Exosome measuring method and exosome measuring kit

Abstract

To provide an exosome measuring method and an exosome measuring kit capable of measuring the concentration of exosomes in a sample more sensitively and easily than a conventional measuring method.SOLUTION: In the method, a measuring chip 100 is prepared including a metal film 120 and a first binding substance 130 immobilized on the metal film and bound to an exosome 140. A sample containing exosomes is provided on the metal film to bind the exosomes contained in the sample to the first binding substance. The exosome is labeled with a fluorescent substance 150 by a second binding substance 131 which binds to the exosome. In a state where the exosome labeled with the fluorescent substance is bound to the first binding substance, the metal film is irradiated with excitation light L1 so that surface plasmon resonance occurs in the metal film, and fluorescence L3 emitted from the fluorescent substance is detected.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for measuring exosomes and a kit for measuring exosomes. [Background technology]

[0002] Exosomes are membrane vesicles with a diameter of about 40 nm to 150 nm that are produced by cells and released extracellularly. Exosomes are covered with a phospholipid bilayer membrane and contain various cellular components such as membrane proteins and microRNA (miRNA). Since different exosomes are released from cells depending on the type and condition of the cells, exosomes are thought to mediate communication between cells and organs.

[0003] In recent years, exosomes secreted into body fluids such as blood and saliva have been attracting attention as diagnostic markers for various diseases such as cancer and infectious diseases. For example, Patent Document 1 discloses an analysis method, an analysis reagent, and an analysis device for detecting exosomes that serve as tumor markers. In this method, an antibody against an antigen contained in exosomes and an antibody against an antigen contained in cells that secrete exosomes are used to detect exosomes based on avidin-biotin interaction. In addition, Patent Document 2 discloses an exosome analysis device and an exosome capture method in which an antibody against exosomes is fixed to a recess in a base part made of a synthetic resin having an uneven structure, and exosomes are captured in the recess. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2013 / 094307 [Patent Document 2] JP 2014-219384 A Summary of the Invention [Problem to be solved by the invention]

[0005] As mentioned above, various analytical methods for exosomes have been developed to date, but there is a demand for methods that can measure exosomes with higher sensitivity and reproducibility.

[0006] An object of the present invention is to provide an exosome measurement method and an exosome measurement kit that can measure the exosome concentration in a sample more sensitively and simply than conventional measurement methods. [Means for solving the problem]

[0007] A method for measuring exosomes according to one embodiment of the present invention includes the steps of: preparing a measurement chip including a metal film and a first binding substance immobilized on the metal film and capable of binding to exosomes; providing a specimen containing exosomes on the metal film and allowing the exosomes contained in the specimen to bind to the first binding substance; labeling the exosomes before or after binding to the first binding substance with a fluorescent substance via a second binding substance that binds to the exosomes; and, while the exosomes labeled with the fluorescent substance are bound to the first binding substance, irradiating the metal film with excitation light so that surface plasmon resonance occurs in the metal film, and detecting fluorescence emitted from the fluorescent substance.

[0008] An exosome measurement kit according to one embodiment of the present invention comprises a measurement chip including a metal film and a first binding substance immobilized on the metal film that binds to exosomes, and a labeling reagent for labeling exosomes with a fluorescent substance, wherein the labeling reagent comprises a second binding substance that binds to the exosomes. Effect of the Invention

[0009] According to the present invention, the concentration of exosomes in a sample can be measured more easily and with higher sensitivity than conventional measurement methods. [Brief description of the drawings]

[0010] [Figure 1A]FIG. 1A is a flowchart showing an example of a method for measuring exosomes according to the present embodiment. [Figure 1B] FIG. 1B is a flowchart showing another example of the exosome measurement method according to the present embodiment. [Diagram 2] FIG. 2A is a schematic cross-sectional view for explaining the configuration of a measurement chip for PC-SPFS, and FIG. 2B is a schematic cross-sectional view for explaining the configuration of a measurement chip for GC-SPFS. [Figure 3A] FIG. 3A is a schematic cross-sectional view showing an example of a measurement chip for PC-SPFS. [Figure 3B] FIG. 3B is a perspective schematic diagram showing another example of the measurement chip for PC-SPFS. [Figure 3C] FIG. 3C is a schematic cross-sectional view showing another example of the measurement chip for PC-SPFS. [Figure 4] Figure 4 is a calibration curve showing the relationship between the number of exosomes and their concentration. [Diagram 5] FIG. 5 is a CD9-positive CD63-positive exosome standard curve. [Figure 6] FIG. 6 is a graph showing the relationship between irradiation energy, signal-to-noise ratio (S / N), and detection limit for CD9-positive CD63-positive exosomes. [Figure 7] FIG. 7 is a standard curve of CD9-positive, PSMA-positive exosomes. [Figure 8] FIG. 8 is a graph showing the relationship between irradiation energy, signal-to-noise ratio (S / N), and detection limit for CD9-positive, PSMA-positive exosomes. [Figure 9] FIG. 9 is a standard curve of CD9-positive CD63-positive exosomes derived from LNCap cells. [Figure 10] FIG. 10 is a graph showing the relationship between irradiation energy, signal-to-noise ratio (S / N), and detection limit for CD9-positive CD63-positive exosomes derived from LNCap cells. [Figure 11]FIG. 11 shows a calibration curve of CD9-positive glycan-bearing exosomes derived from LNCap cells, using a lectin that recognizes glycans on exosomes. [Figure 12] FIG. 12 shows a calibration curve of CD9-positive glycan-bearing exosomes derived from prostate cancer cells, using a lectin that recognizes glycans on exosomes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

[0012] [Method of measuring exosomes] Exosomes are secreted by both normal cells and disease-related cells such as cancer cells. Therefore, in order to use exosomes as a disease marker, a method is required that can distinguish and detect the large amount of exosomes derived from normal cells present in a sample (e.g., blood) from the smaller amount of exosomes derived from disease-related cells. Generally, the concentration of exosomes present in serum is 1.0 × 10 11 pieces / ml(1.0×10 8 It is said that the number of exosomes in a sample is about 1.0 × 10 6 pieces / ml(1.0×10 3 A method capable of detecting exosomes at levels below 100 cells / μl is needed.

[0013] In the exosome measurement method according to the present embodiment, in order to improve the measurement sensitivity, exosomes are measured using surface plasmon-field enhanced fluorescence spectroscopy (hereinafter also referred to as "SPFS"). SPFS excites fluorescent substances with an electric field enhanced by surface plasmon resonance (hereinafter also referred to as "SPR") to emit fluorescence, and therefore can detect targets (exosomes in this embodiment) with higher sensitivity than general fluorescent immunoassays.

[0014] In addition, SPFS can use whole blood as a sample, making it possible to detect disease-related exosomes easily and with fewer steps without the need to purify exosomes in the blood.

[0015] As a result of extensive investigations based on the above-mentioned idea, the inventors discovered that exosomes can be measured with high sensitivity using SPFS, and completed the exosome measurement method according to the present embodiment.

[0016] The exosome measurement method according to the present embodiment will be specifically described below. Fig. 1A is a flow chart showing an example of the exosome measurement method according to the present embodiment, which is a measurement method using a primary reaction and a secondary reaction, and Fig. 1B is a flow chart showing an example of the exosome measurement method according to the present embodiment, which is a measurement method using a primary reaction, a secondary reaction, and a tertiary reaction.

[0017] (Preparation of measurement chip) First, a measurement chip including a metal film and a first binding substance that binds to exosomes is prepared (step S10). In SPFS, an evanescent wave generated when a metal film is irradiated with light (excitation light in this embodiment) is combined with a surface plasmon to generate SPR. Known methods for generating SPR include a method of arranging a prism on one side of a metal film (Kretschmann arrangement) and a method of forming a diffraction grating on a metal film. SPFS employing the former method is called prism coupling (PC)-SPFS, and SPFS employing the latter method is called grating coupling (GC)-SPFS. The exosome measurement method according to this embodiment may employ either PC-SPFS or GC-SPFS.

[0018] As described above, the metal film generates SPR when irradiated with excitation light. The type of metal constituting the metal film is not particularly limited as long as it is a metal that can generate SPR. Examples of the metal constituting the metal film include gold, silver, copper, aluminum, and alloys thereof.

[0019] The first binding substance can bind to exosomes and is immobilized on a metal film to capture exosomes in a sample. Usually, the first binding substance is uniformly immobilized in a predetermined region (reaction field) on the metal film. The type of the first binding substance immobilized on the metal film is not particularly limited as long as it can bind to exosomes, that is, it can bind to the first binding determinant possessed by exosomes. Examples of binding substances include antibodies that can bind to exosomes, nucleic acids that can bind to exosomes, lipids that can bind to exosomes, and proteins other than antibodies that can bind to exosomes, such as lectins that can bind to sugar chains on exosomes. Here, the binding determinant possessed by exosomes is a part of exosomes that the binding substance recognizes when binding to exosomes. For example, when the binding substance is an antibody, the binding determinant is an antigenic determinant (epitope) of an antigen, and when the binding substance is a ligand, the binding determinant is a binding site of a corresponding receptor.

[0020] The first binding determinant of exosomes to which the first binding substance binds is not particularly limited, but includes binding determinants known as exosome markers and binding determinants known as markers of cells secreting exosomes. Examples of binding determinants known as exosome markers include CD9, CD63, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, LAMP1, and the like. Antibodies against these binding determinants are commercially available, and the antibodies can be used as the first binding substance. When a substance (e.g., an antibody) that reacts with the above substances is used as the first binding substance that binds to the binding determinant known as an exosome marker, it binds only to exosomes, and is therefore effective in detecting exosomes in a sample.

[0021] Examples of binding determinants known as markers of cells secreting exosomes include caveolin-1, PSMA, EpCAM, Grypican-1, Survivin, CD91, Tspan8, CD147, EGFR, HER2, CD44, Galactin, and Integrin. Antibodies against these binding determinants are also commercially available, and such antibodies can be used as the first binding substance. When a binding substance (e.g., an antibody) that binds to a binding determinant known as a marker of cells secreting exosomes is used as the first binding substance, it binds only to specific cells and exosomes secreted therefrom, and is therefore effective in detecting exosomes derived from specific cells in a sample.

[0022] When the first binding substance is an antibody, the antibody may be a monoclonal antibody, a polyclonal antibody, or an antibody fragment. The type of the first binding substance immobilized on the metal film may be one type, or two or more types. For example, when the first binding substance immobilized on the metal film is an antibody, the antibody is one or two or more types of monoclonal or polyclonal antibodies.

[0023] The first binding substance is preferably selected in consideration of the combination with the second binding substance, which will be described in relation to the fluorescent label. Preferred combinations of the first binding substance and the second binding substance will be described later.

[0024] The method for immobilizing the binding substance is not particularly limited. For example, a self-assembled monolayer (hereinafter referred to as "SAM") or a polymer film to which a binding substance (e.g., an antibody) is bound may be formed on a metal film. Examples of SAM include HOOC-(CH 2 ) 11Examples of materials constituting the polymer membrane include membranes formed of substituted aliphatic thiols such as -SH. Examples of materials constituting the polymer membrane include polyethylene glycol and MPC polymer. Alternatively, a polymer having a reactive group (or a functional group that can be converted into a reactive group) that can be bound to a binding substance (e.g., an anti-exosome antibody) may be immobilized on a metal membrane, and the binding substance (e.g., an antibody) may be bound to the polymer.

[0025] The measurement chip is preferably a structure with each side having a length of several mm to several cm, but may be a smaller or larger structure that does not fall within the category of a "chip".

[0026] FIG. 2A is a schematic cross-sectional view for explaining the configuration of a measurement chip for PC-SPFS, and FIG. 2B is a schematic cross-sectional view for explaining the configuration of a measurement chip for GC-SPFS. For the sake of convenience, the size and shape of each component in these figures are not accurate. In addition, these figures show an example in which an anti-exosome antibody that recognizes an antigen on an exosome is used as the first binding substance.

[0027] As shown in FIG. 2A, the measurement chip 100 for PC-SPFS has a prism 110, a metal film 120, and an anti-exosome antibody (first binding substance) 130 (layer) that recognizes an antigen on an exosome. The prism 110 is made of a dielectric material that is transparent to the excitation light L1, and has an incident surface 111 on which the excitation light L1 is incident, a deposition surface 112 on which the excitation light L1 is reflected, and an exit surface 113 from which the reflected light L2 is emitted. The shape of the prism 110 is not particularly limited. In the example shown in FIG. 2A, the shape of the prism 110 is a column with a trapezoidal bottom surface. The surface corresponding to one base of the trapezoid is the deposition surface 112, the surface corresponding to one leg is the incident surface 111, and the surface corresponding to the other leg is the exit surface 113. Examples of materials for the prism 110 include resin and glass. The material of the prism 110 is preferably a resin having a refractive index for the excitation light of 1.4 to 1.6 and small birefringence. The metal film 120 is disposed on the film-forming surface 112 of the prism 110. The method for forming the metal film 120 is not particularly limited. Examples of the method for forming the metal film 120 include sputtering, vapor deposition, and plating. The thickness of the metal film 120 is not particularly limited, but is preferably within the range of 30 to 70 nm.

[0028] 2A, when excitation light L1 is irradiated onto the metal film 120 via a prism 110 so that SPR occurs in the metal film 120, an electric field enhanced by SPR is generated near the metal film 120. At this time, if exosomes 140 that have reacted with a second binding substance 131 labeled with a fluorescent substance 150 are bound to an anti-exosome antibody (first binding substance) 130 that recognizes an antigen on the exosome on the metal film 120, the fluorescent substance 150 is excited by the enhanced electric field and emits fluorescence L3.

[0029] As shown in FIG. 2B, the measurement chip 200 for GC-SPFS has a metal film 210 on which a diffraction grating 211 is formed and an anti-exosome antibody (first binding substance) 130 (layer) that recognizes an antigen on an exosome. The method for forming the metal film 210 is not particularly limited. Examples of the method for forming the metal film 210 include sputtering, vapor deposition, and plating. The thickness of the metal film 210 is not particularly limited, but is preferably within a range of 30 to 500 nm. The shape of the diffraction grating 211 is not particularly limited as long as it can generate an evanescent wave. For example, the diffraction grating 211 may be a one-dimensional diffraction grating or a two-dimensional diffraction grating. For example, in the one-dimensional diffraction grating, a plurality of parallel convex portions are formed at a predetermined interval on the surface of the metal film 210. In the two-dimensional diffraction grating, convex portions of a predetermined shape are periodically arranged on the surface of the metal film 210. Examples of the arrangement of the convex portions include a square lattice, a triangular (hexagonal) lattice, and the like. Examples of the cross-sectional shape of the diffraction grating 211 include a rectangular wave shape, a sine wave shape, a sawtooth shape, and the like. The method of forming the diffraction grating 211 is not particularly limited. For example, after forming the metal film 210 on a flat substrate (not shown), an uneven shape may be imparted to the metal film 210. Alternatively, the metal film 210 may be formed on a substrate (not shown) that has previously been imparted with an uneven shape. Either method can form the metal film 210 including the diffraction grating 211.

[0030] As shown in Fig. 2B, when the metal film 210 (diffraction grating 211) is irradiated with excitation light L1 so that SPR occurs in the metal film 210 (diffraction grating 211), an electric field enhanced by SPR is generated near the metal film 210 (diffraction grating 211). At this time, if exosomes 140 that have reacted with second binding substance 131 labeled with fluorescent substance 150 are bound to anti-exosome antibodies (first binding substances) 130 that recognize antigens on exosomes on the metal film 210 (diffraction grating 211), the fluorescent substance 150 is excited by the enhanced electric field and emits fluorescence L3.

[0031] 3A to 3C are schematic cross-sectional views showing an example of a measurement chip for PC-SPFS. As shown in FIG. 3A, the measurement chip 300 has a prism 110 having an incident surface 111, a film-forming surface 112, and an exit surface 113, a metal film 120 formed on the film-forming surface 112 of the prism 110, and a channel cover 310 arranged on the film-forming surface 112 of the prism 110 or the metal film 120. In FIG. 3, the incident surface 111 and the exit surface 113 are located in front of and behind the paper, respectively. The measurement chip 300 further has a channel 320, a liquid injection section 330 connected to one end of the channel 320, and a reservoir section 340 connected to the other end of the channel 320. In this embodiment, the flow channel cover 310 is attached to the metal film 120 (or the prism 110) via an adhesive layer 350 such as a double-sided tape, and the adhesive layer 350 also plays a role in defining the side shape of the flow channel 320. Although omitted in FIG. 3, an anti-exosome antibody (first binding substance) 130 that recognizes an antigen on an exosome is immobilized in a partial region (reaction field) of the metal film 120 exposed in the flow channel 320. The liquid injection section 330 is blocked by a liquid injection section covering film 331, and the storage section 340 is blocked by a storage section covering film 341. The storage section covering film 341 is provided with an air hole 342.

[0032] The flow channel lid 310 is formed of a material transparent to the fluorescence L3. However, a part of the flow channel lid 310 may be formed of a material opaque to the fluorescence L3 as long as it does not interfere with extraction of the fluorescence L3. Examples of materials transparent to the fluorescence L3 include resin. The flow channel lid 310 may be joined to the metal film 120 (or the prism 110) by laser welding, ultrasonic welding, or pressure bonding using a clamp member without using the adhesive layer 350. In this case, the side shape of the flow channel 320 is determined by the flow channel lid 310.

[0033] A pipette tip is inserted into the liquid injection section 330. At this time, the opening of the liquid injection section 330 (a through hole provided in the liquid injection section covering film 331) comes into contact with the outer periphery of the pipette tip without any gap. Therefore, by injecting liquid from the pipette tip into the liquid injection section 330, it is possible to introduce the liquid into the flow channel 320, and by sucking the liquid in the liquid injection section 330 into the pipette tip, it is possible to remove the liquid in the flow channel 320. In addition, by alternately injecting and sucking the liquid, it is also possible to transport the liquid back and forth within the flow channel 320.

[0034] When an amount of liquid that exceeds the volume of the flow channel 320 is introduced from the liquid injector 330 into the flow channel 320, the liquid flows into the reservoir 340 from the flow channel 320. In addition, when the liquid is sent back and forth in the flow channel 320, the liquid also flows into the reservoir 340. The liquid that flows into the reservoir 340 is agitated in the reservoir 340. When the liquid is agitated in the reservoir 340, the concentration of the components (e.g., exosomes, cleaning components, etc.) of the liquid (such as a specimen or cleaning solution) passing through the flow channel 320 becomes uniform, which makes it easier for various reactions to occur in the flow channel 320 and improves the cleaning effect.

[0035] 3B and 3C are schematic diagrams showing an example of a well-shaped measurement chip. FIG. 3B is a schematic diagram of a well-shaped measurement chip 400 with a reaction detection unit on the bottom surface (see, for example, International Publication No. 2012 / 157403). In this chip, the dielectric member 412 is a hexahedron (truncated square pyramid) with a substantially trapezoidal cross section, and the well member 418 is configured in a square shape according to the shape of the dielectric member 412. Then, in a state where a first binding substance that binds to exosomes to be detected is fixed to the ligand fixing region 416 on the metal thin film 414 of the sensor structure 22, a sample solution containing exosomes is supplied into the through-hole 420, and the sensor structure 422 is stirred.

[0036] FIG. 3C shows a well having a reaction detection unit on the side wall surface (see, for example, International Publication No. WO 2018 / 021238). The detection chip 500 has a well body 510 and a side wall member 520. The well body 510 has a storage unit (well) 511 inside. The storage unit 511 is a bottomed recess configured to be able to store liquid, and is open to the outside through a first opening 512 provided at the top and a second opening 513 provided at the side. The side wall member 520 has a prism 521 as an optical element, a metal film 525, and a reaction field 526. The prism 521 is an optical element made of a dielectric material transparent to excitation light, and has an incident surface (not shown), a reflecting surface 523, and an exit surface (not shown). The prism 521 also functions as a side wall constituting the storage unit 511. There is a capture region on metal film 525, and the capture region is a region where a first binding substance for capturing exosomes in a sample is immobilized.

[0037] The type of specimen is not particularly limited as long as it contains exosomes. Examples of specimens include body fluids such as blood (serum, plasma, whole blood), urine, sweat, saliva, breast milk, semen, lymph, cerebrospinal fluid, and tears, as well as dilutions of these body fluids with physiological saline or a buffer solution. In the exosome detection method according to the present embodiment, since exosomes are detected using SPFS, whole blood can also be used as a specimen. Therefore, from the viewpoint of ease of acquisition, serum, plasma, whole blood, or dilutions thereof are preferred as specimens. These specimens may contain microvesicles, impurities, and the like in addition to exosomes. In the present invention, the specimen can be used as is for measurement without removal by centrifugation or filtering, but there is no problem in using a purified liquid after centrifugation or filtering to remove or precipitate microvesicles and impurities for measurement. In addition, the method for removing microvesicles, impurities, and the like is not limited to these.

[0038] The specimen may also be an exosome extract prepared from a body fluid. By extracting exosomes from a body fluid and using them as a specimen, it becomes possible to detect and quantify even trace amounts of exosomes that are difficult to detect in the body fluid itself. There is no particular limitation on the method for extracting exosomes, and any conventionally known method for extracting exosomes can be used. For example, exosomes can be extracted from body fluids by ultracentrifugation, immunoprecipitation, or polymer precipitation.

[0039] A surfactant may be further added to the specimen. Addition of a surfactant can prevent exosome aggregation and reduce background noise and variation in measured values. The surfactant is preferably a surfactant that is widely used in the fields of biology and medicine, and includes, for example, Tween 20, sodium deoxycholate, and Triton-X100. The surfactant does not destroy exosomes, but may be used at a concentration that prevents their aggregation. For example, Tween 20 can be used at a concentration of 0.001% to 5% or less, preferably 0.005% to 1% or less, and more preferably 0.010% to 0.05% or less. In addition, sodium deoxycholate can be used at a concentration of 0.001% to 0.025% or less, preferably 0.002% to 0.020% or less, and more preferably 0.003% to 0.010% or less. In addition, in the case of Triton-X100, it can be used at a concentration of 0.001% to 0.010% or less, preferably 0.002% to 0.007% or less, and more preferably 0.003% to 0.005% or less.

[0040] (First order reaction) Next, a sample is provided on the metal film of the measurement chip, and the exosomes contained in the sample are bound to the first binding substance immobilized on the metal film (first reaction; step S20). The method of providing the sample is not particularly limited. For example, the sample may be provided on the metal film using a pipette with a pipette tip attached to the tip. There is no particular limit to the reaction time of the first reaction, but from the viewpoint of increasing the reaction efficiency between the exosomes and the first binding substance, a longer reaction time is preferable, and is usually 5 minutes or more and 180 minutes or less, preferably 60 minutes or more and 150 minutes or less, and more preferably 100 minutes or more and 120 minutes or less. Usually, after the first reaction is completed, the surface of the metal film is washed with a buffer solution or the like to remove components that are not bound to the first binding substance (washing; step S21). Moreover, after cleaning, the optical blank can be measured (measuring the optical blank; step S2).

[0041] (Secondary reaction) Next, a labeling reagent is provided on the metal film of the measurement chip, and the exosomes bound to the binding substance are labeled with a fluorescent substance via a second binding substance that binds to the exosomes. The type of labeling reagent is not particularly limited as long as the second binding substance labeled with a fluorescent substance can react with the exosomes bound to the first binding substance. For example, the labeling reagent is a second binding substance that is labeled with a fluorescent substance and binds to a second binding determinant possessed by the exosomes (secondary reaction + fluorescent labeling in Figure 1A; step S30). Alternatively, the labeling reagent includes both a second binding substance that binds to a second binding determinant possessed by the exosomes, and a third binding substance that is labeled with a fluorescent substance and binds to the second binding substance bound to the exosomes [secondary reaction in Figure 1B; step S30 and fluorescent labeling (tertiary reaction); step S35].

[0042] The method of providing the labeling reagent is not particularly limited. For example, the labeling reagent may be provided on the metal film using a pipette with a pipette tip attached to the end. Usually, after the second reaction and / or the third reaction is completed, the surface of the metal film is washed with a buffer solution or the like to remove the second binding substance (or the third binding substance) that is not bound to the exosome (washing; steps S31 and S36).

[0043] The type of the second binding substance contained in the labeling reagent is not particularly limited as long as it can bind to exosomes. Examples of the second binding substance include antibodies capable of binding to exosomes, nucleic acids capable of binding to exosomes, lipids capable of binding to exosomes, and proteins other than antibodies capable of binding to exosomes, such as lectins capable of binding to sugar chains on exosomes.

[0044] The second binding determinant of exosomes to which the second binding substance binds is not particularly limited, but includes binding determinants known as exosome markers and binding determinants known as markers of cells secreting exosomes. Examples of binding determinants known as exosome markers include CD9, CD63, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, LAMP1, and the like. Antibodies against these binding determinants are commercially available, and the antibodies can be used as the second binding substance. When a substance (e.g., an antibody) that reacts with the above substances is used as the second binding substance that binds to the binding determinant known as an exosome marker, it binds only to exosomes, so that it is effective for detecting only exosomes even if other things (such as cells) are bound to the metal film.

[0045] Examples of binding determinants known as markers of cells secreting exosomes include caveolin-1, PSMA, EpCAM, Grypican-1, Survivin, CD91, Tspan8, CD147, EGFR, HER2, CD44, Galactin, and Integrin. Antibodies against these binding determinants are also commercially available, and these antibodies can be used as the second binding substance. When a binding substance (e.g., an antibody) that binds to a binding determinant known as a marker of cells secreting exosomes is used as the second binding substance, it binds only to specific cells and exosomes secreted therefrom, and is therefore effective in detecting only exosomes derived from specific cells among various exosomes bound to a metal membrane.

[0046] When the second binding substance is an antibody, the antibody may be a monoclonal antibody, a polyclonal antibody, or an antibody fragment. The type of the second binding substance contained in the labeling reagent may be one type or two or more types. For example, the anti-exosome antibody labeled with a fluorescent substance is one or two or more types of anti-exosome monoclonal antibodies or anti-exosome polyclonal antibodies. In this case, it is preferable that the anti-exosome monoclonal antibody and anti-exosome polyclonal antibody labeled with a fluorescent substance are different from the one or two or more types of anti-exosome monoclonal antibodies immobilized on the metal film.

[0047] The type of the second binding substance contained in the labeling reagent may be the same as or different from the type of the first binding substance immobilized on the metal film. For example, the first binding substance and the second binding substance may both be antibodies, or one may be an antibody and the other a protein other than an antibody.

[0048] The first binding substance immobilized on the metal film and the second binding substance contained in the labeling reagent each bind to a binding determinant possessed by exosomes, but it is preferable that the first binding determinant bound by the first binding substance is different from the second binding determinant bound by the second binding substance. The first binding substance and the second binding substance do not compete for the same binding determinant, but bind to different binding determinants, thereby making it possible to more reliably immobilize exosomes on the metal film and label them with the labeling substance. In addition, the first binding determinant bound by the first binding substance is different from the second binding determinant bound by the second binding substance, so that exosomes to be further measured based on the binding of the second binding substance can be narrowed down from the exosomes detected from the sample based on the binding of the first binding substance.

[0049] For the first binding substance and the second binding substance, at least one of the first binding determinant and the second binding determinant is preferably a binding determinant known as a marker for exosomes. By using a binding substance that binds to a binding determinant known as a marker for exosomes, only exosomes can be detected from substances in a sample. In addition, at least one of the first binding determinant and the second binding determinant is preferably a binding determinant known as a marker for cells that secrete exosomes. By using a binding substance that binds to a binding determinant known as a marker for cells that secrete exosomes, exosomes derived from a specific cell can be detected. In the present invention, it is more preferable that one of the first binding substance and the second binding substance binds to a binding determinant known as a marker for exosomes, and the other binds to a binding determinant known as a marker for cells that secrete exosomes. By using such two types of binding substances in combination, exosomes secreted by a specific cell can be specifically detected. For example, it is known that cancer cells secrete exosomes different from normal cells, and by detecting exosomes derived from cancer cells in body fluids, it becomes possible to use exosomes as a cancer marker.

[0050] The third binding substance contained in the labeling reagent is not particularly limited as long as it can be labeled with a fluorescent substance and can bind to the second binding substance. Examples of the third binding substance include antibodies, nucleic acids, lipids, proteins other than antibodies, and proteins other than antibodies (e.g., lectins) that can bind to the antibodies, nucleic acids, lipids, and proteins other than antibodies used as the second binding substance. For example, by using a combination of the second and third binding substances in which multiple third binding substances bind to one second binding substance, it is possible to bind more fluorescent substances to exosomes than when the second binding substance is directly fluorescently labeled and used, thereby improving the measurement sensitivity. In addition, even when it is difficult to directly label the second binding substance with a fluorescent substance, or when the binding ability of the second binding substance to exosomes is changed, reduced, or lost due to fluorescent labeling, the third binding substance can be used to label exosomes.

[0051] The type of fluorescent substance for labeling the second or third binding substance is not particularly limited as long as it can be used in SPFS. Examples of fluorescent substances include cyanine dyes, Alexa Fluor (registered trademark) dyes from Thermo Scientific, and CF dyes from Biotium. Among commercially available fluorescent dyes, Alexa Fluor dyes and CF dyes have high quantum efficiency for the wavelength of excitation light used in SPFS. In addition, CF dyes do not fade much during fluorescence detection, so fluorescence detection can be performed stably. The method of labeling the binding substance with a fluorescent substance is not particularly limited and can be appropriately selected from known methods. For example, a fluorescent substance may be bound to an amino group or a sulfhydryl group of a binding substance (e.g., an anti-exosome antibody).

[0052] In the above description, exosomes are bound to the first binding substance immobilized on the metal film, and then the exosomes are labeled with a fluorescent substance using a labeling reagent. However, exosomes may be labeled with a fluorescent substance using a labeling reagent before binding the exosomes to the first binding substance immobilized on the metal film. In this case, the specimen and the labeling reagent (second binding substance) may be mixed before providing the specimen on the metal film. A third binding substance that is further fluorescently labeled may be added to the mixture of the specimen and the labeling reagent (second binding substance). Furthermore, the step of binding exosomes to the binding substance immobilized on the metal film and the step of labeling exosomes with a fluorescent substance may be performed simultaneously. In this case, the specimen and the labeling reagent (second binding substance, or second and third binding substances) may be provided simultaneously on the metal film.

[0053] (Fluorescence measurement) Next, the fluorescence indicating the amount of exosomes is measured by SPFS (step S40). Specifically, in a state where exosomes labeled with a fluorescent substance are bound to a binding substance immobilized on a metal film, excitation light is irradiated onto the metal film so that SPR occurs on the metal film, and the fluorescence emitted from the fluorescent substance is measured. Usually, a signal value correlating to the amount of exosomes is calculated by subtracting a previously measured optical blank value from the measured fluorescence value. If necessary, the signal value may be converted into the amount (number / ml) or concentration (μg / ml) of exosomes using a previously prepared calibration curve, etc.

[0054] For example, a calibration curve for exosomes can be obtained by preparing samples with different dilution rates using commercially available exosomes, determining the number and concentration of exosomes in each sample, and determining the signal value using the method of the present invention, and plotting the signal value against the number and concentration of exosomes. In addition, the number and concentration of exosomes in a sample can be measured, and a calibration curve (Figure 4) can be obtained in which the number of exosomes is plotted against the concentration of exosomes. By using such a calibration curve, the amount of exosomes counted as the number can be converted to a concentration (μg / ml). By performing such a conversion, it is also possible to compare with other companies' devices that detect the amount of exosomes as a concentration (μg / ml).

[0055] The method for measuring the number of exosomes is not particularly limited, and for example, a qNano / Nanoparticle Multi-Analyzer (manufactured by Meiwafosis Co., Ltd.) can be used. The method for measuring the concentration of exosomes is also not particularly limited, and for example, it can be measured by protein quantification (BCA method, manufactured by ThermoFischer).

[0056] As shown in FIG. 2A, when the measurement chip 100 for PC-SPFS is used, the excitation light L1 is irradiated onto the metal film 120 via the prism 110. As a result, SPR occurs in the metal film 120, and the fluorescent substance 150 present in the vicinity of the metal film 120 is excited by the enhanced electric field and emits fluorescence L3. The incident angle of the excitation light L1 on the metal film 120 is set so that SPR occurs in the metal film 120, and is preferably a resonance angle or an enhanced angle. Here, the "resonance angle" refers to the incident angle at which the amount of reflected light L2 is minimized when the incident angle of the excitation light L1 on the metal film 120 is scanned. In addition, the "enhanced angle" refers to the incident angle at which the amount of scattered light (plasmon scattered light) of the same wavelength as the excitation light L1 emitted above the metal film 120 (the opposite side of the prism 110) is maximized when the incident angle of the excitation light L1 on the metal film 120 is scanned.

[0057] As shown in FIG. 2B, when the measurement chip 200 for GC-SPFS is used, the excitation light L1 is directly irradiated onto the metal film 210 (diffraction grating 211). As a result, SPR occurs in the metal film 210 (diffraction grating 211), and the fluorescent substance 150 present in the vicinity of the metal film 210 (diffraction grating 211) is excited by the enhanced electric field and emits fluorescence L3. The incident angle of the excitation light L1 with respect to the metal film 210 is set so that SPR occurs in the metal film 210, and an angle at which the intensity of the enhanced electric field formed by SPR is strongest is preferable. The optimal incident angle of the excitation light L1 is appropriately set depending on the pitch of the diffraction grating 211, the wavelength of the excitation light L1, the type of metal constituting the metal film 210, and the like.

[0058] The type of excitation light is not particularly limited, but is usually a laser light. For example, the excitation light is a laser light emitted from a laser light source having an output of 10 μW to 30 mW. The irradiation energy of the excitation light is 7.5 μW / mm 2 More than 30mW / mm 2 Less than 8.5μW / mm 2 More than 10mW / mm 2 Less than 9.5 μW / mm is preferable. 2 More than 5mW / mm 2 More preferably, the irradiation energy of the excitation light is 30 mW / mm 2 By setting the light intensity at 30 mW / mm or less, the fluorescence intensity is increased, the signal-to-noise ratio (S / N) is increased, and it becomes possible to detect smaller amounts of exosomes, thereby improving the detection limit. The wavelength of the excitation light is appropriately set according to the excitation wavelength of the fluorescent substance used. In addition, the light intensity must be set to 30 mW / mm or less. 2 If the radiation exposure exceeds this value, the heat caused by the irradiation will cause dissociation of the antigen-antibody reaction, resulting in a poor signal-to-noise ratio.

[0059] The fluorescence detector is preferably installed in the direction in which the fluorescence intensity is highest with respect to the measurement chip. For example, as shown in FIG. 2A, when a measurement chip 100 for PC-SPFS is used, the direction in which the intensity of the fluorescence L3 is highest is the normal direction of the metal film 120, so the detector is installed directly above the measurement chip. On the other hand, as shown in FIG. 2B, when a measurement chip 200 for GC-SPFS is used, the direction in which the intensity of the fluorescence L3 is highest is a direction that is somewhat inclined with respect to the normal line of the metal film 120, so the detector is installed at a position that is not directly above the measurement chip. The detector is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).

[0060] By following the above procedure, the concentration of exosomes contained in the sample can be measured.

[0061] [Exosome measurement kit] The exosome measurement kit according to the present embodiment is a set of the above measurement chip and the above labeling reagent (containing a fluorescent substance and a second binding substance, and optionally further containing a third binding substance). By preparing the above measurement chip and labeling reagent in advance in this manner, users (medical workers, etc.) can more easily perform the above exosome measurement method. The measurement kit may further contain a surfactant. The use of a surfactant can reduce background noise and variation in measurement values.

[0062] [effect] As described above, according to the exosome measurement method or measurement kit of the present embodiment, exosomes in a sample can be measured easily and with high sensitivity by utilizing SPFS. EXAMPLES

[0063] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0064] Experiment 1: Measurement of exosomes from healthy blood using two types of antibodies against exosomes A measurement chip 300 having the configuration shown in Fig. 3 was prepared. An anti-CD9 monoclonal antibody was immobilized as a first binding substance on a specific region (reaction section) of the metal film 120 (gold thin film) exposed in the flow channel 320.

[0065] Standard exosomes (lyophilized product) derived from healthy individuals purchased from Cosmo Bio Co., Ltd. were hydrated and used as specimen samples. Dilutions were made in a 10-fold series using PBS containing 1% BSA. The sample dilution solution in the device contained a surfactant, and the concentration of the surfactant was 0.05%.

[0066] The number of exosomes in each sample was measured using a qNano / Nanoparticle Multi-Analyzer (manufactured by Meiwafosis Co., Ltd.) for each dilution.

[0067] Furthermore, the signal value correlating with the amount of exosomes was measured for the same specimen as above. A specimen (either diluted blood) was introduced from the liquid injection section 330 into the flow channel 320 using a pipette tip, and the liquid was sent back and forth (primary reaction). The reaction time for the primary reaction was 100 minutes. After removing the specimen from the flow channel 320 from the liquid injection section 330, the inside of the flow channel 320 was washed once with a cleaning solution. Next, a labeled reagent (anti-CD63 monoclonal antibody labeled with an Alexa Fluor dye) was introduced from the liquid injecting section 330 into the flow channel 320 and sent back and forth (secondary reaction). The reaction time for the secondary reaction was 10 minutes. After removing the labeled reagent from the flow channel 320 from the liquid injecting section 330, the flow channel 320 was washed once with a cleaning solution. Next, a measurement solution was introduced from the liquid injecting section 330 into the flow channel 320. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with excitation light (laser light) from the prism 110 side so that the incident angle of the excitation light with respect to the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the amount of irradiation energy was 3.8 mW / mm 2The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate the signal value correlating with the amount of exosomes. The same measurement was performed six times for each sample.

[0068] The measured signal values ​​were plotted against the number of exosomes in the sample (pieces / μl) to obtain a calibration curve for CD9-positive CD63-positive exosomes (Figure 5).

[0069] As is clear from the plot in Figure 5, there is a correlation between the number of exosomes in the sample and the signal value, and the detection limit is 2.1 × 10 2 The number was low at 1 / μl.

[0070] Next, the signal value correlated with the amount of exosomes was calculated in the same manner as above, except for changing the output of the excitation light used for detection. The output of the excitation light used for detection was 5 mW, 15 mW, or 30 mW, and the irradiation energy of each was 3.8 mW / mm 2 , 11.3mW / mm 2 or 22.6mW / mm 2 For each irradiation energy amount, the detection limit for measuring exosomes from the specimen was determined in the same manner as above.

[0071] In addition, the signal-to-noise ratio (S / N) of the above measurements was calculated as follows: the signal value (S) was calculated by subtracting the optical blank from the measurement signal in the presence of exosomes, and the noise value (N) was calculated by subtracting the optical blank from the measurement signal in the absence of exosomes. Next, the S / N ratio was calculated.

[0072] The calculated S / N and detection limit were plotted against the amount of irradiation energy, respectively, to obtain a graph (Figure 6) showing the relationship between irradiation energy, S / N, and detection limit for CD9-positive CD63-positive exosomes derived from healthy individuals.

[0073] In the plot of Figure 6, when the irradiation energy of the excitation light used to measure exosomes from a sample was low, the S / N ratio increased (i.e., the noise to the signal decreased) and the detection limit was improved.

[0074] Experiment 2: Measurement of exosomes associated with prostate cancer using antibodies against exosome-secreting cells and antibodies against exosomes

[0075] A measurement chip 300 having the configuration shown in Fig. 3 was prepared. An anti-PSMA monoclonal antibody was immobilized as a first binding substance on a specific region (reaction portion) of the metal film 120 (thin gold film) exposed in the flow channel 320.

[0076] Standard exosomes (lyophilized product) derived from LNCap cells, a type of prostate cancer cell, purchased from Cosmo Bio Co., Ltd. were hydrated and used as the specimen sample. A 10-fold dilution series was performed using PBS containing 1% BSA. The sample dilution solution in the device contained a surfactant, and the surfactant concentration was 0.05%.

[0077] The number of LNCap cell-derived exosomes in the samples was measured using a qNano / Nanoparticle Multi-Analyzer (manufactured by Meiwafosis Co., Ltd.).

[0078] Furthermore, for the same specimen as above, in order to create a calibration curve of CD9-positive PSMA-positive exosomes derived from LNCap cells, a signal value correlating to the amount of exosomes was measured for each specimen with different dilution rates. A specimen (any diluted culture supernatant) was introduced into the flow channel 320 from the liquid injection section 330 using a pipette tip, and the liquid was sent back and forth (first reaction). The reaction time of the first reaction was 100 minutes. After removing the specimen in the flow channel 320 from the liquid injection section 330, the inside of the flow channel 320 was washed once with a cleaning solution. Next, an anti-CD9 monoclonal antibody labeled with a labeling reagent (Alexa Fluor dye) was introduced into the flow channel 320 from the liquid injection section 330, and the liquid was sent back and forth (second reaction). The reaction time of the second reaction was 10 minutes. After removing the labeling reagent in the flow channel 320 from the liquid injection section 330, the inside of the flow channel 320 was washed once with a cleaning solution. Next, a measurement liquid was introduced into the flow channel 320 from the liquid injection section 330. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with excitation light (laser light) from the prism 110 side so that the incident angle of the excitation light on the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the amount of irradiation energy was 3.8 mW / mm 2 The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate the signal value correlating with the amount of exosomes. The same measurement was performed six times for each sample.

[0079] The measured signal values ​​were plotted against the number of exosomes in the sample (pieces / μl) to obtain a calibration curve for CD9-positive, PSMA-positive exosomes (Figure 7).

[0080] As is clear from the plot in Figure 7, the above method was able to detect exosomes (CD9-positive, PSMA-positive exosomes) derived from specific cells (prostate cancer cells). In addition, there was a correlation between the number of CD9-positive, PSMA-positive exosomes and the signal value, and the detection limit was 1.7 × 10 2 The levels were low at 1 / μl.

[0081] Next, the signal value correlated with the amount of CD9+PSMA+ exosomes was calculated in the same manner as above, except for changing the output of the excitation light used for detection. The output of the excitation light used for detection was 5 mW, 15 mW, or 30 mW, and the irradiation energy of each was 3.8 mW / mm 2 , 11.3mW / mm 2 or 22.6mW / mm 2 For each irradiation energy amount, the detection limit for measuring exosomes from the specimen was determined in the same manner as above.

[0082] In addition, for the above measurements, the signal-to-noise ratio (S / N) was calculated in the same manner as in Experiment 1.

[0083] The calculated S / N and detection limit were plotted against the amount of irradiation energy, respectively, to obtain a graph (FIG. 8) showing the relationship between irradiation energy, S / N, and detection limit for CD9-positive, PSMA-positive exosomes.

[0084] In the plot of Figure 8, when the irradiation energy of the excitation light used to measure CD9-positive, PSMA-positive exosomes was low, the S / N ratio increased (i.e., the noise to the signal decreased) and the detection limit was improved.

[0085] Experiment 3: Measurement of exosomes derived from prostate cancer cells using two types of antibodies against exosomes

[0086] A measurement chip 300 having the configuration shown in Fig. 3 was prepared. An anti-CD9 monoclonal antibody was immobilized as a first binding substance on a specific region (reaction section) of the metal film 120 (gold thin film) exposed in the flow channel 320.

[0087] Standard exosomes (lyophilized product) derived from LNCap cells, a type of prostate cancer cell, purchased from Cosmo Bio Co., Ltd. were hydrated and used as the specimen sample. A 10-fold dilution series was performed using PBS containing 1% BSA. The sample dilution solution in the device contained a surfactant, and the surfactant concentration was 0.05%.

[0088] In order to create a calibration curve for CD9-positive CD63-positive exosomes derived from LNCap cells, a signal value correlating with the amount of exosomes was measured for each of the samples with different dilution rates. A sample (any of the diluted culture supernatants) was introduced into the flow channel 320 from the liquid injection section 330 using a pipette tip, and the liquid was sent back and forth (first reaction). The reaction time of the first reaction was 100 minutes. After removing the sample from the flow channel 320 from the liquid injection section 330, the flow channel 320 was washed once with a cleaning solution. Next, a labeling reagent (anti-CD63 monoclonal antibody labeled with Alexa Fluor dye) was introduced into the flow channel 320 from the liquid injection section 330, and the liquid was sent back and forth (second reaction). The reaction time of the second reaction was 10 minutes. After removing the labeling reagent from the flow channel 320 from the liquid injection section 330, the flow channel 320 was washed once with a cleaning solution. Next, a measurement liquid was introduced into the flow channel 320 from the liquid injection section 330. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with excitation light (laser light) from the prism 110 side so that the incident angle of the excitation light on the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the amount of irradiation energy was 3.8 mW / mm 2 The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate the signal value correlating with the amount of exosomes. The same measurement was performed six times for each sample.

[0089] The number of LNCap cell-derived exosomes in the same samples was measured. The number of exosomes extracted was measured using a qNano / Nanoparticle Multi-Analyzer (Meiwafosis Co., Ltd.). The weight was determined by protein quantification (BCA method, ThermoFischer).

[0090] The measured signal values ​​were plotted against the number of exosomes in the sample (pieces / μl) to obtain a calibration curve for CD9-positive CD63-positive exosomes (Figure 9).

[0091] As is clear from the plot in Figure 9, the above method was able to detect CD9+CD63+ exosomes even in samples derived from specific cells (prostate cancer cells). In addition, there was a correlation between the number of CD9+CD63+ exosomes and the signal value, and the detection limit was 3.2 × 10 0 The number was low at 1 / μl.

[0092] Next, the signal value correlated with the amount of CD9+CD63+ exosomes was calculated in the same manner as above, except for changing the output of the excitation light used for detection. The output of the excitation light used for detection was 5 mW, 15 mW, or 30 mW, and the irradiation energy of each was 3.8 mW / mm 2 , 11.3mW / mm 2 or 22.6mW / mm 2 For each irradiation energy amount, the detection limit for measuring exosomes from the specimen was determined in the same manner as above.

[0093] In addition, for the above measurements, the signal-to-noise ratio (S / N) was calculated in the same manner as in Experiment 1.

[0094] The calculated S / N and detection limit were plotted against the amount of irradiation energy, respectively, to obtain a graph (FIG. 10) showing the relationship between irradiation energy, S / N, and detection limit for CD9-positive CD63-positive exosomes.

[0095] In the plot of Figure 10, when the irradiation energy of the excitation light used to measure CD9-positive CD63-positive exosomes was low, the S / N ratio increased (i.e., the noise to the signal decreased) and the detection limit was improved.

[0096] Experiment 4: Measurement of CD9-positive glycan-bearing exosomes using an antibody against exosomes and WFA lectin that recognizes glycans on exosomes

[0097] A measurement chip 300 having the configuration shown in Fig. 3 was prepared. An anti-CD9 monoclonal antibody was immobilized as a first binding substance on a specific region (reaction section) of the metal film 120 (gold thin film) exposed in the flow channel 320.

[0098] Standard exosomes (lyophilized product) derived from LNCap cells, a type of prostate cancer cell, purchased from Cosmo Bio Co., Ltd. were hydrated and used as the specimen sample. A 10-fold dilution series was performed using PBS containing 1% BSA. The sample dilution solution in the device contained a surfactant, and the surfactant concentration was 0.05%.

[0099] In order to create a calibration curve for CD9-positive glycan-bearing exosomes derived from LNCap cells, a signal value correlating with the amount of exosomes was measured for each of the samples with different dilution rates. A sample (any of the diluted culture supernatants) was introduced into the flow channel 320 from the liquid injection section 330 using a pipette tip, and the liquid was sent back and forth (first reaction). The reaction time for the first reaction was 100 minutes. After removing the sample from the flow channel 320 from the liquid injection section 330, the flow channel 320 was washed once with a cleaning solution. Next, a labeling reagent (WFA lectin labeled with Alexa Fluor dye) was introduced into the flow channel 320 from the liquid injection section 330, and the liquid was sent back and forth (second reaction). The reaction time for the second reaction was 10 minutes. After removing the labeling reagent from the flow channel 320 from the liquid injection section 330, the flow channel 320 was washed once with a cleaning solution. Next, a measurement liquid was introduced into the flow channel 320 from the liquid injection section 330. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with excitation light (laser light) from the prism 110 side so that the incident angle of the excitation light on the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the amount of irradiation energy was 3.8 mW / mm 2 The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate the signal value correlating with the amount of exosomes. The same measurement was performed six times for each sample.

[0100] The number of LNCap cell-derived exosomes in the same samples was measured using the qNano / Nanoparticle Multi-Analyzer (Meiwafosis Co., Ltd.).

[0101] The measured signal values ​​were plotted against the number of exosomes in the sample (pieces / μl) to obtain a calibration curve for CD9-positive glycan-bearing exosomes (FIG. 11).

[0102] As is clear from the plot in Figure 11, the above method was able to detect CD9-positive glycan-bearing exosomes even in samples derived from specific cells (prostate cancer cells). In addition, there was a correlation between the number of CD9-positive glycan-bearing exosomes and the signal value, with the detection limit being 9.9 × 10 1 The number was low at 1 / μl.

[0103] Experiment 5: Measurement of exosomes derived from prostate cancer cells using antibodies against exosome-secreting cells and lectins that recognize glycans on exosomes

[0104] A measurement chip 300 having the configuration shown in Fig. 3 was prepared. An anti-PSMA monoclonal antibody was immobilized as a first binding substance on a specific region (reaction portion) of the metal film 120 (thin gold film) exposed in the flow channel 320.

[0105] Standard exosomes (lyophilized product) derived from LNCap cells, a type of prostate cancer cell, purchased from Cosmo Bio Co., Ltd. were hydrated and used as the specimen sample. A 10-fold dilution series was performed using PBS containing 1% BSA. The sample dilution solution in the device contained a surfactant, and the concentration of the surfactant was 0.05%.

[0106] The number of LNCap cell-derived exosomes in the samples was measured using a qNano / Nanoparticle Multi-Analyzer (manufactured by Meiwafosis Co., Ltd.).

[0107] Furthermore, for the same specimen as above, in order to create a calibration curve of PSMA-positive glycan-bearing exosomes derived from LNCap cells, a signal value correlating to the amount of exosomes was measured for each specimen with different dilution rates. A specimen (any diluted culture supernatant) was introduced into the flow channel 320 from the liquid injection section 330 using a pipette tip, and the liquid was sent back and forth (first reaction). The reaction time of the first reaction was 100 minutes. After removing the specimen in the flow channel 320 from the liquid injection section 330, the inside of the flow channel 320 was washed once with a cleaning solution. Next, WFA lectin labeled with a labeling reagent (Alexa Fluor dye) was introduced into the flow channel 320 from the liquid injection section 330, and the liquid was sent back and forth (second reaction). The reaction time of the second reaction was 10 minutes. After removing the labeling reagent in the flow channel 320 from the liquid injection section 330, the inside of the flow channel 320 was washed once with a cleaning solution. Next, a measurement liquid was introduced into the flow channel 320 from the liquid injection section 330. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with excitation light (laser light) from the prism 110 side so that the incident angle of the excitation light on the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the amount of irradiation energy was 3.8 mW / mm 2 The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate the signal value correlating with the amount of exosomes. The same measurement was performed six times for each sample.

[0108] The measured signal values ​​were plotted against the number of exosomes in the sample (pieces / μl) to obtain a calibration curve for PSMA-positive glycan-bearing exosomes (Figure 12).

[0109] As is clear from the plot in Figure 12, the above method was able to detect exosomes (exosomes bearing PSMA-positive glycans) derived from specific cells (prostate cancer cells). In addition, there was a correlation between the number of exosomes bearing PSMA-positive glycans and the signal value, and the detection limit was 2.5 × 10 2 The number was low at 1 / μl.

[0110] This application claims priority from Japanese Patent Application No. 2019-119824, filed on June 27, 2019. The entire contents of said application are incorporated herein by reference. [Industrial Applicability]

[0111] By using the exosome measurement method or measurement kit according to the present embodiment, exosomes can be measured with high sensitivity and ease. Therefore, the exosome detection method and measurement kit according to the present invention are useful for, for example, clinical tests. [Explanation of symbols]

[0112] 100, 200, 300, 400, 500 measurement chips 110 Prism 111 Incidence plane 112 Film formation surface 113 Exit surface 120 Metal Film 130 Anti-exosome antibody (first binding substance) 131 Anti-exosome antibody (second binding substance) 140 Exosomes 150 Fluorescent Materials 210 Metal Film 211 Diffraction Grating 310 Channel cover 320 Flow Path 330 Liquid injection part 331 Liquid injection part covering film 340 Storage Unit 341 Reservoir Covering Film 342 Vent 350 Adhesive layer 412 Dielectric materials 414 Metallic Thin Film 416 Ligand Fixation Region 418 Well Parts 420 through hole 422 Sensor Structure 510 Well Body 511 Storage unit 520 Side wall member 521 Prism 523 Reflective surface 525 Metal Film 526 Reaction Field L1 excitation light L2 reflected light L3 fluorescence

Claims

1. A step of preparing a measurement chip including a metal film and a first binding substance that binds to exosomes and is immobilized on the metal film; Providing a specimen containing exosomes on the metal film and binding the exosomes contained in the specimen to the first binding substance; labeling the exosomes with a fluorescent substance via a second binding substance that binds to the exosomes before or after binding to the first binding substance; A step of irradiating the metal film with excitation light so that surface plasmon resonance occurs in the metal film while the exosomes labeled with the fluorescent substance are bound to the first binding substance, and detecting fluorescence emitted from the fluorescent substance; A method for measuring exosomes, comprising:

2. The method for measuring exosomes according to claim 1, wherein the first binding substance binds to a first binding determinant possessed by the exosome, and the second binding substance binds to a second binding determinant possessed by the exosome, the first binding determinant and the second binding determinant being different.

3. The method for measuring exosomes according to claim 2 , wherein at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for the exosome.

4. The method for measuring exosomes according to claim 2 or 3, wherein at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for cells that secrete the exosomes.

5. The method for measuring exosomes according to any one of claims 1 to 4, wherein in the labeling step, the exosomes are labeled with a fluorescent substance using a third binding substance that binds to the second binding substance.

6. The irradiation energy of the excitation light is 7.5 μW / mm ２ 10mW / mm or more ２ The method for measuring exosomes according to any one of claims 1 to 5, wherein the method is as follows:

7. The method for measuring exosomes according to any one of claims 1 to 6, wherein a surfactant is added to the specimen before providing the specimen containing exosomes on the metal film.

8. The method for measuring exosomes according to claim 7, wherein the surfactant is at least one selected from the group consisting of Tween 20, sodium deoxycholate, and Triton-X100.

9. The method for measuring exosomes according to any one of claims 1 to 8, wherein the sample is serum, plasma, whole blood, or a dilution thereof.

10. The method for measuring exosomes according to any one of claims 1 to 8, wherein the specimen is an exosome extract.

11. the metal film is disposed on a prism; The excitation light is irradiated onto the metal film through a prism. The method for measuring exosomes according to any one of claims 1 to 10.

12. the metal film includes a diffraction grating; the first binding substance is immobilized on the diffraction grating; The excitation light is irradiated onto the diffraction grating. The method for measuring exosomes according to any one of claims 1 to 10.

13. A measuring chip including a metal film and a first binding substance that binds to exosomes and is immobilized on the metal film; A labeling reagent for labeling exosomes with a fluorescent substance; Including, A kit for measuring exosomes, wherein the labeling reagent comprises a second binding substance that binds to the exosomes.

14. The exosome measurement kit according to claim 13, wherein the first binding substance binds to a first binding determinant possessed by the exosome, and the second binding substance binds to a second binding determinant possessed by the exosome, the first binding determinant and the second binding determinant being different.

15. The exosome measurement kit according to claim 14, wherein at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for the exosome.

16. The exosome measurement kit according to claim 14 or 15, wherein at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for cells that secrete the exosome.

17. The exosome measurement kit according to any one of claims 13 to 16, wherein the labeling reagent further comprises a third binding substance that binds to the second binding substance.

18. The exosome measurement kit according to any one of claims 13 to 17, further comprising a surfactant to be added to a specimen.

19. The exosome measurement kit according to claim 18, wherein the surfactant is at least one selected from the group consisting of Tween 20, sodium deoxycholate, and Triton-X100.

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