WO2020262374A1 - Exosome measurement method and exosome measurement kit - Google Patents

Abstract

The present invention provides an exosome measurement method that makes it possible to measure the exosome concentration in a sample with higher sensitivity and more easily compared with existing measurement methods. In the measurement method according to the present invention, a measurement chip including a metallic film and a first binding substance that is immobilized on the metallic film and that binds to exosomes is prepared. A sample containing exosomes is provided on the metallic film, causing the exosomes contained in the sample to bind to the first binding substance. The exosomes are labelled with a fluorescent substance via a second binding substance that binds to the exosomes. In a state where the exosomes labelled with the fluorescent substance are bound to the first binding substance, the metallic film is irradiated with excitation light so as to cause surface plasmon resonance on the metallic film, and fluorescence radiated from the fluorescent substance is detected.

Description

Exosome measurement method and exosome measurement kit

The present invention relates to an exosome measuring method and an exosome measuring kit.

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 and contain a variety of cellular components such as membrane proteins and microRNAs (miRNAs). Exosomes are thought to mediate communication between cells and organs, because different exosomes are released from cells depending on the type and state of the cells.

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 an exosome as a tumor marker. In this method, an antibody against an antigen possessed by an exosome and an antibody against an antigen possessed by a cell secreting the exosome are used to detect the exosome based on an avidin-biotin interaction. Further, Patent Document 2 discloses an exosome analysis device and a method for capturing exosomes, in which an antibody against exosomes is fixed in a recess of a base portion made of a compatible resin having an uneven structure and the exosome is captured in the recess. There is.

International Publication No. 2013/094307 Japanese Unexamined Patent Publication No. 2014-219384

As mentioned above, various methods for analyzing exosomes have been developed so far, but there is a demand for a method capable of measuring exosomes with higher sensitivity and higher reproducibility.

An object of the present invention is to provide a method for measuring exosomes and a kit for measuring exosomes, which can measure the concentration of exosomes in a sample more sensitively and easily than the conventional measuring methods.

The method for measuring an exosome according to an embodiment of the present invention includes a step of preparing a measuring chip containing a metal film and a first binding substance that binds to the exosome, which is immobilized on the metal film, and the metal film. A step of providing a sample containing an exosome on the sample and binding the exosome contained in the sample to the first binding substance, and the exosome before or after binding to the first binding substance. A step of labeling with a fluorescent substance via a second binding substance that binds to the exosome, and a surface with the metal film in a state where the exosome labeled with the fluorescent substance is bound to the first binding substance. It includes a step of irradiating the metal film with excitation light so as to cause plasmon resonance and detecting the fluorescence emitted from the fluorescent substance.

An exosome measurement kit according to an embodiment of the present invention comprises a metal membrane, a measurement chip immobilized on the metal membrane, and a first binding substance that binds to the exosome, and the exosome is labeled with a fluorescent substance. The labeling reagent comprises a second binding substance that binds to the exosome.

According to the present invention, the concentration of exosomes in a sample can be measured more sensitively and easily than the conventional measuring method.

FIG. 1A is a flowchart showing an example of a method for measuring exosomes according to the present embodiment. FIG. 1B is a flowchart showing another example of the method for measuring exosomes according to the present embodiment. FIG. 2A is a schematic cross-sectional view for explaining the configuration of the measuring chip for PC-SPFS, and FIG. 2B is a schematic cross-sectional view for explaining the configuration of the measuring chip for GC-SPFS. FIG. 3A is a schematic cross-sectional view showing an example of a measuring chip for PC-SPFS. FIG. 3B is a schematic perspective view showing another example of the measuring chip for PC-SPFS. FIG. 3C is a schematic cross-sectional view showing another example of the measuring chip for PC-SPFS. FIG. 4 is a calibration curve showing the relationship between the number of exosomes and the concentration. FIG. 5 is a CD9-positive CD63-positive exosome calibration curve. FIG. 6 is a graph showing the relationship between irradiation energy, signal-to-noise ratio (S / N), and detection limit for CD9-positive and CD63-positive exosomes. FIG. 7 is a calibration curve of CD9-positive PSMA-positive exosomes. 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. FIG. 9 is a calibration curve of CD9-positive and CD63-positive exosomes derived from LNCaP cells. FIG. 10 is a graph showing the relationship between irradiation energy, signal-to-noise ratio (S / N), and detection limit for CD9-positive and CD63-positive exosomes derived from LNCaP cells. FIG. 11 is a calibration curve of a CD9-positive sugar chain-carrying exosome derived from LNCaP cells using a lectin that recognizes sugar chains on exosomes. FIG. 12 is a calibration curve of a CD9-positive sugar chain-carrying exosome derived from prostate cancer cells using a lectin that recognizes sugar chains on exosomes.

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

[Measurement method of exosomes]
Exosomes are secreted by normal cells as well as by disease-related cells such as cancer cells. Therefore, in order to use exosomes as disease markers, it is possible to distinguish between a large amount of normal cell-derived exosomes present in a sample (for example, blood) and a smaller amount of disease-related cell-derived exosomes. Method is required. Generally, the concentration of exosomes present in serum is said to be about 1.0 × 10 ¹¹ (1.0 × 10 ⁸ / μl), and among them, exosomes related to diseases (for example, cancer). To detect exosomes, a method capable of detecting exosomes of 1.0 × 10 ⁶ / ml (1.0 × 10 ³ / μl) or less is required.

In the method for measuring exosomes 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”). To do. SPFS is a target (exosome in this embodiment) rather than a general fluorescence immunoassay because it excites a fluorescent substance by an electric field enhanced by surface plasmon resonance (hereinafter, also referred to as “SPR”) to emit fluorescence. Can be detected with high sensitivity.

In addition, since whole blood can be used as a sample for SPFS, disease-related exosomes can be easily detected with a small number of procedures without purifying exosomes in blood.

Based on the above idea, the present inventors have found that exosomes can be measured with high sensitivity by SPFS as a result of repeated diligent studies, and completed the method for measuring exosomes according to the present embodiment.

Hereinafter, the method for measuring exosomes according to the present embodiment will be specifically described. FIG. 1A is a flowchart showing a measurement method using a primary reaction and a secondary reaction, which is an example of a method for measuring exosomes according to the present embodiment, and FIG. 1B is a method for measuring exosomes according to the present embodiment. It is a flowchart which shows the measurement method using the primary reaction, the secondary reaction and the tertiary reaction which are an example.

(Preparation of measuring tip)
First, a measuring chip containing a metal film and a first binding substance that binds to an exosome is prepared (step S10). In SPFS, SPR is generated by combining an evanescent wave generated when a metal film is irradiated with light (excitation light in the present embodiment) and a surface plasmon. As a method for generating SPR, a method of arranging a prism on one surface of a metal film (Kretschmann arrangement), a method of forming a diffraction grating on the metal film, and the like are known. The SPFS adopting the former method is referred to as prism coupling (PC) -SPFS, and the SPFS adopting the latter method is referred to as lattice coupling (GC) -SPFS. As the method for measuring exosomes according to the present embodiment, either PC-SPFS or GC-SPFS may be adopted.

As described above, the metal film causes 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 capable of causing SPR. Examples of metals constituting the metal film include gold, silver, copper, aluminum and alloys thereof.

The first binding substance can bind to exosomes and is immobilized on a metal membrane to capture exosomes in a sample. Usually, the first binding substance is uniformly immobilized on a predetermined region (reaction field) on the metal film. The type of the first binding substance immobilized on the metal membrane is not particularly limited as long as it can bind to the exosome, that is, it binds to the first binding determinant of the exosome. Examples of binding agents 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 an exosome is a part of the exosome that the binding substance recognizes when it binds to the exosome. For example, when the binding substance is an antibody, the binding determinant is the antigenic determinant (epitope) of the antigen, and when the binding substance is a ligand, the binding determinant is the binding site of the corresponding receptor.

The first binding determinant of the exosome to which the first binding substance binds is not particularly limited, but a binding determinant known as an exosome marker or a binding determinant known as a marker of a cell secreting an exosome is used. included. 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 to these binding determinants are commercially available, and the antibodies can be used as the first binding substance. When a substance that reacts with the above substances (for example, an antibody) is used as the first binding substance that binds to a binding determinant known as an exosome marker, it binds only to the exosome, so that the exosome in the sample can be detected. It is valid.

Binding determinants known as markers for cells that secrete exosomes include caveolin-1, PSMA, EpCAM, Glypican-1, Survivin, CD91, Tspan8, CD147, and EGFR. , HER2, CD44, Galactin, Integrin and the like. Antibodies to these binding determinants are also commercially available, and the antibodies can be used as the first binding substance. When a binding substance (eg, an antibody) that binds to a binding determinant known as a cell marker that secretes exosomes is used as the first binding substance, it binds only to a specific cell and the exosome secreted from the cell, so that the sample It is effective in detecting exosomes derived from specific cells inside.

When the first binding substance is an antibody, the antibody may be a monoclonal antibody, a polyclonal antibody, or a fragment of the antibody. Further, 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 membrane is an antibody, the antibody is one type or two or more types of monoclonal antibody or polyclonal antibody.

It should be noted that the first binding substance is preferably selected in consideration of the combination with the second binding substance described in relation to the fluorescent label. The preferred combination of the first binding substance and the second binding substance will be described later.

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 (for example, an antibody) is bound may be formed on the metal film. Examples of SAMs include membranes formed with substituted aliphatic thiols such as HOOC- (CH ₂ ) _11- SH. Examples of materials constituting the polymer membrane include polyethylene glycol and MPC polymer. Further, a polymer having a reactive group (or a functional group that can be converted into a reactive group) capable of binding to a binding substance (for example, an anti-exosome antibody) is immobilized on a metal membrane, and the binding substance (for example, an antibody) is attached to this polymer. May be combined.

The measuring chip is preferably a structure in which each piece has a length of several mm to several cm, but may be a smaller structure or a larger structure that is not included in the category of “chip”.

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

As shown in FIG. 2A, the measuring 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 transparent to the excitation light L1, and includes an incident surface 111 on which the excitation light L1 is incident, a film forming surface 112 on which the excitation light L1 is reflected, and an exit surface 113 on which the reflected light L2 is emitted. Has. 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 pillar body having a trapezoidal bottom surface. The surface corresponding to one base of the trapezoid is the film forming 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 prism 110 include resin and glass. The material of the prism 110 is preferably a resin having a refractive index of 1.4 to 1.6 with respect to excitation light and a small birefringence. The metal film 120 is arranged on the film forming surface 112 of the prism 110. The method for forming the metal film 120 is not particularly limited. Examples of methods 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 in the range of 30 to 70 nm.

As shown in FIG. 2A, when the metal film 120 is irradiated with the excitation light L1 via the prism 110 so that the SPR is generated in the metal film 120, an electric field enhanced by the SPR is generated in the vicinity of the metal film 120. At this time, the anti-exosome antibody (first binding substance) 130 that recognizes the antigen on the exosome on the metal film 120 is bound to the exosome 140 that has reacted with the second binding substance 131 labeled with the fluorescent substance 150. Then, the fluorescent substance 150 is excited by the enhanced electric field and emits fluorescent L3.

As shown in FIG. 2B, the measuring chip 200 for GC-SPFS is a layer of a metal film 210 on which a diffraction grating 211 is formed and an anti-exosome antibody (first binding substance) 130 (which recognizes an antigen on an exosome). ). The method for forming the metal film 210 is not particularly limited. Examples of methods 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 in the 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 a one-dimensional diffraction grating, a plurality of convex portions parallel to each other are formed at predetermined intervals on the surface of the metal film 210. In the two-dimensional diffraction grating, convex portions having a predetermined shape are periodically arranged on the surface of the metal film 210. Examples of the arrangement of convex parts include a square grid, a triangular (hexagonal) grid, and the like. Examples of the cross-sectional shape of the diffraction grating 211 include a rectangular wave shape, a sinusoidal shape, and a sawtooth shape. 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), the metal film 210 may be given an uneven shape. Further, the metal film 210 may be formed on a substrate (not shown) to which a concave-convex shape is previously provided. With either method, the metal film 210 including the diffraction grating 211 can be formed.

As shown in FIG. 2B, when the metal film 210 (diffraction grating 211) is irradiated with the excitation light L1 so that SPR is generated in the metal film 210 (diffraction grating 211), the electric field enhanced by the SPR is generated by the metal film 210 (diffraction grating 211). It occurs in the vicinity of the grating 211). At this time, the exosome 140 that reacted with the second binding substance 131 labeled with the fluorescent substance 150 on the anti-exosome antibody (first binding substance) 130 that recognizes the antigen on the exosome on the metal film 210 (diffraction lattice 211). Is bound, the fluorescent substance 150 is excited by the enhanced electric field and emits fluorescent L3.

3A to 3C are schematic cross-sectional views showing an example of a measuring chip for PC-SPFS. As shown in FIG. 3A, the measuring chip 300 includes a prism 110 having an incident surface 111, a film forming surface 112, and an emitting surface 113, a metal film 120 formed on the film forming surface 112 of the prism 110, and the prism 110. It has a flow path lid 310 arranged on the film forming surface 112 or the metal film 120. In FIG. 3, the entrance surface 111 and the exit surface 113 are present in front of and behind the paper surface, respectively. The measuring tip 300 also has a flow path 320, a liquid injection unit 330 connected to one end of the flow path 320, and a storage unit 340 connected to the other end of the flow path 320. In the present embodiment, the flow path lid 310 is adhered to the metal film 120 (or prism 110) via an adhesive layer 350 such as double-sided tape, and the adhesive layer 350 serves to define the side surface shape of the flow path 320. Also responsible. Although omitted in FIG. 3, an anti-exosome antibody (first binding substance) that recognizes an antigen on an exosome is present in a part of the metal film 120 (reaction field) exposed in the flow path 320. 130 is fixed. The liquid injection section 330 is closed by the liquid injection section covering film 331, and the storage section 340 is closed by the storage section covering film 341. The storage portion covering film 341 is provided with a ventilation hole 342.

The flow path lid 310 is made of a material transparent to fluorescent L3. However, a part of the flow path lid 310 may be made of a material opaque to the fluorescent L3 as long as it does not interfere with the removal of the fluorescent L3. Examples of materials that are transparent to fluorescent L3 include resins. The flow path lid 310 may be joined to the metal film 120 (or prism 110) by laser welding, ultrasonic welding, crimping using a clamp member, or the like without using the adhesive layer 350. In this case, the side surface shape of the flow path 320 is defined by the flow path lid 310.

A pipette tip is inserted into the liquid injection unit 330. At this time, the opening of the liquid injection portion 330 (the through hole provided in the liquid injection portion covering film 331) comes into contact with the outer periphery of the pipette tip without a gap. Therefore, the liquid can be introduced into the flow path 320 by injecting the liquid into the liquid injection section 330 from the pipette tip, and the liquid in the liquid injection section 330 is sucked into the pipette tip to enter the flow path 320. Liquid can be removed. Further, by alternately injecting and sucking the liquid, the liquid can be reciprocated in the flow path 320.

When an amount of liquid exceeding the volume of the flow path 320 is introduced into the flow path 320 from the liquid injection unit 330, the liquid flows into the storage unit 340 from the flow path 320. Further, when the liquid is reciprocated in the flow path 320, the liquid flows into the storage unit 340. The liquid that has flowed into the storage unit 340 is agitated in the storage unit 340. When the liquid is agitated in the reservoir 340, the concentration of the liquid (sample, cleaning liquid, etc.) component (for example, exosome, cleaning component, etc.) passing through the flow path 320 becomes uniform, and various reactions occur in the flow path 320. It is more likely to occur and the cleaning effect is enhanced.

3B and 3C are schematic views showing an example of a well-shaped measuring chip, respectively. FIG. 3B is a schematic view of a well-shaped measuring chip 400 having 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 having a substantially trapezoidal cross section (a quadrangular pyramid shape), and the well member 418 is formed in a square shape according to the shape of the dielectric member 412. Then, in a state where the first binding substance that binds to the exosome to be detected is fixed in the ligand fixing region 416 on the metal thin film 414 of the sensor structure 22, the sample solution containing the exosome is supplied into the through hole 420. The sensor structure 422 is agitated.

Further, FIG. 3C shows a well in which the reaction detection unit is on the side wall surface (see, for example, International Publication No. 2018/021238). The detection tip 500 has a well body 510 and a side wall member 520. The well body 510 has an accommodating portion (well) 511 inside the well body 510. The accommodating portion 511 is a bottomed recess configured to accommodate the liquid, and is opened to the outside by a first opening 512 provided at the upper part and a second opening 513 provided at the side portion. 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 emitting surface (not shown). The prism 521 also functions as a side wall constituting the accommodating portion 511. There is a capture region on the metal membrane 525, and the capture region is a region where a first binding substance for capturing exosomes in a sample is immobilized.

The type of sample is not particularly limited as long as it contains exosomes. Examples of samples include blood (serum, plasma, whole blood), urine, sweat, saliva, breast milk, semen, lymph, cerebrospinal fluid, tears, and other body fluids, as well as saline and buffers. Contains diluted diluent. In the method for detecting exosomes according to the present embodiment, since exosomes are detected using SPFS, whole blood can also be used as a sample. Therefore, from the viewpoint of availability and the like, serum, plasma, whole blood or a diluted solution thereof is preferable as a sample. In addition to exosomes, these samples may also contain microvesicles, impurities, and the like. In the present invention, the sample can be used for measurement as it is without centrifugation or removal by filtering, but purification is performed after centrifugation or filtering is performed to remove or precipitate microvesicles and contaminants. There is no problem even if the liquid is used for measurement. Further, the method for removing icrovesicles and impurities is not limited to these.

Further, the sample may be an exosome extract prepared from a body fluid. By extracting exosomes from body fluids and using them as samples, it is possible to detect and quantify even trace amounts of exosomes that were difficult to detect with body fluids themselves. The method for extracting exosomes is not particularly limited, and conventionally known methods for extracting exosomes can be adopted. For example, exosomes can be extracted from body fluids by ultracentrifugation, immunoprecipitation or polymer precipitation.

A surfactant may be further added to the sample. By adding a surfactant, it is possible to prevent exosome aggregation and reduce background noise and variation in measured values. The surfactant is preferably a surfactant widely used in the field of biomedicine, and includes, for example, Tween 20, sodium deoxycholate, and Triton-X100. Surfactants do not destroy exosomes, but may be used in concentrations that prevent their aggregation. For example, in the case of Tween 20, it 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 the case of sodium deoxycholate, it is 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. can do. Further, in the case of Triton-X100, it is 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. be able to.

(Primary reaction)
Next, the sample is provided on the metal film of the measurement chip, and the exosome contained in the sample is bound to the first binding substance immobilized on the metal film (primary reaction; step S20). The method of providing the sample is not particularly limited. For example, a sample may be provided on a metal membrane using a pipette with a pipette tip attached to the tip. The reaction time of the primary reaction is not particularly limited, but from the viewpoint of increasing the reaction efficiency between the exosome and the first binding substance, a longer reaction time is preferable, and usually 5 minutes or more and 180 minutes or less, preferably. It is 60 minutes or more and 150 minutes or less, more preferably 100 minutes or more and 120 minutes or less. Usually, after the primary 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).
Further, after cleaning, the optical blank can be measured (measure the optical blank; step S2).

(Secondary reaction)
Next, a labeling reagent is provided on the metal membrane of the measurement chip, and the exosome bound to the binding substance is labeled with the fluorescent substance via the second binding substance that binds to the exosome. The type of labeling reagent is not particularly limited as long as the exosome bound to the first binding substance can react with the second binding substance labeled with the fluorescent substance. For example, the labeling reagent is a fluorescent substance-labeled second binding substance that binds to the second binding determinant of the exosome (secondary reaction of FIG. 1A + fluorescent labeling; step S30). Alternatively, the labeling reagent is a fluorescent substance-labeled third binding substance that binds to a second binding substance that binds to the second binding determinant of the exosome and a second binding substance that binds to the exosome. Both are included [secondary reaction of FIG. 1B; step S30 and fluorescent labeling (third reaction); step S35].

The method of providing the labeling reagent is not particularly limited. For example, a pipette with a pipette tip attached to the tip may be used to provide the labeling reagent on the metal membrane. Usually, after the secondary reaction and / or the tertiary reaction is completed, the surface of the metal membrane is washed with a buffer solution or the like to remove a second binding substance (or a third binding substance) that is not bound to exosomes. Remove (cleaning; steps S31 and S36).

The type of the second binding substance contained in the labeling reagent is not particularly limited as long as it can bind to the exosome. Examples of second binding agents 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. Is included.

The second binding determinant possessed by the exosome to which the second binding substance binds is not particularly limited, but a binding determinant known as an exosome marker or a binding determinant known as a marker for cells secreting exosomes is used. included. 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 to these binding determinants are commercially available, and the antibodies can be used as a second binding substance. When a substance that reacts with the above substances (for example, an antibody) is used as a second binding substance that binds to a binding determinant known as an exosome marker, it binds only to the exosome, so that even if it is not an exosome on a metal membrane. Even if (cells, etc.) are bound, it is effective in detecting only exosomes.

Binding determinants known as markers for cells that secrete exosomes include caveolin-1, PSMA, EpCAM, Glypican-1, Survivin, CD91, Tspan8, CD147, and EGFR. , HER2, CD44, Galactin, Integrin and the like. Antibodies to these binding determinants are also commercially available, and the antibodies can be used as a second binding substance. When a binding substance (eg, an antibody) that binds to a binding determinant known as a marker for cells secreting exosomes is used as the second binding substance, it binds only to a specific cell and the exosome secreted from the cell. It is effective in detecting only exosomes derived from specific cells from various exosomes bound on a metal membrane.

When the second binding substance is an antibody, the antibody may be a monoclonal antibody, a polyclonal antibody, or a fragment of the antibody. In addition, the type of the second binding substance contained in the labeling reagent may be one type or two or more types. For example, a fluorescently labeled anti-exosome antibody is one or more anti-exosome monoclonal antibodies or anti-exosome polyclonal antibodies. In this case, the fluorescent substance-labeled anti-exosome monoclonal antibody and anti-exosome polyclonal antibody may be different from one or more anti-exosome monoclonal antibodies immobilized on the metal membrane. preferable.

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, both the first binding substance and the second binding substance may be antibodies, one may be an antibody, and the other may be a protein other than an antibody.

The first binding substance immobilized on the metal membrane and the second binding substance contained in the labeling reagent each bind to the binding determinant possessed by the exosome, and the binding of the first binding substance It is preferable that the first binding determinant and the second binding determinant to which the second binding substance binds are different. Rather than competing for the same binding determinant, the first binding substance and the second binding substance bind to different binding determinants, thereby more reliably fixing the exosome to the metal membrane and labeling with the labeling substance. It becomes possible to do. Further, since the first binding determinant to which the first binding substance binds and the second binding determinant to which the second binding substance binds are different, it is detected from the sample based on the binding of the first binding substance. The exosomes to be measured can be further narrowed down based on the binding of the second binding substance.

For the first binding substance and the second binding substance, it is preferable that at least one of the first binding determinant and the second binding determinant is a binding determinant known as an exosome marker. Only exosomes can be detected from substances in a sample by using a binding substance that binds to a binding determinant known as an exosome marker. In addition, it is preferable that at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for cells secreting exosomes. Exosomes derived from specific cells can be detected by using a binding substance that binds to a binding determinant known as a marker for cells that secrete exosomes. In the present invention, one of the first binding substance and the second binding substance is bound to a binding determinant known as an exosome marker, and the other is bound to a binding determinant known as a marker for cells secreting exosomes. It is more preferable to combine them. By using such two kinds of binding substances in combination, exosomes secreted by a specific cell can be specifically detected. For example, cancer cells are known to secrete exosomes different from normal cells, and by detecting exosomes derived from cancer cells in body fluids, exosomes can be used as cancer markers.

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, and proteins other than antibodies (eg, lectins) that can bind to antibodies, nucleic acids, lipids, proteins other than antibodies, etc. used as the second binding substance. Is done. For example, when a combination of second and third binding substances is used in which a plurality of third binding substances are bound to one second binding substance, the second binding substance is directly fluorescently labeled. It is possible to improve the measurement sensitivity by binding more fluorescent substances to exosomes than if they were present. In addition, when it is difficult to directly label the second binding substance with a fluorescent substance, or when the binding property of the second binding substance to the exosome is changed, decreased or lost due to the fluorescent labeling, the third binding substance is also used. Can be used to label exosomes.

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 materials include cyanine dyes, Thermo Scientific's Alexa Fluor® dyes, and Biotium's CF dyes. Among the commercially available fluorescent dyes, Alexa Fluor dye and CF dye have high quantum efficiency with respect to the wavelength of the excitation light used in SPFS. Further, since the CF dye does not cause much fading at the time of fluorescence detection, it is possible to stably perform fluorescence detection. The method for 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 (for example, an anti-exosome antibody).

In the above description, the exosome was bound to the first binding substance immobilized on the metal film, and then the exosome was labeled with a fluorescent substance using a labeling reagent, but the first binding substance immobilized on the metal film was used. The exosome may be labeled with a fluorescent substance using a labeling reagent before binding the exosome to the binding substance. In this case, the sample and the labeling reagent (second binding substance) may be mixed before the sample is provided on the metal film. Further, a fluorescently labeled third binding substance can be added to the mixture of the sample and the labeling reagent (second binding substance). Further, the step of binding the exosome to the binding substance immobilized on the metal film and the step of labeling the exosome with a fluorescent substance may be performed at the same time. In this case, the sample and the labeling reagent (second binding substance, or second and third binding substances) may be provided simultaneously on the metal film.

(Fluorescence measurement)
Next, fluorescence indicating the amount of exosomes is measured by SPFS (step S40). Specifically, in a state where an exosome labeled with a fluorescent substance is bound to a binding substance immobilized on the metal film, the metal film is irradiated with excitation light so that SPR occurs on the metal film, thereby fluorescing. Measure the fluorescence emitted by the substance. Usually, a pre-measured optical blank value is subtracted from the measured fluorescence value to calculate a signal value that correlates with the amount of exosomes. If necessary, the signal value may be converted into the amount (number / ml) or concentration (μg / ml) of exosomes by using a calibration curve prepared in advance.

For example, for the calibration curve, samples having different dilution rates are prepared using commercially available exosomes, the number and concentration of exosomes in each sample are obtained, and the signal value is obtained by the method of the present invention, and the signal value is the number of exosomes. The calibration curve of exosomes can be obtained by plotting against or concentration. It is also possible to measure the number and concentration of exosomes in the sample, respectively, and obtain a calibration curve (FIG. 4) 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 a number can be converted into a concentration (μg / ml). By performing such conversion, it is possible to compare with other companies' devices that detect the amount of exosomes as a concentration (μg / ml).

The method for measuring the number of exosomes is not particularly limited, but for example, a qNano / nanoparticle multianalyzer (manufactured by Meiwaforsis 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 Thermo Fisher).

As shown in FIG. 2A, when the measuring chip 100 for PC-SPFS is used, the excitation light L1 is applied to the metal film 120 via the prism 110. As a result, SPR occurs in the metal film 120, and the fluorescent substance 150 existing in the vicinity of the metal film 120 is excited by the enhanced electric field to emit fluorescent L3. The angle of incidence of the excitation light L1 on the metal film 120 is set so that SPR occurs in the metal film 120, but is preferably a resonance angle or an enhancement angle. Here, the "resonance angle" means an incident angle when the amount of reflected light L2 is minimized when the incident angle of the excitation light L1 with respect to the metal film 120 is scanned. Further, the "enhanced angle" is scattered light having the same wavelength as the excitation light L1 emitted above the metal film 120 (opposite the prism 110) when the incident angle of the excitation light L1 with respect to the metal film 120 is scanned. It means the incident angle when the amount of light (Prismon scattered light) is maximized.

As shown in FIG. 2B, when the measuring chip 200 for GC-SPFS is used, the excitation light L1 is directly applied to 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 existing in the vicinity of the metal film 210 (diffraction grating 211) is excited by the enhanced electric field to emit fluorescent L3. The angle of incidence of the excitation light L1 on the metal film 210 is set so that SPR is generated in the metal film 210, but the angle at which the intensity of the enhanced electric field formed by the SPR is the strongest is preferable. The optimum incident angle of the excitation light L1 is appropriately set according to 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.

The type of excitation light is not particularly limited, but is usually 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, 7.5μW / mm ² or more 30 mW / mm ² or less, preferably 8.5μW / mm ² or more 10 mW / mm ² or less, 9.5μW / mm ² or more 5 mW / mm ² or less Is more preferable. By setting the irradiation energy of the excitation light to 30 mW / mm ² or less, the fluorescence intensity is increased, the signal-to-noise ratio (S / N) is increased, and a smaller amount of exosomes can be detected. Can be improved. The wavelength of the excitation light is appropriately set according to the excitation wavelength of the fluorescent substance used. Further, when the amount of light exceeds 30 mW / mm ² , the dissociation of the antigen-antibody reaction due to the heat of irradiation proceeds, so that the signal-to-noise ratio deteriorates.

It is preferable that the fluorescence detector is installed in the direction in which the fluorescence intensity is highest with respect to the measuring chip. For example, as shown in FIG. 2A, when the measuring 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 that the detector is a measuring chip. It will be installed directly above. On the other hand, as shown in FIG. 2B, when the measuring chip 200 for GC-SPFS is used, the direction in which the intensity of the fluorescence L3 is highest is a direction inclined to some extent with respect to the normal of the metal film 120, so that it can be detected. The vessel is installed at a position not directly above the measuring chip. The detector is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).

By the above procedure, the concentration of exosomes contained in the sample can be measured.

[Exosome measurement kit]
The exosome measurement kit according to the present embodiment contains the above-mentioned measurement chip and the above-mentioned labeling reagent (containing a fluorescent substance and a second binding substance, and optionally further containing a third binding substance). It is a set. By setting the above-mentioned measuring chip and labeling reagent in advance in this way, a user (medical worker or the like) can more easily perform the above-mentioned exosome measuring method. In addition, the measurement kit may further contain a surfactant. By further using a surfactant, it is possible to reduce background noise and variation in measured values.

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

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Experiment 1: Measurement of exosomes from healthy subject blood using antibodies against two types of 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 in a specific region (reaction part) of the metal film 120 (gold thin film) exposed in the flow path 320.

Standard exosomes (lyophilized) derived from healthy individuals purchased from Cosmo Bio Co., Ltd. were hydrated and used as sample samples. The dilution was a 10-fold dilution series, and PBS containing 1% BSA was used for dilution.
The sample diluent in the apparatus contained a surfactant, and the concentration of the surfactant was 0.05%.

The number of exosomes in the sample was measured for each of the samples with different dilution rates. A qNano / nanoparticle multi-analyzer (manufactured by Meiwaforsis Co., Ltd.) was used for the measurement.

Furthermore, for the same sample as above, the signal value that correlates with the amount of exosomes was measured. A sample (one of the diluted blood) was introduced into the flow path 320 from the liquid injection section 330 by a pipette tip, and the liquid was reciprocally sent (primary reaction). The reaction time of the primary reaction was 100 minutes. After removing the sample in the flow path 320 from the liquid injection unit 330, the inside of the flow path 320 was washed once with a washing liquid. Next, a labeling reagent (an anti-CD63 monoclonal antibody labeled with Alexa Fluor dye was introduced into the flow path 320 from the liquid injection section 330 and reciprocally sent (secondary reaction). The reaction time of the secondary reaction was 10 minutes. After removing the labeling reagent in the flow path 320 from the liquid injection unit 330, the inside of the flow path 320 was washed once with a cleaning liquid. Then, the measurement liquid was introduced into the flow path 320 from the liquid injection unit 330. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with the excitation light (laser light) from the prism 110 side so that the incident angle of the excitation light on the metal film 120 became the enhancement angle. The output of the excitation light used for the detection was 5 mW, and the irradiation energy amount was 3.8 mW / mm ^{2. The} optical blank value measured in advance from the obtained fluorescence value was obtained. The signal value that correlates with the amount of exosomes was calculated. The same measurement was performed 6 times for each sample.

The measured signal value was plotted against the number of exosomes (pieces / μl) in the sample to obtain a calibration curve (FIG. 5) of CD9-positive and CD63-positive exosomes.

As is clear from the plot of FIG. 5, there was a correlation between the number of exosomes in the sample and the signal value, and the detection limit was as low as 2.1 × 10 ² / μl.

Next, the signal value that correlates with the amount of exosomes was calculated in the same manner as above except that the output of the excitation light used for detection was changed. The output of the excitation light used for the detection was 5 mW, 15 mW or 30 mW, and the irradiation energy amounts were 3.8 mW / mm ² , 11.3 mW / mm ² or 22.6 mW / mm ² , respectively. For each irradiation energy amount, the detection limit when measuring exosomes from a sample was determined in the same manner as described above.

For the above measurement, the signal-to-noise ratio (S / N) was calculated as follows. As the signal value (S), the value obtained by subtracting the optical blank from the measurement signal in the presence of the exosome was obtained, and as the noise value (N), the value obtained by subtracting the optical blank from the measurement signal in the absence of the exosome was obtained. Next, the S / N ratio was calculated.

The calculated S / N and detection limit are plotted against the amount of irradiation energy, respectively, and a graph showing the relationship between the irradiation energy, S / N and the detection limit for the CD9-positive CD63-positive exosome derived from a healthy subject (FIG. 6) was obtained.

In the plot of FIG. 6, when the irradiation energy of the excitation light used when measuring the exosome from the sample is low, the S / N becomes large (that is, the noise to the signal is reduced) and the detection limit is also improved.

Experiment 2: Measurement of prostate cancer-related exosomes using antibodies against exosome-secreting cells and antibodies against exosomes

A measuring chip 300 having the configuration shown in FIG. 3 was prepared. An anti-PSMA monoclonal antibody was immobilized as a first binding substance in a specific region (reaction part) of the metal film 120 (gold thin film) exposed in the flow path 320.

Standard exosomes (lyophilized) derived from LNCaP cells, which are prostate cancer cells purchased from Cosmo Bio, were hydrated and used as sample samples. The dilution was a 10-fold dilution series, and PBS containing 1% BSA was used for dilution.
The sample diluent in the apparatus contained a surfactant, and the concentration of the surfactant was 0.05%.

The number of LNCaP cell-derived exosomes in the sample was measured. A qNano / nanoparticle multi-analyzer (manufactured by Meiwaforsis Co., Ltd.) was used for the measurement.

Furthermore, for the same samples as above, in order to prepare a calibration curve for CD9-positive PSMA-positive exosomes derived from LNCaP cells, signal values correlating with the amount of exosomes were measured for each of the samples having different dilution ratios. A sample (one of the diluted culture supernatants) was introduced into the flow path 320 from the liquid injection section 330 by a pipette tip, and the liquid was reciprocally sent (primary reaction). The reaction time of the primary reaction was 100 minutes. After removing the sample in the flow path 320 from the liquid injection unit 330, the inside of the flow path 320 was washed once with a washing liquid. Next, an anti-CD9 monoclonal antibody labeled with a labeling reagent (Alexa Fluor dye) was introduced into the flow path 320 from the liquid injection section 330 and reciprocally sent to the liquid (secondary reaction). The reaction time of the secondary reaction was 10 minutes. After removing the labeling reagent in the flow path 320 from the liquid injection section 330, the inside of the flow path 320 was washed once with a washing liquid. Next, the measuring liquid was introduced into the flow path 320 from the liquid injection unit 330. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with the excitation light (laser light) from the prism 110 side so that the angle of incidence of the excitation light on the metal film 120 became the enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for the 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 that correlates with the amount of exosomes. The same measurement was performed 6 times for each sample.

The measured signal value was plotted against the number of exosomes (pieces / μl) in the sample to obtain a calibration curve (FIG. 7) of CD9-positive PSMA-positive exosomes.

As is clear from the plot of FIG. 7, exosomes (CD9-positive PSMA-positive exosomes) derived from specific cells (prostate cancer cells) could be detected by the above method. In addition, there was a correlation between the number of CD9-positive PSMA-positive exosomes and the signal value, and the detection limit was as low as 1.7 × 10 ² // μl.

Next, a signal value correlating with the amount of CD9-positive PSMA-positive exosomes was calculated in the same manner as above except that the output of the excitation light used for detection was changed. The output of the excitation light used for the detection was 5 mW, 15 mW or 30 mW, and the irradiation energy amounts were 3.8 mW / mm ² , 11.3 mW / mm ² or 22.6 mW / mm ² , respectively. For each irradiation energy amount, the detection limit when measuring exosomes from a sample was determined in the same manner as described above.

For the above measurement, the signal-to-noise ratio (S / N) was calculated in the same manner as in Experiment 1.

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

In the plot of FIG. 8, when the irradiation energy of the excitation light used when measuring the CD9-positive PSMA-positive exosome is low, the S / N becomes large (that is, the noise to the signal is reduced) and the detection limit is also improved. did.

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

A measuring chip 300 having the configuration shown in FIG. 3 was prepared. An anti-anti-CD9 monoclonal antibody was immobilized as a first binding substance in a specific region (reaction part) of the metal film 120 (gold thin film) exposed in the flow path 320.

Standard exosomes (lyophilized) derived from LNCaP cells, which are prostate cancer cells purchased from Cosmo Bio, were hydrated and used as sample samples. The dilution was a 10-fold dilution series, and PBS containing 1% BSA was used for dilution.
The sample diluent in the apparatus contained a surfactant, and the concentration of the surfactant was 0.05%.

In order to prepare a calibration curve of CD9-positive and CD63-positive exosomes derived from LNCaP cells, signal values correlating with the amount of exosomes were measured for each of the samples having different dilution ratios. A sample (one of the diluted culture supernatants) was introduced into the flow path 320 from the liquid injection section 330 by a pipette tip, and the liquid was reciprocally sent (primary reaction). The reaction time of the primary reaction was 100 minutes. After removing the sample in the flow path 320 from the liquid injection unit 330, the inside of the flow path 320 was washed once with a washing liquid. Next, a labeling reagent (anti-CD63 monoclonal antibody labeled with Alexa Fluor dye) was introduced into the flow path 320 from the liquid injection section 330 and reciprocally sent to the liquid (secondary reaction). The reaction time of the secondary reaction was 10 minutes. After removing the labeling reagent in the flow path 320 from the liquid injection section 330, the inside of the flow path 320 was washed once with a washing liquid. Next, the measuring liquid was introduced into the flow path 320 from the liquid injection unit 330. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with the excitation light (laser light) from the prism 110 side so that the angle of incidence of the excitation light on the metal film 120 became the enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for the 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 that correlates with the amount of exosomes. The same measurement was performed 6 times for each sample.

For the same sample as above, the number of LNCaP cell-derived exosomes in the sample was measured. The number of extracted exosomes was measured using a qNano / nanoparticle multi-analyzer (manufactured by Meiwaforsis Co., Ltd.). Weight was determined by protein quantification (BCA method, manufactured by Thermo Fisher).

The measured signal value was plotted against the number of exosomes (pieces / μl) in the sample to obtain a calibration curve (FIG. 9) of CD9-positive and CD63-positive exosomes.

As is clear from the plot of FIG. 9, CD9-positive and CD63-positive exosomes could be detected even in a sample derived from a specific cell (prostate cancer cell) by the above method. Also, a correlation exists between the number and the signal value of the CD9 positive CD63 positive exosomes, the detection limit was as low as 3.2 × 10 ⁰ cells / [mu] l.

Next, a signal value correlating with the amount of CD9-positive and CD63-positive exosomes was calculated in the same manner as above except that the output of the excitation light used for detection was changed. The output of the excitation light used for the detection was 5 mW, 15 mW or 30 mW, and the irradiation energy amounts were 3.8 mW / mm ² , 11.3 mW / mm ² or 22.6 mW / mm ² , respectively. For each irradiation energy amount, the detection limit when measuring exosomes from a sample was determined in the same manner as described above.

For the above measurement, the signal-to-noise ratio (S / N) was calculated in the same manner as in Experiment 1.

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

In the plot of FIG. 10, when the irradiation energy of the excitation light used when measuring the CD9-positive and CD63-positive exosomes is low, the S / N becomes large (that is, the noise to the signal is reduced) and the detection limit is also improved. did.

Experiment 4: Measurement of CD9-positive sugar chain-carrying exosomes using antibodies against exosomes and WFA lectins that recognize sugar chains on exosomes

A measuring chip 300 having the configuration shown in FIG. 3 was prepared. An anti-CD9 monoclonal antibody was immobilized as a first binding substance in a specific region (reaction part) of the metal film 120 (gold thin film) exposed in the flow path 320.

Standard exosomes (lyophilized) derived from LNCaP cells, which are prostate cancer cells purchased from Cosmo Bio, were hydrated and used as sample samples. The dilution was a 10-fold dilution series, and PBS containing 1% BSA was used for dilution.
The sample diluent in the apparatus contained a surfactant, and the concentration of the surfactant was 0.05%.

In order to prepare a calibration curve of CD9-positive sugar chain-bearing exosomes derived from LNCaP cells, signal values correlating with the amount of exosomes were measured for each of the samples having different dilution ratios. A sample (one of the diluted culture supernatants) was introduced into the flow path 320 from the liquid injection section 330 by a pipette tip, and the liquid was reciprocally sent (primary reaction). The reaction time of the primary reaction was 100 minutes. After removing the sample in the flow path 320 from the liquid injection unit 330, the inside of the flow path 320 was washed once with a washing liquid. Next, a labeling reagent (WFA lectin labeled with Alexa Fluor dye) was introduced into the flow path 320 from the liquid injection section 330 and reciprocally fed (secondary reaction). The reaction time of the secondary reaction was 10 minutes. After removing the labeling reagent in the flow path 320 from the liquid injection section 330, the inside of the flow path 320 was washed once with a washing liquid. Next, the measuring liquid was introduced into the flow path 320 from the liquid injection unit 330. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with the excitation light (laser light) from the prism 110 side so that the angle of incidence of the excitation light on the metal film 120 became the enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for the 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 that correlates with the amount of exosomes. The same measurement was performed 6 times for each sample.

For the same sample as above, the number of LNCaP cell-derived exosomes in the sample was measured. The number of extracted exosomes was measured using a qNano / nanoparticle multi-analyzer (manufactured by Meiwaforsis Co., Ltd.).

The measured signal value was plotted against the number of exosomes (pieces / μl) in the sample to obtain a calibration curve (FIG. 11) of CD9-positive sugar chain-carrying exosomes.

As is clear from the plot of FIG. 11, CD9-positive sugar chain-carrying exosomes could be detected even in a sample derived from a specific cell (prostate cancer cell) by the above method. In addition, there was a correlation between the number of CD9-positive sugar chain-carrying exosomes and the signal value, and the detection limit was as low as 9.9 × 10 ¹ / μl.

Experiment 5: Measurement of exosomes derived from prostate cancer cells using antibodies against exosome-secreting cells and lectins that recognize sugar chains on exosomes

A measuring chip 300 having the configuration shown in FIG. 3 was prepared. An anti-PSMA monoclonal antibody was immobilized as a first binding substance in a specific region (reaction part) of the metal film 120 (gold thin film) exposed in the flow path 320.

Standard exosomes (lyophilized) derived from LNCaP cells, which are prostate cancer cells purchased from Cosmo Bio, were hydrated and used as sample samples. The dilution was a 10-fold dilution series, and PBS containing 1% BSA was used for dilution.
The sample diluent in the apparatus contained a surfactant, and the concentration of the surfactant was 0.05%.

The number of LNCaP cell-derived exosomes in the sample was measured. A qNano / nanoparticle multi-analyzer (manufactured by Meiwaforsis Co., Ltd.) was used for the measurement.

Furthermore, for the same samples as above, in order to prepare a calibration curve for PSMA-positive sugar chain-bearing exosomes derived from LNCap cells, signal values correlating with the amount of exosomes were measured for each of the samples having different dilution ratios. A sample (one of the diluted culture supernatants) was introduced into the flow path 320 from the liquid injection section 330 by a pipette tip, and the liquid was reciprocally sent (primary reaction). The reaction time of the primary reaction was 100 minutes. After removing the sample in the flow path 320 from the liquid injection unit 330, the inside of the flow path 320 was washed once with a washing liquid. Next, the WFA lectin labeled with the labeling reagent (Alexa Fluor dye) was introduced into the flow path 320 from the liquid injection section 330 and reciprocally fed (secondary reaction). The reaction time of the secondary reaction was 10 minutes. After removing the labeling reagent in the flow path 320 from the liquid injection section 330, the inside of the flow path 320 was washed once with a washing liquid. Next, the measuring liquid was introduced into the flow path 320 from the liquid injection unit 330. In this state, the fluorescence value was measured by SPFS. That is, the metal film 120 was irradiated with the excitation light (laser light) from the prism 110 side so that the angle of incidence of the excitation light on the metal film 120 became the enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for the 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 that correlates with the amount of exosomes. The same measurement was performed 6 times for each sample.

The measured signal value was plotted against the number of exosomes (pieces / μl) in the sample to obtain a calibration curve (FIG. 12) of PSMA-positive sugar chain-carrying exosomes.

As is clear from the plot of FIG. 12, exosomes (PSMA-positive sugar chain-carrying exosomes) derived from specific cells (prostate cancer cells) could be detected by the above method. In addition, there was a correlation between the number of PSMA-positive sugar chain-carrying exosomes and the signal value, and the detection limit was as low as 2.5 × 10 ² / μl.

This application claims priority based on Japanese Patent Application No. 2019-11824 filed on June 27, 2019. All the contents described in the application specification are incorporated in the application specification.

By using the exosome measurement method or measurement kit according to the present embodiment, exosomes can be measured with high sensitivity and easily. Therefore, the exosome detection method and measurement kit according to the present invention are useful for, for example, clinical examinations.

100, 200, 300, 400, 500 Measuring chip 110 Prism 111 Incident surface 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 Exosome 150 Fluorescent material 210 Metal film 211 Diffraction grating 310 Flow path lid 320 Flow path 330 Liquid injection part 331 Liquid injection part coating film 340 Storage part 341 Storage part coating film 342 Ventilation hole 350 Adhesive layer 421 Dielectric member 414 Metal thin film 416 Grating fixation region 418 Well member 420 Through hole 422 Sensor structure 510 Well body
511 containment
520 Side wall member
521 prism
523 Reflective surface
525 metal film
526 Reaction field
L1 excitation light L2 reflected light L3 fluorescence

Claims (19)

1. A step of preparing a measuring chip containing a metal film and a first binding substance that binds to an exosome, which is immobilized on the metal film.
   A step of providing a sample containing an exosome on the metal film and binding the exosome contained in the sample to the first binding substance.
   A step of labeling the exosome before or after binding to the first binding substance with a fluorescent substance via a second binding substance that 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 so that surface plasmon resonance occurs on the metal film, and the metal film is emitted from the fluorescent substance. The process of detecting the fluorescence to be generated and
   A method for measuring exosomes, including.
2. The first binding substance binds to the first binding determinant of the exosome, and the second binding substance binds to the second binding determinant of the exosome. The method for measuring an exosome according to claim 1, wherein the binding determinant of 1 is different from the second binding determinant.
3. The method for measuring an exosome 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 an exosome 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 secreting the exosome.
5. The measurement of an exosome according to any one of claims 1 to 4, wherein in the labeling step, the exosome is labeled with a fluorescent substance using a third binding substance that binds to the second binding substance. Method.
6. The method for measuring an exosome according to any one of claims 1 to 5, wherein the irradiation energy of the excitation light is 7.5 μW / mm ² or more and 10 mW / mm ² or less.
7. The method for measuring exosomes according to any one of claims 1 to 6, wherein a surfactant is added to the sample before the sample containing the exosome on the metal film is provided.
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 sample is an exosome extract.
11. The metal film is arranged on the prism and
    The excitation light is applied to the metal film via a prism.
    The method for measuring an exosome according to any one of claims 1 to 10.
12. The metal film includes a diffraction grating and
    The first binding substance is immobilized on the diffraction grating and is immobilized on the diffraction grating.
    The excitation light irradiates the diffraction grating.
    The method for measuring an exosome according to any one of claims 1 to 10.
13. A measuring chip containing a metal membrane and a first binding substance that binds to an exosome, which is immobilized on the metal membrane.
    Labeling reagents for labeling exosomes with fluorescent substances,
    Including
    An exosome measurement kit comprising the labeling reagent with a second binding substance that binds to the exosome.
14. The first binding substance binds to the first binding determinant of the exosome, and the second binding substance binds to the second binding determinant of the exosome. The exosome measurement kit according to claim 13, wherein the binding determinant of 1 is different from the second binding determinant.
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 secreting 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 the sample.
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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