JP7623124B2 - Method and kit for measuring contents of extracellular vesicles - Google Patents

Description

The present invention relates to a method and kit for measuring the contents of extracellular vesicles.

Extracellular vesicles (EVs) are a general term for membrane vesicles produced by cells and released outside the cells. They are called exosomes, microvesicles, etc., depending on their size, origin, and mechanism of development. For convenience, extracellular vesicles are referred to based on their size and sedimentation rate as microvesicles (or large EVs) that sediment at 10,000 x g, and exosomes (or small EVs) that do not sediment at 10,000 x g. However, a clear definition has not yet been established, making it difficult to strictly distinguish between them.

It is known that cells release different extracellular vesicles depending on their type and condition, and therefore, extracellular vesicles are thought to mediate communication between cells and organs.

Extracellular vesicles 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, analysis reagent, and analysis device for detecting extracellular vesicles that serve as tumor markers. In this method, an antibody against an antigen possessed by extracellular vesicles and an antibody against an antigen possessed by cells secreting extracellular vesicles are used to detect extracellular vesicles based on avidin-biotin interaction. Patent Document 2 discloses an extracellular vesicle analysis device and an extracellular vesicle capture method in which an antibody against extracellular vesicles is fixed to a recess in a base part made of synthetic resin or the like having an uneven structure, and the extracellular vesicles are captured in the recess.

Extracellular vesicles have a phospholipid bilayer membrane and encapsulate various cellular components such as proteins and nucleic acids inside. In recent years, it has been reported that the inclusions contained in extracellular vesicles may also be disease markers. A common method for measuring the inclusions contained in extracellular vesicles is to extract the extracellular vesicles from a biological specimen, such as cell culture supernatant or blood, and then dissolve the phospholipid membrane of the extracellular vesicles to release the inclusions and measure them using an ELISA method or the like. Reasons for extracting and then measuring the extracellular vesicles rather than using the biological specimen itself include the removal of impurities in the specimen and the concentration of the extracellular vesicles, and various methods such as ultracentrifugation have been reported for extracting extracellular vesicles (e.g., Non-Patent Document 1).

International Publication No. 2013/094307 JP 2014-219384 A

Takahiro Ochiya and Yusuke Yoshioka (eds.), "Exosomes: Changing Medical Care," Kagaku Dojin Co., Ltd., 2018

As mentioned above, various methods for detecting the inclusions of extracellular vesicles have been researched, but it has been difficult to measure the inclusions with high sensitivity from small amounts of specimens. Conventional methods for extracting extracellular vesicles (e.g., ultracentrifugation) contain a large amount of impurities in the extract, which can affect the measurement results as contaminants. Furthermore, attempts to increase the purity of extracellular vesicles result in a loss of extracellular vesicles, making it difficult to accurately measure the inclusions in the specimen.

Furthermore, in order to measure the inclusions in extracellular vesicles, it is necessary to release the inclusions from the extracellular vesicles. However, if an elution solution is added to the sample to elute the inclusions from the extracellular vesicles, the sample will be diluted. Therefore, conventional methods such as the ELISA method have a problem of insufficient sensitivity when measuring inclusions using small amounts of sample or samples with low concentrations of extracellular vesicles. Therefore, there is a demand for a method for measuring inclusions with high sensitivity without being affected by impurities, even when small amounts of sample or samples with low concentrations of extracellular vesicles are used.

The present invention aims to provide a method for measuring inclusions in extracellular vesicles, which is capable of measuring inclusions in a highly sensitive and simple manner, even when using a small amount of specimen or a biological specimen with a low concentration of extracellular vesicles, without being affected by contaminants. Another aim of the present invention is to provide a kit for measuring inclusions in extracellular vesicles from a biological specimen.

The method for measuring inclusions in 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 binding to inclusions in extracellular vesicles; releasing inclusions from extracellular vesicles contained in a specimen; providing the inclusions on the metal film to bind the inclusions contained in the specimen to the first binding substance; labeling the inclusions before or after binding to the first binding substance with a fluorescent substance via a second binding substance that binds to the inclusions; and irradiating the metal film with excitation light so that surface plasmon resonance occurs in the metal film while the inclusions labeled with the fluorescent substance are bound to the first binding substance, and detecting the fluorescence emitted from the fluorescent substance.

The exosome inclusion measurement kit according to one embodiment of the present invention includes an elution solution for eluting the inclusions of extracellular vesicles from the extracellular vesicles, a measurement chip including a metal membrane and a first binding substance immobilized on the metal membrane that binds to the inclusions, and a labeling reagent for labeling the inclusions with a fluorescent substance.

The method and kit of the present invention for measuring the inclusions of extracellular vesicles are not easily affected by contaminants, and therefore it is possible to measure the inclusions with high sensitivity without extracting extracellular vesicles from the specimen. Furthermore, even if a series of operations such as the release of inclusions from extracellular vesicles and dilution of the specimen are performed automatically within the device, it is possible to easily measure the inclusions without insufficient sensitivity.

FIG. 1 is a flowchart showing an example of a method for measuring contents of extracellular vesicles according to the present embodiment. FIG. 2A is a schematic cross-sectional view for explaining the configuration of a measurement chip for PC-SPFS. FIG. 2B is a schematic cross-sectional view for explaining the configuration of a measurement chip for GC-SPFS. FIG. 3A is a schematic cross-sectional view showing an example of a measurement chip for PC-SPFS. FIG. 3B is a schematic perspective view showing another example of the measurement chip for PC-SPFS. FIG. 3C is a schematic cross-sectional view showing another example of the measurement chip for PC-SPFS. FIG. 4 is a flowchart showing another example of the method for measuring contents of extracellular vesicles according to the present embodiment. FIG. 5 is a flowchart showing yet another example of the method for measuring contents of extracellular vesicles according to this embodiment. FIG. 6 is a bar graph showing the measurement results of Example 1. FIG. 7 is a bar graph showing the measurement results of Example 2. FIG. 8 is a bar graph showing the measurement results of Comparative Example 1. FIG. 9 is a bar graph showing the measurement results of Example 3. FIG. 10 is a bar graph showing the measurement results of Example 4.

The following describes in detail the embodiments of the present invention with reference to the drawings.

[Method for measuring contents of extracellular vesicles]
Extracellular vesicles are secreted by both normal cells and disease-related cells such as cancer cells, and it is known that the contents of the vesicles vary depending on the type and condition of the cells that secreted the vesicles. In order to measure the contents of the vesicles, the phospholipid membrane of the vesicles must be decomposed to release the contents before measurement.

In the method for measuring the inclusions in extracellular vesicles according to the present embodiment, inclusions are measured using surface plasmon-field enhanced fluorescence spectroscopy (hereinafter also referred to as "SPFS") in order to improve measurement sensitivity. 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 (inclusions in extracellular vesicles in this embodiment) with higher sensitivity than general fluorescent immunoassays.

Conventional methods used to determine the presence and/or concentration of specific inclusions of extracellular vesicles in a specimen all involve the loss of the extracellular vesicles themselves or their inclusions, or the decrease in concentration due to dilution, during the process leading up to the preparation of a specimen containing the inclusions of the extracellular vesicles. For example, when an elution solution is added to a specimen containing extracellular vesicles to elute the inclusions from the extracellular vesicles, the specimen is diluted by the elution solution. Furthermore, when the extracellular vesicles in the specimen are extracted or concentrated in consideration of the dilution of the specimen by the elution solution, the loss of the extracellular vesicles is likely to occur, and the reproducibility of the measurement of the inclusions may decrease.

On the other hand, since the measurement method of the present invention is based on the SPFS system, the elution of the inclusions from the extracellular vesicles in the sample using an elution solution and the dilution of the sample can be automated within the SPFS device, thereby suppressing the variation in the measurement values that may occur due to the elution process and dilution, and making it possible to easily measure the inclusions in extracellular vesicles.

Based on the above idea, the inventors conducted extensive research and discovered that specific inclusions in extracellular vesicles can be measured with high sensitivity using SPFS, and completed a method for measuring inclusions in extracellular vesicles according to the present embodiment.

The method for measuring contents of extracellular vesicles of the present invention includes the following steps 1 to 5.
Step 1 of preparing a measurement chip including a metal film and a first binding substance immobilized on the metal film and capable of binding to an inclusion of an extracellular vesicle;
A step 2 of releasing encapsulated substances from extracellular vesicles contained in a specimen;
Step 3: providing the inclusion on the metal film and allowing the inclusion contained in the sample to bind to the first binding substance;
A step 4 of labeling the inclusion with a fluorescent substance via a second binding substance that binds to the inclusion before or after binding to the first binding substance;
Step 5: while the inclusion labeled with the fluorescent substance is bound to the first binding substance, irradiating the metal film with excitation light so as to generate surface plasmon resonance in the metal film, and detecting the fluorescence emitted from the fluorescent substance.

Steps 1 to 5 of the method for measuring contents of extracellular vesicles according to this embodiment will be specifically described below. Figure 1 is a flow chart showing an example of the method for measuring extracellular vesicles according to this embodiment, which uses a primary reaction and a secondary reaction.

In the present invention, "extracellular vesicles" is a general term for membrane vesicles that are produced by cells and released outside the cells, and are called exosomes, microvesicles, etc. depending on their size, origin, mechanism of development, etc. Extracellular vesicles are roughly classified into exosomes derived from endosomes and microvesicles derived from plasma membranes, but in the present invention, the term "extracellular vesicles" refers to all extracellular membrane vesicles, including exosomes and microvesicles.

In the present invention, "inclusions of extracellular vesicles" refers to substances contained in extracellular vesicles in a specimen that can be markers for diseases, etc., and are to be distinguished from substances present in a free state in the specimen. Known inclusions of extracellular vesicles include nucleic acids such as DNA, mRNA, and miRNA, and proteins (broadly speaking, including polypeptides and peptides), and fragments of these can also be disease markers, etc. In addition, in the present invention, "inclusions of extracellular vesicles" are also simply referred to as "inclusions."

Specific examples of inclusions of extracellular vesicles include, but are not limited to, heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90).

(Step 1: Preparation of measurement chip)
A measurement chip including a metal film and a first binding substance that binds to the inclusion of extracellular vesicles is prepared (step S01 in FIG. 1). 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 surface 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 method for measuring extracellular vesicles according to this embodiment may employ either PC-SPFS or GC-SPFS.

As described above, the metal film generates SPR when irradiated with excitation light. There are no particular limitations on the type of metal that constitutes the metal film, so long as it is a metal that can generate SPR. Examples of metals that constitute the metal film include gold, silver, copper, aluminum, and alloys of these.

The first binding substance is not particularly limited as long as it can bind to the target substance contained in the extracellular vesicles and can be immobilized on the metal film. 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 the inclusion, that is, it can bind to the first binding determinant possessed by the inclusion. Examples of binding substances include antibodies that can bind to the inclusion, nucleic acids that can bind to the inclusion, lipids that can bind to the inclusion, lectins that can bind to the inclusion, and other proteins that can bind to the inclusion. Here, the binding determinant possessed by the inclusion is a part of the inclusion that the binding substance recognizes when binding to the inclusion. For example, when the binding substance is an antibody, the binding determinant is an 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.

For example, when measuring HSP70 as an inclusion, an antibody against the binding determinant of HSP70 is commercially available, and the antibody can be used as the first binding substance. If a substance that binds to a binding determinant specific to a specific target inclusion is used as the first binding substance, the first binding substance will bind only to the specific inclusion, and is therefore effective in detecting the inclusion in the sample.

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.

It is preferable to select the first binding substance 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.

The method of immobilizing the binding substance is not particularly limited. For example, a self-assembled monolayer (hereinafter referred to as "SAM") or a polymeric film to which a binding substance (e.g., an antibody) is bound may be formed on a metal film. Examples of SAMs include films formed with substituted aliphatic thiols such as HOOC-(CH ₂ ) ₁₁ -SH. Examples of materials constituting the polymeric film 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) capable of binding to a binding substance (e.g., an antibody) may be immobilized on a metal film, and the binding substance (e.g., an antibody) may be bound to this polymer.

The measurement chip is preferably a structure with each piece measuring a few mm to a few cm in length, but may be a smaller or larger structure that does not fall within the category of a "chip".

Figure 2A is a schematic cross-sectional view for explaining the configuration of a measurement chip for PC-SPFS, and Figure 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-inclusion antibody that recognizes an antigenic determinant on the inclusion is used as the first binding substance.

As shown in FIG. 2A, the measurement chip 100 for PC-SPFS has a prism 110, a metal film 120, and an anti-inclusion antibody (first binding substance) 130 (layer) that recognizes an antigen determinant on the inclusion. 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 film-forming 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 base. 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 the 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 for the excitation light and small birefringence. The metal film 120 is disposed on the film formation surface 112 of the prism 110. The method of forming the metal film 120 is not particularly limited. Examples of the method of 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.

As shown in FIG. 2A, when the metal film 120 is irradiated with excitation light L1 through the 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 the inclusion 140 that has reacted with the second binding substance 131 labeled with the fluorescent substance 150 is bound to the antibody (first binding substance) 130 that recognizes an antigenic determinant on the inclusion on the metal film 120, the fluorescent substance 150 is excited by the enhanced electric field and emits fluorescence L3.

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-inclusion antibody (first binding substance) 130 (layer) that recognizes an antigen determinant on the inclusion. The method of forming the metal film 210 is not particularly limited. Examples of the method of 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 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 parallel convex portions are formed at a predetermined interval on the surface of the metal film 210. In a 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.

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 the inclusion 140 that has reacted with the second binding substance 131 labeled with the fluorescent substance 150 is bound to the anti-inclusion antibody (first binding substance) 130 that recognizes an antigenic determinant on the inclusion on the metal film 210 (diffraction grating 211), the fluorescent substance 150 is excited by the enhanced electric field and emits fluorescence L3.

Figures 3A to 3C are schematic cross-sectional views showing an example of a measurement chip for PC-SPFS. As shown in 3A, the measurement chip 300 has a prism 110 having an incident surface 111, a deposition surface 112, and an exit surface 113, a metal film 120 formed on the deposition surface 112 of the prism 110, and a channel cover 310 arranged on the deposition surface 112 of the prism 110 or the metal film 120. In Figure 3A, 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 storage 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 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. 3A, an anti-inclusion antibody (first binding substance) 130 that recognizes an antigenic determinant on the inclusion 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 has an air hole 342.

The flow channel lid 310 is formed of a material that is transparent to the fluorescence L3. However, a part of the flow channel lid 310 may be formed of a material that is opaque to the fluorescence L3 as long as it does not interfere with the extraction of the fluorescence L3. Examples of materials that are 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.

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 gaps. Therefore, liquid can be introduced into the flow path 320 by injecting liquid from the pipette tip into the liquid injection section 330, and liquid in the flow path 320 can be removed by sucking the liquid in the liquid injection section 330 into the pipette tip. In addition, liquid can be sent back and forth within the flow path 320 by alternately injecting and sucking the liquid.

When an amount of liquid introduced from the liquid injection section 330 into the flow path 320 exceeds the volume of the flow path 320, the liquid flows from the flow path 320 into the storage section 340. Liquid also flows into the storage section 340 when liquid is sent back and forth within the flow path 320. The liquid that flows into the storage section 340 is agitated within the storage section 340. When the liquid is agitated within the storage section 340, the concentration of the components (e.g., extracellular vesicles, inclusions, cleaning components, etc.) of the liquid (such as a specimen or cleaning solution) passing through the flow path 320 becomes uniform, making it easier for various reactions to occur within the flow path 320 and improving the cleaning effect.

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. WO 2012/157403). In this chip, the dielectric member 412 is a hexahedron (quadratic pyramid shape) with a substantially trapezoidal cross section, and the well member 418 is configured as a rectangular parallelepiped to match the shape of the dielectric member 412. Then, in a state where a first binding substance that binds to the inclusion 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 the inclusion is supplied into the through hole 420, and the supplied sample solution is stirred.

Also, FIG. 3C shows a well in which the reaction detection unit is on the sidewall surface (see, for example, International Publication No. WO 2018/021238). The measurement chip 500 has a well body 510 and a sidewall 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 sidewall 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 that is 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 sidewall constituting the storage unit 511. There is a capture region on the metal film 525, which is an area where a first binding substance is immobilized to capture the contents in the sample.

(Step 2: Release of encapsulated material from extracellular vesicles)
Before the actual measurement, the contents of the extracellular vesicles contained in the sample are released (step S10).

The type of specimen is not particularly limited as long as it contains extracellular vesicles. 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 buffer solutions. Furthermore, the specimen may be a cell culture supernatant containing extracellular vesicles released from cultured cells, and a dilution thereof. In the method for measuring the contents of extracellular vesicles according to the present embodiment, the contents are detected using SPFS, so whole blood can also be used as the specimen. Therefore, from the viewpoint of ease of acquisition, cell culture supernatant, serum, plasma, whole blood, urine, saliva, cell lysate, or dilutions thereof are preferred as specimens.

The specimen may also be extracellular vesicles isolated from body fluids. By using isolated extracellular vesicles as the specimen, it is possible to select specific extracellular vesicles from among multiple types of extracellular vesicles present in body fluids and measure only the contents thereof. In addition, when free inclusions may be present in the specimen, it is possible to selectively measure the substance contained in the extracellular vesicles without detecting the free substance. There is no particular limitation on the method for isolating extracellular vesicles, but as described below, extracellular vesicles can be captured in the SPFS device using a binding substance specific to extracellular vesicles. In addition, conventionally known methods for extracting extracellular vesicles can also be adopted. For example, extracellular vesicles can be isolated and extracted from body fluids using ultracentrifugation, immunoprecipitation, polymer precipitation, or an SPFS device.

The method for releasing the inclusions from the extracellular vesicles is not particularly limited as long as it decomposes the phospholipid membrane of the extracellular vesicles but does not damage the inclusions. The most common method is to add an elution solution to the sample. There is no particular limit to the elution solution as long as it can decompose the phospholipid membrane of the extracellular vesicles without damaging the inclusions of interest. The elution solution can be selected according to the inclusions to be measured, but a membrane dissolving agent such as a surfactant that is widely used in the fields of biology and medicine is preferable. From the viewpoint of suppressing the effect on the inclusions, it is preferable that the surfactant is a non-denaturing surfactant, and specific examples include nonionic surfactants such as Triton (registered trademark) X-100 (compound name: t-octylphenoxypolyethoxyethanol) and Tween 20 (compound name: polyoxyethylene sorbitan monolaurate), and anionic surfactants such as sodium deoxycholate.

The concentration of the eluent used is not particularly limited, so long as it degrades the phospholipid membrane of the extracellular vesicles but does not degrade the inclusions to be measured. In fact, it varies depending on the type of eluent, the type of inclusions, and even the treatment time (i.e., the time the eluent is in contact with the sample). For example, when the surfactant is Triton (registered trademark) X-100, its concentration in the eluent is 0.1 to 20% by mass, preferably 0.1 to 10% by mass, and more preferably 0.5 to 5% by mass. When the surfactant is Tween 20, its concentration in the eluent is 10 to 30% by mass, preferably 15 to 25% by mass, and more preferably 15 to 20% by mass.

There is no particular limit to the time for which the eluate is in contact with the specimen, and it is usually 0.5 to 30 minutes, preferably 3 to 10 minutes. A time of 0.5 minutes or more is sufficient for the phospholipid membrane of the extracellular vesicles to be dissolved and the contents to be released into the specimen.

The release of the inclusions from the extracellular vesicles can be carried out in a container such as a test tube, but can also be carried out in an SPFS device. In particular, when an elution solution is used, it is preferable to simultaneously release the inclusions and dilute the sample in the device. Specifically, the concentration and amount of the elution solution added can be adjusted taking into account the concentration of the surfactant or the like at which the release of the inclusions is achieved and the dilution rate of the sample.

(Step 3: Primary reaction)
Next, a sample containing the extracellular vesicles released from the inclusions is provided on the metal film of the measurement chip, and the inclusions are bound to the first binding substance immobilized on the metal film (step S20 in FIG. 1). 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 primary reaction, but from the viewpoint of increasing the reaction efficiency between the inclusions 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 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 (step S21 in FIG. 1; washing).
After cleaning, the optical blank can be measured (step S22 in FIG. 1; measuring the optical blank).

(Step 4: Secondary reaction)
Next, a labeling reagent is provided on the metal film of the measurement chip, and the inclusion bound to the first binding substance is labeled with a fluorescent substance via the second binding substance bound to the inclusion (step S30 in FIG. 1). 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 inclusion bound to the first binding substance. For example, the labeling reagent is a second binding substance labeled with a fluorescent substance that binds to the second binding determinant possessed by the inclusion. Alternatively, the labeling reagent includes both a second binding substance that binds to the second binding determinant possessed by the inclusion, and another third binding substance labeled with a fluorescent substance that binds to the second binding substance bound to the inclusion.

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. Typically, after the second and/or third reactions are completed, the surface of the metal film is washed with a buffer solution or the like to remove the second binding substance (or other binding substances) that are not bound to the inclusions (step S31 in FIG. 1).

The type of the second 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 inclusion. Examples of the second binding substance include antibodies capable of binding to the inclusion, nucleic acids capable of binding to the inclusion, lipids capable of binding to the inclusion, lectins capable of binding to the inclusion, and other proteins capable of binding to the inclusion.

The second binding determinant of the inclusion to which the second binding substance binds is not particularly limited, but includes binding determinants known as inclusion markers. For example, when measuring HSP70 as an inclusion, antibodies against the binding determinant are commercially available, and such antibodies can be used as the second binding substance. When a substance that binds to a binding determinant specific to a specific target inclusion is used as the second binding substance, the second binding substance binds only to the specific inclusion, and is therefore effective in detecting only the target inclusion, even if something other than the inclusion (such as extracellular vesicles) is bound to the metal film.

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-inclusion antibody labeled with a fluorescent substance is one or two or more types of anti-inclusion monoclonal antibodies or anti-inclusion polyclonal antibodies. In this case, it is preferable that the anti-inclusion monoclonal antibody and anti-inclusion polyclonal antibody labeled with a fluorescent substance are different from the one or two or more types of anti-inclusion monoclonal antibodies immobilized on the metal film.

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, or one may be an antibody and the other a protein other than an antibody.

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 the target inclusion, 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. By binding to different binding determinants rather than competing for the same binding determinant, it becomes possible to more reliably immobilize the inclusion on the metal film and label it with the labeling substance. In addition, by making the first binding determinant bound by the first binding substance and the second binding determinant bound by the second binding substance different, it is possible to narrow down the inclusions to be further measured based on the binding of the second binding substance from among the inclusions detected from the sample based on the binding of the first binding substance.

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 other binding substances include antibodies, nucleic acids, lipids, lectins, other proteins, etc. 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 second and third binding substances such that multiple third binding substances bind to one second binding substance, it is possible to bind more fluorescent substances to the inclusions than when the second binding substance is directly fluorescently labeled and used, thereby improving the measurement sensitivity. In addition, the inclusions can be labeled using the third binding substance 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 the inclusions is changed, reduced, or lost due to fluorescent labeling.

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-extracellular vesicle antibody).

In the above description, the inclusions are bound to the first binding substance immobilized on the metal film, and then the inclusions are labeled with a fluorescent substance using a labeling reagent. However, the inclusions may be labeled with a fluorescent substance using a labeling reagent before the inclusions are bound 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 the specimen is provided on the metal film. In addition, a further fluorescently labeled (third) binding substance may be added to the mixture of the specimen and the labeling reagent (second binding substance). Furthermore, the step of binding the inclusions to the binding substance immobilized on the metal film and the step of labeling the inclusions 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 on the metal film simultaneously.

(Step 5: Fluorescence measurement)
Next, the fluorescence indicating the amount of inclusions is measured by SPFS (step S40 in FIG. 1). Specifically, in a state where the inclusions labeled with a fluorescent substance are bound to the binding substance immobilized on the metal film, the metal film is irradiated with excitation light so that SPR occurs in the metal film, and the fluorescence emitted from the fluorescent substance is measured. Usually, a signal value correlating to the amount of inclusions is calculated by subtracting a previously measured optical blank value from the measured fluorescence value. If necessary, the signal value may be converted to the amount (number/ml) or concentration (μg/ml) of inclusions using a previously prepared calibration curve or the like.

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 material 150 present in the vicinity of the metal film 120 is excited by the enhanced electric field and emits fluorescence 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, and is preferably a resonance angle or an enhanced angle. Here, the "resonance angle" refers to the angle of incidence at which the amount of reflected light L2 is minimized when the angle of incidence of the excitation light L1 on the metal film 120 is scanned. In addition, the "enhanced angle" refers to the angle of incidence 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 angle of incidence of the excitation light L1 on the metal film 120 is scanned.

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 angle of incidence of the excitation light L1 with respect to the metal film 210 is set so that SPR occurs in the metal film 210, and it is preferable that the angle be such that the intensity of the enhanced electric field formed by SPR is the strongest. The optimal angle of incidence 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, etc.

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 with an output of 10 μW to 30 mW. The irradiation energy of the excitation light is 7.5 μW/mm ² or more and 30 mW/mm ² or less, preferably 8.5 μW/mm ² or more and 10 mW/mm ² or less, and more preferably 9.5 μW/mm ² or more and 5 mW/mm ² or less. By setting the irradiation energy of the excitation light to 30 mW/mm ² or less, the fluorescence intensity is increased to increase the signal-to-noise ratio (S/N), and it becomes possible to detect a smaller amount of inclusions, so that the detection limit can be improved. The wavelength of the excitation light is appropriately set according to the excitation wavelength of the fluorescent substance used. In addition, when the amount of light exceeds 30 mW/mm ² , dissociation of the antigen-antibody reaction due to the heat of irradiation progresses, and the signal-to-noise ratio deteriorates.

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

(Isolation of extracellular vesicles)
In the measurement method of the present invention, it is not necessary to isolate extracellular vesicles from a specimen, but as described above in relation to step 2 of the method of the present invention, it is also possible to isolate extracellular vesicles from a biological specimen using an SPFS device and elute the inclusions from the isolated extracellular vesicles. Next, a method for isolating extracellular vesicles from a biological specimen using an SPFS device will be specifically described. Figure 4 is a flow chart showing a measurement method in which the above-mentioned steps 3 to 5 are carried out after isolating extracellular vesicles from a biological specimen using an SPFS device.

The method for isolating extracellular vesicles from a biological sample using the SPFS device includes S10' (preparation of the measurement chip), S20' (primary reaction), and S21' (washing) in FIG. 4, which correspond to steps 1 and 3 of the inclusion measurement method of the present invention. The sample used is the same as the sample used in the inclusion measurement method of the present invention.

Steps S01', S20' and S21' in FIG. 4 can be carried out substantially in the same manner as steps 1 and 3 of the inclusion measurement method of the present invention described above, except that the first binding substance is changed to a substance capable of binding to extracellular vesicles. Examples of binding substances used here include antibodies capable of binding to extracellular vesicles, nucleic acids capable of binding to extracellular vesicles, lipids capable of binding to extracellular vesicles, lectins capable of binding to extracellular vesicles, and other proteins capable of binding to extracellular vesicles. The binding determinant possessed by the extracellular vesicles is a part of the extracellular vesicles that the binding substance recognizes when binding to the extracellular vesicles. 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 the corresponding receptor.

The first binding determinant of the extracellular vesicles to which the first binding substance binds is not particularly limited, but includes binding determinants known as markers of extracellular vesicles and binding determinants known as markers of cells secreting extracellular vesicles. Examples of binding determinants known as markers of extracellular vesicles 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 such 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 a marker of extracellular vesicles, it binds only to extracellular vesicles, and is therefore effective in detecting extracellular vesicles in a sample.

Binding determinants known as markers of cells secreting extracellular vesicles include caveolin-1, PSMA, EpCAM, Grypican-1, Survivin, CD91, Tspan8, CD147, EGFR, HER2, CD44, Galactin, Integrin, etc. 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 extracellular vesicles is used as the first binding substance, it binds only to specific cells and extracellular vesicles secreted therefrom, and is therefore effective in detecting extracellular vesicles derived from specific cells in a sample.

Furthermore, before carrying out step S20', a surfactant may be added to the sample. Adding a surfactant makes it possible to prevent aggregation of extracellular vesicles. The surfactant is preferably a surfactant that is widely used in the fields of biology and medicine, and examples of such surfactants include Tween 20, sodium deoxycholate, and Triton (registered trademark) X-100. The surfactant does not destroy the extracellular vesicles, but may be used at a concentration that prevents 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 addition, in the case of sodium deoxycholate, it 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. Additionally, Triton (registered trademark) X-100 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.

After steps S01', S20' and S21' in FIG. 4 are performed, the extracellular vesicles are captured in a state bound to the first binding substance immobilized on the measurement chip. The extracellular vesicles bound to the chip in this way can be subjected to step 2 of the method of the present invention to release the inclusions (step S10 in FIG. 4). When the inclusions are eluted using the elution solution described above, an elution solution containing the eluted inclusions is obtained, and this elution solution is collected (step S11 in FIG. 4). In the measurement chip shown in FIG. 3A, the elution solution containing the inclusions can be collected by aspirating the elution solution in the liquid injection section 330 into a pipette tip. The collected elution solution containing the inclusions can then be subjected to steps 3 to 5 of the method of the present invention for measurement of the inclusions by SPFS.

In addition, the eluate containing the inclusions eluted from the extracellular vesicles recovered by the above-mentioned method can also be used as a sample for analysis by a method other than SPFS. For example, if the inclusions are miRNA, the recovered eluate can be used as a sample to analyze by RT-PCR to measure the miRNA concentration.

(Extracellular vesicle recovery)
Furthermore, in the measurement method of the present invention, the extracellular vesicles isolated from a specimen using the SPFS device can be collected and used as a specimen. Next, a method of isolating and collecting extracellular vesicles from a specimen using the SPFS device and measuring the contents thereof will be specifically described. Figure 5 is a flow chart showing a measurement method in which the above-mentioned steps 1 to 5 are carried out after collecting extracellular vesicles from a biological specimen using the SPFS device.

First, steps similar to steps S01', S20', and S21' in FIG. 4 described above are performed to capture extracellular vesicles that bind to the first binding substance immobilized on the measurement chip. Next, a dissociation solution is used to dissociate the extracellular vesicles from the first binding substance, liberating the extracellular vesicles in the dissociation solution (dissociation step, S50' in FIG. 5), and the dissociation solution containing the dissociated extracellular vesicles is collected and processed (processing step, step S60' in FIG. 5), to obtain isolated extracellular vesicles.

(Dissociation step)
The dissociation liquid for dissociating the extracellular vesicles from the first binding substance and releasing them in the dissociation liquid (S50' in FIG. 5) is not particularly limited as long as it is a liquid that loosens the bond between the first binding substance and the extracellular vesicles and dissociates the extracellular vesicles from the first binding substance. The type of dissociation liquid varies depending on the type of the first binding substance, but examples include acidic solutions, basic solutions, salt solutions, and surfactant solutions. Acidic solutions or salt solutions are preferred because of their ease of dissociation and less damage to membrane proteins.

Examples of acidic substances contained in the acidic solution include glycine and citric acid, with glycine being preferred. The pH of the acidic solution is 1.5 to 4.0, with 2.0 to 3.0 being preferred. A pH of 4.0 or less allows sufficient dissociation of the binding substance from the extracellular vesicles.

Examples of salts contained in the salt solution include NaCl and KCl, with NaCl being preferred. The salt concentration of the salt solution is 0.1 M to 5.0 M, with around 1 M being preferred. If the salt concentration is 0.1 M or higher, dissociation of the binding substance from the extracellular vesicles is possible.

The method of providing the dissociation liquid is not particularly limited. For example, a pipette with a pipette tip attached to the end may be used to provide the dissociation liquid onto the metal film. In the measurement chip shown in FIG. 3A, the dissociation liquid can be introduced into the flow channel 320 by injecting the dissociation liquid from the pipette tip into the liquid injection section 330, and the dissociation liquid can be sent back and forth within the flow channel 320 by alternately injecting and suctioning the liquid.

The time for which the dissociation solution is in contact with the extracellular vesicles bound to the first binding substance varies depending on the type and concentration of the dissociation solution, as well as the type of the first binding substance, but is usually from 0.5 minutes to 30 minutes, preferably from 5 minutes to 15 minutes, and more preferably from 10 minutes to 15 minutes. Although it depends on the conditions, a contact time of 10 minutes or more makes it possible to dissociate the binding substance and the extracellular vesicles to a sufficient level.

(Recovery process)
Next, the dissociation liquid containing the dissociated extracellular vesicles is collected and processed (step S60' in FIG. 5) to obtain isolated extracellular vesicles. Since the extracellular vesicles are free in the dissociation liquid, they are collected together with the dissociation liquid. In the measurement chip shown in FIG. 3A, the dissociation liquid in which the extracellular vesicles are free can be collected by aspirating the dissociation liquid in the liquid injection unit 330 into the pipette tip.

Because components of the dissociation solution may damage extracellular vesicles or interfere with further analysis of the extracellular vesicles in the long term, the collected dissociation solution is treated to remove or suppress the effects of the dissociation solution on the extracellular vesicles.

When the dissociation solution is an acidic solution, in order to suppress the effect of the acid on the extracellular vesicles and subsequent analysis, the recovered dissociation solution is neutralized with a basic solution to a pH of 6.0 to 8.0, preferably 7.0 to 7.4. The type of basic substance contained in the basic solution used for neutralization varies depending on the type of acidic substance contained in the dissociation solution, but typically includes NaOH and carbonate buffer. The pH of the basic solution is typically 9.0 to 12.0, preferably 10.0 to 11.0. If the pH is 9.0 or higher, it is possible to neutralize the acidic substances in the dissociation solution, and if neutralization is possible, there is no problem with a high pH.

When the dissociation liquid is a salt solution, the collected dissociation liquid is diluted to suppress the influence of salts on the extracellular vesicles and subsequent analysis. The dilution liquid used for dilution is not particularly limited as long as it is a solution that can suppress the influence of salt substances in the dissociation liquid without damaging the extracellular vesicles, but it is preferable that it does not affect further analysis that may be performed thereafter. Examples of dilution liquids include water, physiological saline, and buffers, and examples of buffers include PBS and TBS. The dilution liquid is mixed with the collected dissociation liquid. The dilution ratio at this time can be determined according to the salt concentration of the dissociation liquid, but is usually 2 to 10 times, preferably 3 to 6 times, and more preferably 5 times. If the dilution ratio is 2 times or more, it is possible to suppress the influence of salt substances in the dissociation liquid. The dilution ratio can also be determined according to the concentration of the collected extracellular vesicles and the sensitivity of the analysis that may be performed next.

The neutralized or diluted dissociation solution can be used in the method of the present invention or other analytical methods as a sample for measuring the contents of extracellular vesicles.

[Extracellular vesicle contents measurement kit]
The measurement kit for measuring the inclusions of extracellular vesicles according to the present embodiment is a set including the above-mentioned elution solution for eluting the inclusions from extracellular vesicles, 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). By preparing the above-mentioned elution solution, measurement chip, and labeling reagent in advance, the user (such as a medical professional) can more easily perform the above-mentioned measurement method for the inclusions of extracellular vesicles.

[effect]
As described above, in the method and kit for measuring the contents of extracellular vesicles according to the present embodiment, it is possible to measure the contents of extracellular vesicles with high sensitivity while suppressing the influence of contaminants, without the need to separate or concentrate the extracellular vesicles from the specimen by using SPFS. In addition, even if a series of operations such as elution of the contents from the extracellular vesicles and dilution of the specimen are automatically performed in the device, it is possible to easily measure the contents without insufficient sensitivity.

In addition, because SPFS can use whole blood as a sample, it is possible to develop diagnostic methods and diagnostic kits based on the inclusions of extracellular vesicles in blood that can be easily performed with fewer steps, without the need for processes such as extracting extracellular vesicles.

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

Example 1
As a specimen, hydrated 5637 cell-derived exosomes (lyophilized product) purchased from Cosmobio Co., Ltd. (although hydrated, the lyophilized product is prepared with PBS, making it a buffer system. Hereinafter, this will be referred to as "buffer system") were prepared. The exosome concentration of the specimen was 1.6 x ¹⁰⁶ particles/μl. Furthermore, PBS containing 1% BSA was prepared as a diluent, and a diluent containing 1% Triton (registered trademark) X-100 was prepared as an eluent.

The measurement chip shown in Figure 3A was prepared, and anti-HSP70 antibody 1 (an antibody against heat shock protein 70 that recognizes a different epitope from anti-HSP70 antibody 2 described below) was immobilized as the first binding substance on a specific region (reaction area) of the metal film (thin gold film) exposed in the flow channel, and prepared for SPFS measurement. Note that anti-HSP70 antibody 1 is an antibody that specifically binds to heat shock protein 70 (HSP70).

For the above samples, HSP70, which is an inclusion of exosomes, was eluted from the exosomes, and the signal value correlating to the amount of HSP70 was measured by SPFS. Specifically, the elution solution (dilution solution as a control) and the sample were added to an empty well of a measurement cartridge containing reagents related to the measurement, and a pipette tip was used to aspirate and discharge the sample and elution solution (dilution solution) in the well for approximately 30 seconds, stirring and mixing the sample and elution solution (dilution solution) in the well. The sample was diluted by this mixing (dilution rate in the device was 3 times), and in systems using elution solution, the inclusions were eluted from the exosomes at the same time as dilution, and a sample for measurement was obtained.

Next, the measurement specimen, which is the specimen with the contents dissolved and/or diluted, was introduced into the flow path from the liquid injection part by a pipette tip and sent back and forth (primary reaction). The reaction time of the primary reaction was 100 minutes. The specimen in the flow path was collected from the liquid injection part and discharged into an empty well of the cartridge, and then the flow path was washed once with a washing solution. Next, a labeling reagent (anti-HSP70 antibody 2 labeled with Alexa Fluor dye (an antibody against heat shock protein 70 that recognizes a different epitope from the anti-HSP70 antibody 1 described above) was introduced into the flow channel from the liquid injector, and the liquid was sent back and forth (secondary reaction). The reaction time for the secondary reaction was 10 minutes. After removing the labeling reagent from the flow channel from the liquid injector, the flow channel was washed once with a cleaning solution. Next, a measurement solution was introduced into the flow channel from the liquid injector. In this state, the fluorescence value was measured by SPFS. That is, the metal film was irradiated with excitation light (laser light) from the prism side so that the incident angle of the excitation light on the metal film 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/ ^mm2 . The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate a signal value correlated with the amount of HSP70 contained in the extracellular vesicles. The results are shown in Table 1 below and FIG. 6.

As is clear from the results in Table 1 and Figure 6, the surfactant added to the diluent eluted the contents of extracellular vesicles (exosomes) and made it possible to detect them using SPFS.

Example 2
A sample was prepared by adding 5637 cell-derived exosomes to human serum, with the exosome concentration being 2.0 x ¹⁰⁶ particles/μl.

Except for using the above sample, HSP70, which is an inclusion of exosomes, was eluted from the exosomes in the same manner as in Example 1, and the signal value correlating with the amount of HSP70 was measured by SPFS. The results are shown in Table 2 below and Figure 7.

As is clear from the results in Table 2 and Figure 7, the contents eluted from extracellular vesicles (exosomes) in the specimens could be measured using SPFS without being affected by the various impurities in the serum.

Comparative Example 1
Using the same specimen, first binding substance, and second binding substance as in Example 1, the contents eluted from extracellular vesicles (exosomes) in the specimen were measured by ELISA.

A specimen with an exosome concentration of 1.6 × ¹⁰⁶ particles/μl was prepared in the same manner as in Example 1. Furthermore, PBS containing 1% BSA was prepared as a diluent, and a diluent containing 1% TRITON (registered trademark) X-100 was prepared as an eluent.

70 μl of elution solution was added to 35 μl of sample, mixed, and left to stand at 37°C for 30 minutes to elute the inclusions (HSP70), obtaining a sample for inclusion measurement. A control sample was also prepared in which a diluent containing no elution solution was added instead of the elution solution.

Measurement of HSP70 (inclusions) in the above samples was performed by ELISA. Specifically, anti-HSP70 antibody 1 was immobilized in the wells of a microplate as a capture antibody (primary antibody), and a blocking treatment was further performed to reduce non-specific binding of HSP70 to the wells. The sample was added to the wells of the prepared microplate and left to stand at 37°C for 1 hour to allow HSP70 to bind to the capture antibody. The sample was then removed, and the wells were washed with a washing solution.

Anti-HSP70 antibody 2 labeled with biotin was added to the wells as a detection antibody (secondary antibody) and left to stand at 37°C for 1 hour to allow the detection antibody to bind to HSP70. The detection specimen was then removed and the wells were washed with a washing solution. Next, streptavidin labeled with horseradish peroxidase (HRP) was added to the wells to cause the biotin in the wells to develop color, and when sufficient color development had occurred, a stop solution was added to stop the colorimetric reaction. The absorbance (wavelength 450 nm) of the colored wells was measured using an ELISA plate reader to obtain a signal value. The results are shown in Table 3 below and Figure 8.

As is clear from the results in Table 3 and Figure 8, for a sample with an exosome concentration of 1.6 x ¹⁰⁶ particles/μl, which can be measured by the SPFS-based method of the present invention (Example 1), the ELISA method only gave a signal value similar to that of the control without elution, even when the encapsulated substances were eluted. Therefore, it can be seen that the method of the present invention has a higher measurement sensitivity than the ELISA method.

Example 3
The same specimen as in Example 1 was prepared, exosomes were isolated using an SPFS device, and the contents of the isolated exosomes were measured.

As a specimen, hydrated 5637 cell-derived exosomes (lyophilized product) purchased from Cosmobio Co., Ltd. (although hydrated, the lyophilized product is prepared with PBS, making it a buffer system. Hereinafter, this will be referred to as "buffer system") were prepared. The exosome concentration of the specimen was 1.6 x ¹⁰⁶ particles/μl. Furthermore, PBS containing 1% BSA was prepared as a diluent, and a diluent containing 1% Triton (registered trademark) X-100 was prepared as an eluent.

The measurement chip shown in Figure 3A was prepared, and anti-CD9 monoclonal antibody was immobilized as an exosome-binding substance in a specific region (reaction area) of the metal film (thin gold film) exposed in the flow channel.

The specimen was introduced into the flow path from the liquid injection part using a pipette tip, and the liquid was sent back and forth (primary reaction). The reaction time for the primary reaction was 100 minutes. The specimen in the flow path was collected from the liquid injection part and discharged into an empty well of the cartridge, and then the inside of the flow path was washed once with a cleaning solution.

After removing the washing solution from inside the flow path, an elution solution (dilution solution as a control) was introduced into the flow path from the liquid injection section, and the liquid was pumped back and forth to elute the contents of the exosomes captured in the measurement chip (elution process). The elution process lasted for 10 minutes. The elution solution (dilution solution as a control) containing the contents of the exosomes in the flow path was collected and used as the measurement sample.

Using a different measurement chip to which anti-HSP70 antibody 1 was immobilized, the signal value correlated with the amount of HSP70 was measured by SPFS for the measurement samples obtained above in the same manner as in Example 1. The results are shown in Table 4 below and Figure 9.

As is clear from the results in Table 4 and Figure 9, it was possible to capture extracellular vesicles (exosomes) using the SPFS device, elute the inclusions from the captured extracellular vesicles in the flow channel, and measure the inclusions. Therefore, the method of the present invention makes it possible to continuously capture extracellular vesicles in a sample, release the inclusions from the extracellular vesicles, and measure the inclusions.

Example 4
The same specimen as in Example 1 was prepared, and HSP70 antigen was added thereto to a concentration of 10 ng/ml to obtain a specimen containing exosomes and free HSP70. Exosomes were isolated from the obtained specimen using an SPFS device, and the contents of the isolated exosomes were measured.

Except for using the above sample, exosomes were captured on the measurement chip in the same manner as in Example 3, and the contents of the captured exosomes were eluted, and the eluate containing the exosome contents in the flow channel (dilution solution was used as a control) was collected to obtain a measurement sample.

The signal value correlated with the amount of HSP70 was measured by SPFS for the measurement samples obtained above, in the same manner as in Example 1. The results are shown in Table 5 below and Figure 10.

As is clear from the results in Table 5 and Figure 10, the measurement method of the present invention was able to selectively measure substances encapsulated in extracellular vesicles, even when free encapsulated substances were present in the sample.

By using the method and kit for measuring inclusions in extracellular vesicles according to the present embodiment, it is possible to measure the inclusions with high sensitivity while suppressing the influence of contaminants, without extracting the extracellular vesicles from the specimen. Furthermore, even if a series of operations such as the release of the inclusions from the extracellular vesicles and dilution of the specimen are performed automatically within the device, it is possible to easily measure the inclusions without insufficient sensitivity. Therefore, the method and kit for measuring inclusions in extracellular vesicles according to the present invention are useful for the development of novel disease markers and diagnostic kits.

100, 200, 300, 400, 500 Measurement chip 110 Prism 111 Incident surface 112 Film-forming surface 113 Exit surface 120 Metal film 130 Anti-inclusion antibody (first binding substance)
131 Anti-inclusion antibody (second binding substance)
140 Inclusion 150 Fluorescent substance 210 Metal film 211 Diffraction grating 310 Flow channel cover 320 Flow channel 330 Liquid injecting section 331 Liquid injecting section covering film 340 Storage section 341 Storage section covering film 342 Vent 350 Adhesive layer 412 Dielectric member 414 Metal thin film 416 Ligand fixing region 418 Well member 420 Through hole 422 Sensor structure 510 Well body
511 Storage section
520 Side wall member
521 Prism
523 Reflective Surface
525 Metal Film
526 Reaction field
L1 Excitation light L2 Reflected light L3 Fluorescence

Claims (14)

Preparing a first measuring chip and a second measuring chip including a metal film and a first binding substance immobilized on the metal film and capable of binding to an inclusion of an extracellular vesicle;
preparing an apparatus capable of mounting the first measuring chip and the second measuring chip;
Providing a sample to the first measurement chip installed in the device to isolate extracellular vesicles contained in the sample;
Releasing encapsulated matter from the extracellular vesicles in the first measurement chip installed in the device ;
providing the inclusion on the metal film of the second measurement chip installed in the device and allowing the inclusion to bind to the first binding substance;
A step of labeling the inclusion with a fluorescent substance via a second binding substance that binds to the inclusion before or after binding to the first binding substance in the device ;
a step of irradiating the metal film with excitation light so as to generate surface plasmon resonance in the metal film while the inclusion labeled with the fluorescent substance is bound to the first binding substance in the device , and detecting fluorescence emitted from the fluorescent substance;
A method for measuring contents of extracellular vesicles, comprising: The measurement method according to claim 1, wherein the sample is a cell culture supernatant, serum, plasma, whole blood, urine, saliva, cell lysate, or a dilution thereof. The measurement method according to claim 1 or 2, wherein the inclusion is at least one selected from a nucleic acid, a protein, or a fragment thereof. The measurement method according to any one of claims 1 to 3, in which the release of the inclusions is carried out using an elution solution. The measurement method according to claim 4, wherein the elution solution contains a non-denaturing surfactant. The measurement method according to claim 5, wherein the non-denaturing surfactant is at least one selected from the group consisting of Triton (registered trademark) X-100, Tween 20, and sodium deoxycholate. The measurement method according to any one of claims 1 to 6, wherein the first binding substance binds to a first binding determinant possessed by the inclusion, the second binding substance binds to a second binding determinant possessed by the inclusion, and the first binding determinant and the second binding determinant are different. The measurement method according to any one of claims 1 to 7, wherein the irradiation energy of the excitation light is 7.5 μW/mm ² or more and 30 mW/mm ² or less. the metal film is disposed on a prism;
The excitation light is irradiated onto the metal film through a prism.
The measurement method according to any one of claims 1 to 8. 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 measurement method according to any one of claims 1 to 8. An elution solution for eluting the contents of the extracellular vesicles from the extracellular vesicles;
A first measuring chip including a first metal film and a binding substance that binds to the extracellular vesicles and is immobilized on the first metal film;
A second measuring chip including a second metal film and a first binding substance immobilized on the second metal film and capable of binding to the inclusion;
A labeling reagent for labeling the inclusion with a fluorescent substance;
Including,
A kit for measuring contents of extracellular vesicles. The measuring kit according to claim 11 , wherein the inclusion is at least one selected from the group consisting of a nucleic acid, a protein, and a fragment thereof. The measurement kit according to claim 11 or 12 , wherein the elution solution is a non-denaturing surfactant. The measurement kit according to claim 13 , wherein the non-denaturing surfactant is at least one selected from the group consisting of Triton (registered trademark) X-100, sodium deoxycholate, and Tween 20.

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