD-L1 antibody (MBL antibody labeled by JSR) was used instead of an alkaline phosphatase-labeled anti-CSV antibody (catalog number: H00007431-M08, Abnova labeled by JSR) in the MS300 / Carboxyl (MSP-S300-CA, JSR Life Sciences) dispersion (antibody sensitized by JSR) (hereinafter, this evaluation system may be referred to as "PD-L1 / CSV evaluation system"). The results are shown in Figure 11. In Figure 11, "Capture" refers to the capture molecule, and "Detector" refers to the detection molecule.

[0089] Figure 11 compares the intensities of the "CSV / PD-L1 evaluation system" and the "PD-L1 / CSV evaluation system," suggesting that an evaluation system can be constructed even if the combination of capture molecules and detection molecules is reversed. [Industrial Applicability]

[0090] According to the exosome separation and detection method and separation and detection kit of the present embodiment, exosomes can be separated and used for cancer diagnosis and companion diagnosis.

Claims

1. A capture molecule comprising a substance that specifically binds to an antigen expressed on the surface of a cancer cell; A detection molecule including a substance that specifically binds to an antigen expressed on the surface of a cancer cell and a labeling substance; an antigen expressed on the cancer cell surface in at least one of the capturing molecule and the detecting molecule is cell surface vimentin; An exosome isolation and detection kit for diagnosing early stage cancer.

2. A capture molecule comprising a substance that specifically binds to an antigen expressed on the surface of a cancer cell; A detection molecule including a substance that specifically binds to an antigen expressed on the surface of a cancer cell and a labeling substance; an antigen expressed on the cancer cell surface in at least one of the capturing molecule and the detecting molecule is cell surface vimentin; An exosome isolation and detection kit for companion diagnostic use.

3. The kit described in claim 1 for diagnosing early stage lung cancer.

4. The kit according to any one of claims 1 to 3, wherein the substance that specifically binds to an antigen expressed on the cell surface is an anti-CSV antibody.

5. The kit according to any one of claims 1 to 4, wherein the detection molecule has a substance that specifically binds to an antigen expressed on the surface of cancer cells, which is an anti-CSV antibody, and the capture molecule has a substance that specifically binds to an antigen expressed on the surface of cancer cells, which is an anti-CSV antibody, an anti-EpCAM antibody, or an anti-PD-L1 antibody.

6. The kit according to any one of claims 1 to 5, wherein the capture molecule is immobilized on a magnetic particle.

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