JP2022067460A - Measuring method for inclusion of extracellular vesicle, and measuring kit - Google Patents

Abstract

To provide a measuring method for an inclusion of an extracellular vesicle capable of measuring the inclusion with a sample method with high sensitivity without being influenced by a foreign substance even if a little biological samples or a biological of a low extracellular vesicle concentration is used, and a measuring kit.SOLUTION: A measuring method includes: preparing a measurement chip fixed to a metal film and containing a first bond substance bonded to an inclusion of an extracellular vesicle S01; discharging the inclusion from the extracellular vesicle contained in a sample S10; providing the sample containing the inclusion on the metal film; bonding the inclusion contained in the sample to the first bond substance; marking the inclusion with a fluorescent substance via a second bond substance bonded to the inclusion; irradiating the metal film with excitation light in such a manner that surface plasmon resonance occurs on the metal film; and detecting fluorescent light emitted from the fluorescent substance.SELECTED DRAWING: Figure 1

Description

The present invention relates to methods and kits for measuring extracellular vesicle inclusions.

Extracellular vesicle (hereinafter, sometimes abbreviated as "EV") is a general term for membrane vesicles produced by cells and released extracellularly, and their size, origin, and mechanism of development. These are called exosomes, microvesicles, and the like. For convenience, extracellular vesicles are microvesicles (or large EV) that settle at 10,000 xg and do not settle at 10,000 xg, based on the difference in size and sedimentation rate. Is mainly referred to as an exosome (or small EV). However, no clear definition has been established yet, and it is difficult to make a strict distinction between them.

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

Extracellular vesicles secreted into body fluids such as blood and saliva are 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 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 a cell secreting extracellular vesicles are used to detect extracellular vesicles based on an avidin-biotin interaction. .. Further, Patent Document 2 describes an extracellular vesicle analysis device in which an antibody against extracellular vesicles is immobilized in a recess of a base portion made of a synthetic resin having an uneven structure and the extracellular vesicle is captured in the recess. A method for capturing extracellular vesicles is disclosed.

Extracellular vesicles have a phospholipid bilayer membrane, which contains various cellular components such as proteins and nucleic acids. In recent years, it has been reported that inclusions contained in extracellular vesicles may also be a disease marker. As a method for measuring inclusions contained in extracellular vesicles, extracellular vesicles are extracted from a biological sample such as a cell culture supernatant or blood, and then a phospholipid membrane of the extracellular vesicles is formed. After dissolving and releasing the inclusions, it is common to measure by the ELISA method or the like. Reasons for measuring after extracting extracellular vesicles instead of using the biological sample itself include removal of impurities in the sample, concentration of extracellular vesicles, etc., and extraction of extracellular vesicles. As a method for doing so, various methods such as an ultracentrifugation method have been reported (for example, Non-Patent Document 1).

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

Takahiro Ochiya, Yusuke Yoshioka ed., "Exosomes that change medical care", Kagaku-Dojin Co., Ltd., 2018

As described above, methods for detecting inclusions of various extracellular vesicles have been studied so far, but it has been difficult to measure inclusions from a small amount of sample with high sensitivity. In the conventional method for extracting extracellular vesicles (for example, ultracentrifugation method), there are many impurities contained in the extract, and the impurities may affect the measurement result as impurities. In addition, when trying to increase the purity of extracellular vesicles, loss of extracellular vesicles occurs, and it becomes difficult to accurately measure the inclusions in the sample.

Furthermore, in order to measure the inclusions of extracellular vesicles, it is necessary to release the inclusions from the extracellular vesicles. At this time, the eluent is added to the sample to remove the inclusions from the extracellular vesicles. When eluted, the sample will be diluted. Therefore, in the conventional method such as the ELISA method, insufficient sensitivity becomes a problem in the measurement of inclusions using a small amount of sample or a sample having a low extracellular vesicle concentration. Therefore, there is a demand for a method for measuring inclusions with high sensitivity without being affected by impurities even if a small amount of sample or a sample having a low extracellular vesicle concentration is used.

INDUSTRIAL APPLICABILITY The present invention can measure inclusions with high sensitivity and a simple method without being affected by impurities, even if a small amount of sample or a biological sample having a low extracellular vesicle concentration is used. It is an object of the present invention to provide a method for measuring inclusions of vesicles. It is also an object of the present invention to provide a kit for measuring extracellular vesicle inclusions from a biological sample.

The method for measuring the inclusions of exosomes according to the embodiment of the present invention is a measuring chip containing a metal membrane and a first binding substance immobilized on the metal membrane and bound to the inclusions of extracellular vesicles. The step of preparing the inclusions, the step of releasing the inclusions from the extracellular vesicles contained in the sample, the step of providing the inclusions on the metal film, and the inclusions contained in the sample being the first. A step of binding to the binding substance, a step of 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 a step. In a state where the inclusions labeled with the fluorescent substance are bound to the first binding substance, the metal film is irradiated with excitation light so that surface plasmon resonance occurs in the metal film, and the metal film is subjected to excitation light. Includes a step of detecting emitted fluorescence.

The kit for measuring the inclusions of exosomes according to the embodiment of the present invention is immobilized on the eluate, the metal membrane, and the metal membrane for elution of the inclusions of extracellular vesicles from the extracellular vesicles. Further, it contains a measuring chip containing a first binding substance that binds to the inclusions, and a labeling reagent for labeling the inclusions with a fluorescent substance.

Since the method and kit of the present invention for measuring extracellular vesicle inclusions are not easily affected by impurities, the inclusions should be measured with high sensitivity without extracting extracellular vesicles from a sample. Is possible. In addition, even if a series of operations such as release of inclusions from extracellular vesicles and dilution of specimens are automatically performed in the device, inclusions can be easily measured without causing insufficient sensitivity. Become.

FIG. 1 is a flowchart showing an example of a method for measuring inclusions of extracellular vesicles according to the present embodiment. FIG. 2A is a schematic cross-sectional view for explaining the configuration of a measuring chip for PC-SPFS. FIG. 2B is a schematic cross-sectional view for explaining the configuration of a 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 flowchart showing another example of the method for measuring the inclusions of extracellular vesicles according to the present embodiment. FIG. 5 is a flowchart showing still another example of the method for measuring the inclusions of extracellular vesicles according to the present 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.

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

[Measurement method of extracellular vesicle inclusions]
It is known that extracellular vesicles are secreted by both normal cells and disease-related cells such as cancer cells, and their inclusions differ depending on the type and state of the cells that secreted the extracellular vesicles. Has been done. In order to measure the inclusions of extracellular vesicles, the phospholipid membrane of the extracellular vesicles must be decomposed and the inclusions must be released before measurement.

In the method for measuring the inclusions of extracellular vesicles according to the present embodiment, in order to improve the measurement sensitivity, surface plasmon-field enhanced Fluorescence Spectroscopy (hereinafter, also referred to as “SPFS”). Measure inclusions using. Since SPFS excites a fluorescent substance to emit fluorescence by an electric field enhanced by surface plasmon resonance (hereinafter, also referred to as “SPR”), it is more targeted than a general fluorescence immunoassay method (in the present embodiment, extracellular). Vesicle inclusions) can be detected with high sensitivity.

All conventional methods used to determine the presence and / or concentration of specific inclusions of extracellular vesicles in a sample are in the process of preparing a sample containing inclusions of extracellular vesicles. , The extracellular vesicle itself and its inclusions may be lost, or the concentration may decrease due to dilution. For example, when an eluate is added to a sample containing extracellular vesicles to elute inclusions from the extracellular vesicles, the sample is diluted with the eluate. Further, when the extracellular vesicles in the sample are extracted or concentrated in consideration of the dilution of the sample by the eluate, the loss of extracellular vesicles is likely to occur, and the reproducibility of the measurement of inclusions may be lowered. ..

On the other hand, since the measuring method of the present invention is based on the SPFS system, it is possible to automate the elution of inclusions from extracellular vesicles in the sample using the eluate and the dilution of the sample in the SPFS device. Therefore, it is possible to suppress the variation in measured values that may occur due to the elution treatment and dilution, and to easily measure the inclusions of extracellular vesicles.

As a result of diligent studies based on the above idea, the present inventors have found that a specific inclusion of extracellular vesicles can be measured with high sensitivity by SPFS, and the extracellular vesicles according to the present embodiment have been found. Completed the method for measuring inclusions in.

The method for measuring the inclusions of extracellular vesicles of the present invention includes the following steps 1 to 5.
Step 1 of preparing a measuring chip containing a metal membrane and a first binding substance that binds to the inclusions of extracellular vesicles immobilized on the metal membrane.
Step 2 to release inclusions from extracellular vesicles contained in the sample,
Step 3 of providing the inclusions on the metal film and binding the inclusions contained in the sample to the first binding substance.
The step 4 of labeling the inclusion before or after binding to the first binding substance with a fluorescent substance via the second binding substance that binds to the inclusion.
In a state where the inclusions labeled with the fluorescent substance are bound to the first binding substance, the metal film is irradiated with excitation light so that surface plasmon resonance occurs in the metal film, and the fluorescent substance is used. Step 5 to detect the emitted fluorescence.

Hereinafter, steps 1 to 5 of the method for measuring the inclusions of extracellular vesicles according to the present embodiment will be specifically described. FIG. 1 is a flowchart showing a measurement method using a primary reaction and a secondary reaction, which is an example of a method for measuring extracellular vesicles according to the present embodiment.

In the present invention, "extracellular vesicle" is a general term for membrane vesicles produced by cells and released extracellularly, and is called exosomes, microvesicles, etc. depending on their size, origin of development, mechanism of development, and the like. It is a thing. Although extracellular vesicles are roughly classified into endosome-derived exosomes and plasma membrane-derived microvesicles, in the present invention, the entire extracellular membrane vesicles including exosomes and microvesicles are referred to as “extracellular vesicles”. "Vesicle".

In the present invention, the "inclusion of extracellular vesicles" means a substance contained in the extracellular vesicles in the sample and which can be a marker for diseases and the like, and is a substance existing in the sample in a free state. Is distinct from. Nucleic acids such as DNA, mRNA, and miRNA, and proteins (generally including polypeptides and peptides in a broad sense) are known as inclusions of extracellular vesicles, and these fragments are also disease markers. And so on. In the present invention, the "encapsulation of extracellular vesicles" is also simply referred to as "inclusion".

Specific examples of the 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 measuring chip)
A measuring chip containing a metal membrane and a first binding substance that binds to the inclusions 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) and a surface plasmon are combined to generate SPR. 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. SPFS adopting the former method is referred to as prism coupling (PC) -SPFS, and SPFS adopting the latter method is referred to as lattice coupling (GC) -SPFS. As the method for measuring extracellular vesicles according to this embodiment, either PC-SPFS or GC-SPFS may be adopted.

As mentioned 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 that can cause SPR. Examples of metals constituting the metal film include gold, silver, copper, aluminum and alloys thereof.

The first binding substance is not particularly limited as long as it can bind to the substance of interest contained in the extracellular vesicle and can be immobilized on the metal membrane. Normally, 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 film is not particularly limited as long as it can bind to the inclusions, that is, one that binds to the first binding determinant of the inclusions. Examples of binding agents include antibodies that can bind to inclusions, nucleic acids that can bind to inclusions, lipids that can bind to inclusions, lectins that can bind to inclusions, and other proteins that can bind to inclusions. Here, the binding determinant possessed by the inclusion is a part of the inclusion that is recognized when the binding substance binds to the inclusion. 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.

For example, when HSP70 is measured 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. When a substance that binds to a binding determinant unique to a specific inclusion as a target is used as the first binding substance, the first binding substance binds only to the specific inclusion, so that the inclusion in the sample is detected. It is effective to do.

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.

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 membrane to which a binding substance (for example, an antibody) is bound may be formed on the metal membrane. Examples of SAMs include membranes formed of 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) that can bind to a binding substance (for example, an antibody) is immobilized on a metal film, and the binding substance (for example, an antibody) is bound to this polymer. You may let me.

The measuring chip is preferably a structure having a length of several mm to several cm in each piece, but may be a smaller structure or a larger structure 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 illustration, the size and shape of each component in these figures is not accurate. In addition, these figures show an example of using an anti-inclusion antibody that recognizes an antigenic determinant on an inclusion as a first binding substance.

As shown in FIG. 2A, the measuring chip 100 for PC-SPFS is a layer of an anti-inclusion antibody (first binding agent) 130 (that recognizes an antigenic determinant on a prism 110, a metal film 120 and an inclusion. ). The prism 110 is made of a dielectric material 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 emission 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 prism having a trapezoidal bottom surface. The surface corresponding to one bottom 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 the 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 occurs 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 inclusion 140 that reacted with the second binding substance 131 labeled with the fluorescent substance 150 bound to the antibody (first binding substance) 130 that recognizes the antigenic determinant on the inclusion on the metal film 120. Then, the fluorescent substance 150 is excited by the enhanced electric field and emits fluorescent L3.

As shown in FIG. 2B, the measurement chip 200 for GC-SPFS is an anti-inclusion antibody (first binding substance) that recognizes an antigenic determinant on a metal film 210 and an inclusion on which a diffraction grating 211 is formed. It has 130 (layers). 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 protrusions 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 convex arrangements include square grids, triangular (hexagonal) grids, and the like. Examples of the cross-sectional shape of the diffraction grating 211 include a rectangular wave shape, a sinusoidal 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 plate-shaped 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 occurs in the metal film 210 (diffraction grating 211), the electric field enhanced by the SPR becomes the metal film 210 (diffraction lattice 211). It occurs in the vicinity of the grating 211). At this time, the anti-inclusion antibody (first binding substance) 130 that recognizes the antigenic determinant on the inclusion on the metal film 210 (diffraction lattice 211) is labeled with the fluorescent substance 150 with the second binding substance 131. When the reacted inclusions 140 are 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 3A, the measuring chip 300 is composed of 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 a prism 110. It has a flow path lid 310 arranged on the film surface 112 or the metal film 120. In FIG. 3A, the entrance surface 111 and the exit surface 113 are present in front of and behind the paper surface, respectively. The measuring chip 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. 3A, an anti-inclusion antibody (first) that recognizes an antigenic determinant on an inclusion is in a part of the region (reaction field) of the metal film 120 exposed in the flow path 320. (Binding substance) 130 is immobilized. 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 into 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 components (for example, extracellular vesicles, inclusions, cleaning components, etc.) of the liquid (specimen, washing liquid, etc.) passing through the flow path 320 becomes uniform, and the flow path becomes uniform. Various reactions are likely to occur within 320, 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 diagram 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 (square pyramid trapezoid) having a substantially trapezoidal cross section, and the well member 418 is formed into a rectangular parallelepiped according to the shape of the dielectric member 412. Then, in a state where the first binding substance that binds to the inclusions to be detected is fixed to the ligand fixing region 416 on the metal thin film 414 of the sensor structure 22, the sample solution containing the inclusions is supplied into the through hole 420. And stir the supplied sample solution.

Also, FIG. 3C is a well in which the reaction detector is on the side wall surface (see, eg, International Publication No. 2018/021238). The measuring 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 film 525, and the capture region is a region where a first binding substance for capturing inclusions in a sample is immobilized.

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

The type of the sample is not particularly limited as long as it contains extracellular vesicles. Examples of specimens 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 buffer solutions. Contains diluted diluent. Further, the sample may be a cell culture supernatant containing extracellular vesicles released from the cultured cells and a diluted solution thereof. In the method for measuring the inclusions of extracellular vesicles according to the present embodiment, since the inclusions are detected using SPFS, whole blood can also be used as a sample. Therefore, from the viewpoint of availability and the like, cell culture supernatant, serum, plasma, whole blood, urine, saliva, cell lysate or a dilution thereof are preferable as a sample.

Further, the sample may be an extracellular vesicle isolated from a body fluid or the like. By using isolated extracellular vesicles as a sample, it is possible to select specific extracellular vesicles from multiple types of extracellular vesicles present in body fluids and measure only their inclusions. Become. In addition, when free inclusions may be present in the sample, it is possible to selectively measure the substances contained in the extracellular vesicles without detecting the free substances. The method for isolating the extracellular vesicle is not particularly limited, but as described later, the extracellular vesicle can be captured in the device of SPFS by using a binding substance specific to the extracellular vesicle. Further, a conventionally known method for extracting extracellular vesicles can also be adopted. For example, extracellular vesicles can be isolated and extracted from body fluids using a supercentrifugation method, an immunoprecipitation method, a polymer precipitation method, or an SPFS device.

The method for releasing the inclusions from the extracellular vesicles is not particularly limited as long as it is a method that decomposes the phospholipid membrane of the extracellular vesicles but does not damage the inclusions. The most common method is to add the eluate to the sample. The eluate is not particularly limited as long as it can decompose the phospholipid membrane of extracellular vesicles without damaging the inclusions of interest. The eluate can be selected according to the inclusions to be measured, but a membrane solubilizer such as a surfactant widely used in the fields of biology and medicine is preferable. The surfactant is preferably a non-modified surfactant from the viewpoint of suppressing the influence on the inclusions, and as a specific example, the nonionic surfactant Triton® X-100 (Compound name). : T-octylphenoxypolyethoxyethanol), Tween20 (compound name: polyoxyethylene sorbitan monolaurate) and the like, and sodium deoxycholate which is an anionic surfactant and the like can be mentioned.

The concentration of the eluate used is not particularly limited as long as it decomposes the phospholipid membrane of the extracellular vesicle but does not decompose the inclusions to be measured. In fact, it depends on the type of eluate, the type of inclusions, and the treatment time (that is, the time for contacting the eluate with the sample). For example, when the surfactant is Triton® X-100, its concentration in the eluate is 0.1 to 20% by mass, preferably 0.1 to 10% by mass, more preferably 0. It is 5 to 5% by mass. When the surfactant is Tween 20, the concentration in the eluate is 10 to 30% by mass, preferably 15 to 25% by mass, and more preferably 15 to 20% by mass.

The time for contacting the eluate with the sample is not particularly limited, and is usually 0.5 to 30 minutes, preferably 3 to 10 minutes. 0.5 minutes or more is sufficient to dissolve the phospholipid membrane of the extracellular vesicles and release the inclusions into the specimen.

Release of inclusions from extracellular vesicles can be performed in a container such as a test tube, but can also be performed in a SPFS device. In particular, when an eluate is used, it is preferable to release the inclusions and dilute the sample at the same time in the apparatus. Specifically, the concentration and the amount of the eluate can be adjusted in consideration of the concentration of the surfactant or the like that achieves the release of the inclusions and the dilution rate of the sample.

(Step 3: Primary reaction)
Next, a sample in which the inclusions of extracellular vesicles are released is provided on the metal membrane of the measurement chip, and the inclusions are bound to the first binding substance immobilized on the metal membrane (FIG. 1). 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 inclusions and the first binding substance, a longer reaction time is preferable, and usually 5 minutes or more and 180 minutes or less is preferable. Is 60 minutes or more and 150 minutes or less, more preferably 100 minutes or more and 120 minutes or less. Normally, 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).
Further, after cleaning, the optical blank can be measured (step S22 in FIG. 1; measurement of the optical blank).

(Step 4: Secondary reaction)
Next, a labeling reagent is provided on the metal film of the measuring chip, and the inclusions bound to the first binding substance are labeled with a fluorescent substance via the second binding substance bound to the inclusions (FIG. 1). Step S30). The type of labeling reagent is not particularly limited as long as the inclusions 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 fluorescently labeled second binding agent that binds to the second binding determinant of the inclusion. Alternatively, the labeling reagent is a second binding substance that binds to the second binding determinant of the inclusion, and another fluorescent substance labeled that binds to the second binding substance bound to the inclusion. Contains both of the binding substances of.

The method for 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 a labeling reagent on a metal membrane. Normally, after the secondary reaction and / or the tertiary reaction is completed, the surface of the metal film is washed with a buffer solution or the like to remove a second binding substance (or other binding substance) that is not bound to the inclusions. Remove (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 be bound to inclusions. Examples of the second binding agent include antibodies that can bind to inclusions, nucleic acids that can bind to inclusions, lipids that can bind to inclusions, lectins that can bind to inclusions, and other proteins that can bind to inclusions. ..

The second binding determinant of the inclusion to which the second binding substance binds is not particularly limited, but includes a binding determinant known as a marker of the inclusion. For example, when HSP70 is measured as an inclusion, an antibody against the binding determinant is commercially available, and the antibody can be used as a second binding substance. When a substance that binds to a binding determinant unique to a specific inclusion as a target is used as the second binding substance, the second binding substance binds only to the specific inclusion, so that the inclusion is even on the metal film. It is effective in detecting only the inclusions of the target even if other substances (such as extracellular vesicles) are bound.

When the second 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 second binding substance contained in the labeling reagent may be one type or two or more types. For example, a fluorescent substance-labeled anti-inclusion antibody is one or more anti-inclusion monoclonal antibodies or anti-inclusion polyclonal antibodies. In this case, the fluorescent substance-labeled anti-inclusion monoclonal antibody and anti-inclusion polyclonal antibody may be different from one or more anti-inclusion 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, the first binding substance and the second binding substance may both be antibodies, or one may be an antibody and the other may be 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 the binding determinants of the target inclusions, but the first one. It is preferable that the first binding determinant to which the binding substance binds 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 inclusions to the metal film and labeling with the labeling substance. It will be possible to carry out. Further, the difference between the first binding determinant to which the first binding substance binds and the second binding determinant to which the second binding substance binds causes detection from the sample based on the binding of the first binding substance. It is possible to further narrow down the inclusions to be measured based on the binding of the second binding substance from the inclusions.

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 agents include antibodies, nucleic acids, lipids, and proteins other than antibodies that can bind to antibodies, nucleic acids, lipids, lectins, other proteins, etc. used as the second binding agent. For example, if a combination of second and third binding substances is used such that 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 the inclusions than if they were. 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 inclusions is changed, reduced or lost due to the fluorescent labeling, the third binding is also possible. The substance can be used to label the inclusions.

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 Alexa Fluor® dyes, and Biotium CF dyes. Alexa Fluor dyes and CF dyes have the highest quantum efficiency with respect to the wavelength of the excitation light used in SPFS among the fluorescent dyes on the market. 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 anticellular 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, but the inclusions are immobilized on the metal film. The inclusions may be labeled with a fluorescent substance using a labeling reagent before binding the inclusions to the binding substance of 1. 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, another fluorescently labeled (third) binding substance can be added to the mixture of the sample and the labeling reagent (second binding substance). Further, a step of binding the inclusions to the binding substance immobilized on the metal film and a step of labeling the inclusions 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 membrane.

(Step 5: Fluorescence measurement)
Next, fluorescence indicating the amount of inclusions is measured by SPFS (step S40 in FIG. 1). Specifically, in a state where the inclusions labeled with the 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. The fluorescence emitted from the fluorescent substance is measured. Usually, the measured fluorescence value is subtracted from the pre-measured optical blank value to calculate the signal value that correlates with the amount of inclusions. If necessary, the signal value may be converted into the amount of inclusions (number / ml), concentration (μg / ml), or the like by using a calibration curve prepared in advance.

As shown in FIG. 2A, when the measuring chip 100 for PC-SPFS is used, the excitation light L1 irradiates 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 incident angle of the excitation light L1 with respect to 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 beam emitted from a laser light source having an output of 10 μW to 30 mW. The irradiation energy of the excitation light is 7.5 μW / mm ² 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 9.5 μW / mm ² or more and 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, it is possible to increase the fluorescence intensity, increase the signal-to-noise ratio (S / N), and detect a smaller amount of inclusions. The limit can be raised. 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 irradiation heat proceeds, so that the signal-to-noise ratio deteriorates.

The fluorescence detector is preferably 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 the measuring chip. It is 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 fluorescent L3 is highest is the direction inclined to some extent with respect to the normal of the metal film 120, so that it can be detected. The device is installed in a position not directly above the measuring chip. The detector is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).

(Isolation of extracellular vesicles)
In the measuring method of the present invention, it is not necessary to isolate extracellular vesicles from the sample, but as described above in relation to step 2 of the method of the present invention, extracellular vesicles are used from the biological sample using the SPFS device. Vesicles can also be isolated and inclusions eluted from the isolated extracellular vesicles. Next, a method for isolating extracellular vesicles from a biological sample using an SPFS device will be specifically described. FIG. 4 is a flowchart showing a measurement method for carrying out the above-mentioned steps 3 to 5 after isolating extracellular vesicles from a biological sample using an SPFS device.

The method for isolating extracellular vesicles from a biological sample using the SPFS device corresponds to steps 1 and 3 of the inclusion measuring method of the present invention, S10'(preparation of measuring chip) in FIG. Includes S20'(first-order reaction) and S21' (washing). The sample used is the same as the sample used in the method for measuring inclusions of the present invention.

Steps S01', S20'and S21' in FIG. 4 can be carried out substantially in the same manner as in steps 1 and 3 of the inclusion measuring method of the present invention described above, provided that the first binding substance is used. To a substance that can bind to extracellular vesicles. Examples of binding agents used herein are antibodies that can bind to extracellular vesicles, nucleic acids that can bind to extracellular vesicles, lipids that can bind to extracellular vesicles, lectins that can bind to extracellular vesicles, and extracellular vesicles. Contains other proteins that can bind to vesicles. The binding determinant possessed by the extracellular vesicle is a part of the extracellular vesicle recognized when the binding substance binds to the extracellular vesicle. 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 extracellular vesicle to which the first binding substance binds is not particularly limited, but the binding determinant known as a marker for extracellular vesicles and the cell secreting the extracellular vesicle. Includes binding determinants known as markers of. Binding determinants known as markers for extracellular vesicles include CD9, CD63, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, LAMP1 and the like. Will be. 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-mentioned substance (for example, an antibody) is used as the first binding substance that binds to the binding determinant known as a marker of extracellular vesicles, it binds only to the extracellular vesicles and therefore in the sample. It is effective in detecting extracellular vesicles.

Binding determinants known as markers for cells that secrete extracellular vesicles include caveolin-1, PSMA, EpCAM, Grypican-1, Survivin, CD91, Tspan8, CD147, EGFR, HER2, CD44, Galactin, Integrin and the like can be mentioned. 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 extracellular vesicles is used as the first binding substance, it is applied to specific cells and the extracellular vesicles secreted from the specific cells. Since it binds only, it is effective in detecting extracellular vesicles derived from specific cells in a sample.

Further, a surfactant may be added to the sample before carrying out step S20'. By adding a surfactant, it becomes possible to prevent the aggregation of extracellular vesicles. The surfactant is preferably a surfactant widely used in the field of biology and medicine, and includes, for example, Tween 20, sodium deoxycholate, and Triton® X-100. The surfactant does not destroy the extracellular vesicles, but may be used at a concentration that prevents the aggregation of the extracellular vesicles. 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. In the case of Triton® X-100, 0.001% to 0.010% or less, preferably 0.002% to 0.007% or less, more preferably 0.003% to 0.005% or less. Can be used at the concentration of.

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

In addition, the eluate containing inclusions eluted from extracellular vesicles collected by the above-mentioned method can also be used as a sample for analysis by a method other than SPFS. For example, when the inclusion is miRNA, it is also possible to analyze the collected eluate as a sample by the RT-PCR method and measure the miRNA concentration.

(Recovery of extracellular vesicles)
Further, in the measuring method of the present invention, extracellular vesicles isolated from a sample can be recovered using an SPFS device and used as a sample. Next, a method of isolating and recovering extracellular vesicles from a sample using an SPFS device and measuring the inclusions thereof will be specifically described. FIG. 5 is a flowchart showing a measurement method for carrying out the above-mentioned steps 1 to 5 after collecting extracellular vesicles from a biological sample using an SPFS device.

First, the same steps as the steps S01', S20'and S21' of FIG. 4 described above are carried out to capture extracellular vesicles that bind to the first binding substance immobilized on the measuring chip. Next, using the dissociation solution, the extracellular vesicles are dissociated from the first binding substance, the extracellular vesicles are released into the dissociation solution (dissociation step, S50'in FIG. 5), and the dissociated extracellular vesicles are released. The dissociation solution containing the above is recovered and treated (treatment step, step S60'in FIG. 5) to obtain isolated extracellular vesicles.

(Dissociation process)
The dissociation solution for dissociating the extracellular vesicles from the first binding substance and releasing it into the dissociation solution (S50'in FIG. 5) loosens the binding between the first binding substance and the extracellular vesicles. , There is no particular limitation as long as it is a liquid that dissociates extracellular vesicles from the first binding substance. The type of dissociation solution differs depending on the type of the first binding substance, and examples thereof include an acidic solution, a basic solution, a salt solution, and a surfactant solution, which may cause dissociation and damage to membrane proteins. An acidic solution or a salt solution is preferable because the amount is small.

Examples of the acidic substance contained in the acidic solution include glycine and citric acid, and glycine is preferable. The pH of the acidic solution is 1.5 to 4.0, preferably 2.0 to 3.0. When the pH is 4.0 or less, sufficient dissociation between the binding substance and the extracellular vesicle is possible.

Examples of the salts contained in the salt solution include NaCl and KCl, and NaCl is preferable. The salt concentration of the salt solution is 0.1 M to 5.0 M, preferably around 1 M. When the salt concentration is 0.1 M or more, the binding substance and the extracellular vesicle can be dissociated.

The method of providing the dissociation solution is not particularly limited. For example, a pipette with a pipette tip attached to the tip may be used to provide the dissociation solution on the metal membrane. In the measurement tip shown in FIG. 3A, the dissociation liquid is introduced into the flow path 320 by injecting the dissociation liquid into the liquid injection unit 330 from the pipette tip, and the liquid is alternately injected and sucked to flow. It is also possible to reciprocate the dissociated liquid in the path 320.

The time for contacting the dissociation solution with the extracellular vesicles bound to the first binding substance varies depending on the type and concentration of the dissociation solution, the type of the first binding substance, and the like, but is usually 0.5. It is minutes or more and 30 minutes or less, preferably 5 minutes or more and 15 minutes or less, and more preferably 10 minutes or more and 15 minutes or less. Depending on the conditions, if the contact time is 10 minutes or more, it is possible to dissociate the binding substance and the extracellular vesicle at a sufficient level.

(Recovery process)
Next, the dissociated solution containing the dissociated extracellular vesicles is collected and treated (step S60'in FIG. 5) to obtain isolated extracellular vesicles. Since extracellular vesicles are released in the dissociation fluid, they are collected together with the dissociation fluid. In the measuring tip shown in FIG. 3A, the released dissociation solution of extracellular vesicles can be recovered by sucking the dissociation solution in the liquid injection section 330 into the pipette tip.

Since the components of the dissociation fluid may damage the extracellular vesicles in the long term and interfere with further analysis of the extracellular vesicles, the extracellular vesicles are smaller than the recovered dissociation fluid. Treatment is performed to remove or suppress the effect of the dissociation solution on the vesicles.

When the dissociation solution is an acidic solution, the recovered dissociation solution is neutralized with a basic solution to suppress the effect of the acid on extracellular vesicles and subsequent analysis, and the pH is 6.0 to 8.0. The pH is preferably 7.0 to 7.4. The type of the basic substance contained in the basic solution used for neutralization varies depending on the type of the acidic substance contained in the dissociation solution, but usually includes NaOH, a carbonic acid buffer and the like. The pH of the basic solution is usually pH 9.0 to 12.0, preferably 10.0 to 11.0. When the pH is 9.0 or more, the acidic substance in the dissociation solution can be neutralized, and if the neutralization is possible, there is no problem due to the high pH.

If the dissociation solution is a salt solution, dilute the recovered dissociation solution to reduce the effect of salts on extracellular vesicles and subsequent analysis. The diluent used for dilution is not particularly limited as long as it is a solution capable of suppressing the influence of the salt substance in the dissociation solution without damaging the extracellular vesicles, but further analysis that can be performed thereafter can be performed. It is preferable that it does not affect. Examples of the diluent include water, physiological saline, a buffer and the like, and examples of the buffer include PBS and TBS. The diluted solution is mixed with the recovered dissociated solution. The dilution ratio at this time can be determined according to the salt concentration of the dissociation solution, 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 the salt substance in the dissociation liquid. The dilution rate can also be determined according to the concentration of the recovered extracellular vesicles, the sensitivity of the analysis that can be performed next, and the like.

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 inclusions of extracellular vesicles.

[Measurement kit for extracellular vesicle inclusions]
The kit for measuring the inclusions of extracellular vesicles according to the present embodiment includes the above-mentioned eluate for eluting inclusions from the extracellular vesicles, the above-mentioned measuring chip, and the above-mentioned labeling reagent (fluorescent substance). , A second binding substance and, if desired, a third binding substance) and a set. By setting the above-mentioned eluate, measuring chip, and labeling reagent in advance in this way, the user (medical worker, etc.) can more easily perform the above-mentioned measuring method of the inclusions of extracellular vesicles. ..

[effect]
As described above, in the method and kit for measuring the inclusions of extracellular vesicles according to the present embodiment, SPFS is used to contaminate the extracellular vesicles without the need to separate or concentrate the extracellular vesicles from the sample. It is possible to measure the inclusions of extracellular vesicles with high sensitivity while suppressing the influence of. In addition, even if a series of operations such as elution of inclusions from extracellular vesicles and dilution of specimens are automatically performed in the device, inclusions can be easily measured without causing insufficient sensitivity. Become.

In addition, since whole blood can be used as a sample for SPFS, extracellular vesicles in blood can be easily performed with a small number of procedures without performing a step such as extraction of extracellular vesicles. It is possible to construct a diagnostic method, a diagnostic kit, etc. based on the inclusions of.

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

Example 1
The sample is a hydrated 5637 cell-derived exosome (lyophilized product) purchased from Cosmo Bio Co., Ltd. (although it is hydrated, the lyophilized product is prepared with PBS, so it is a buffer system. (Hereinafter referred to as "buffer system") was prepared. The exosome concentration of the sample is 1.6 × ¹⁰⁶ particles / μl. Further, PBS containing 1% BSA was prepared as a diluent, and a diluted solution containing 1% Triton® X-100 was prepared as an eluate.

The measurement chip shown in FIG. 3A was prepared, and the anti-HSP70 antibody 1 (heat shock) was used as the first binding substance in a specific region (reaction part) of the metal film (gold thin film) exposed in the flow path. An antibody against protein 70, which recognizes an epitope different from that of anti-HSP70 antibody 2 described later), was prepared for SPFS measurement. The anti-HSP70 antibody 1 is an antibody that specifically binds to the heat shock protein 70 (HSP70).

For the above sample, HSP70, which is an inclusion thereof, was eluted from the exosome, and the signal value corresponding to the amount of HSP70 was measured by SPFS. Specifically, the eluate (diluted solution as a control) and the sample are added to the empty well of the measurement cartridge containing the reagent related to the measurement, and suction and discharge are performed in the well using a pipette tip for about 30 seconds. The sample in the well and the eluate (diluted solution) were stirred and mixed. The sample was diluted by the mixing (dilution rate in the device was 3 times), and in the system using the eluate, the inclusions were eluted from the exosome at the same time as the dilution to obtain a sample for measurement.

Next, a measurement sample, which is a sample in which inclusions were eluted and / or diluted, was introduced into the flow path from the liquid injection section by a pipette tip and reciprocated (primary reaction). The reaction time of the primary reaction was 100 minutes. The sample in the flow path was collected from the liquid injection section and discharged into an empty well of the cartridge, and then the inside of the flow path was washed once with a washing liquid. Next, a labeling reagent (anti-HSP70 antibody 2 labeled with Alexa Fluor dye (antibody against heat shock protein 70 that recognizes an epitope different from the above-mentioned anti-HSP70 antibody 1) is applied from the liquid injection section into the flow path. (Secondary reaction). The reaction time of the secondary reaction was 10 minutes. After removing the labeling reagent in the flow path from the liquid injection part, the inside of the flow path was washed once with a washing solution. Then, the measurement liquid was introduced into the flow path from the liquid injection part. In this state, the fluorescence value was measured by SPFS. That is, the prism side so that the incident angle of the excitation light on the metal film becomes the enhanced angle. The metal film was irradiated with excitation light (laser light), and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the irradiation energy amount 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 HSP70, which is an inclusion of extracellular vesicles. The results are shown in Tables 1 and 6 below. ..

As is clear from the results in Table 1 and FIG. 6, the inclusions were eluted from the extracellular vesicles (exosomes) by the detergent added to the diluted solution, and could be detected using SPFS.

Example 2
A sample in which 5637 cell-derived exosomes were added to human serum and the concentration of the exosomes was 2.0 × 10 ⁶ particles / μl was prepared.

In the same manner as in Example 1 except that the above sample was used, HSP70, which is an inclusion thereof, was eluted from the exosome, and the signal value correlated with the amount of HSP70 was measured by SPFS. The results are shown in Table 2 and FIG. 7 below.

As is clear from the results in Table 2 and FIG. 7, inclusions eluted from extracellular vesicles (exosomes) in the sample are measured using SPFS without being affected by various impurities in the serum. I was able to.

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

Similar to Example 1, a sample having an exosome concentration of 1.6 × 10 ⁶ particles / μl was prepared. Further, PBS containing 1% BSA was prepared as a diluent, and a diluted solution containing 1% TRITON (registered trademark) X-100 was prepared as an eluate.

70 μl of the eluate was added to 35 μl of the sample and mixed, and the mixture was allowed to stand at 37 ° C. for 30 minutes to elute the inclusions (HSP70) to obtain a sample for measuring the inclusions. Instead of the eluate, a control sample to which a diluted solution containing no eluate was added was also prepared.

The measurement of HSP70 (inclusions) in the above sample was carried out by the ELISA method. Specifically, the anti-HSP70 antibody 1 was immobilized in the wells of the microplate as a supplementary antibody (primary antibody), and further subjected to blocking treatment to reduce the non-specific binding of HSP70 into the wells. I prepared a plate. The sample was added to the well of the prepared microplate and allowed to stand at 37 ° C. for 1 hour to bind HSP70 to the supplementary antibody. Then, the sample was removed and the well was washed with a washing solution.

As a detection antibody (secondary antibody), biotin-labeled anti-HSP70 antibody 2 was added to the well and allowed to stand at 37 ° C. for 1 hour to bind the detection antibody to HSP70. Then, the detected sample was removed, and the well was washed with a washing solution. Next, streptavidin labeled with horseradish peroxidase (HRP) was added to the wells to develop the color of biotin in the wells, and when the color was sufficiently developed, a stop solution was added to stop the colorimetric reaction. The absorbance (wavelength 450 nm) of the well developed with an ELISA plate reader was measured, and a signal value was obtained. The results are shown in Table 3 and FIG. 8 below.

As is clear from the results in Table 3 and FIG. 8, for a sample having an exosome concentration of 1.6 × 10 ⁶ particles / μl, which can be measured by the method of the present invention based on SPFS (Example 1), the ELISA method used the ELISA method. Elution of inclusions yielded only signal values similar to those of controls without elution. Therefore, it can be seen that the method of the present invention has higher measurement sensitivity than the ELISA method.

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

The sample is a hydrated 5637 cell-derived exosome (lyophilized product) purchased from Cosmo Bio Co., Ltd. (although it is hydrated, the lyophilized product is prepared with PBS, so it is a buffer system. (Hereinafter referred to as "buffer system") was prepared. The exosome concentration of the sample is 1.6 × ¹⁰⁶ particles / μl. Further, PBS containing 1% BSA was prepared as a diluent, and a diluted solution containing 1% Triton® X-100 was prepared as an eluate.

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

A sample was introduced into the flow path from the liquid injection part by a pipette tip and reciprocated (primary reaction). The reaction time of the primary reaction was 100 minutes. The sample in the flow path was collected from the liquid injection section and discharged into an empty well of the cartridge, and then the inside of the flow path was washed once with a washing liquid.

After removing the washing liquid in the flow path, an eluate (a diluted solution was used as a control) was introduced into the flow path from the liquid injection section, and the solution was reciprocated to elute the inclusions from the exosomes captured in the measurement chip. (Elution treatment). The elution treatment time was 10 minutes. The eluate containing the inclusions of exosomes (the control was a diluted solution) in the flow path was collected and used as a sample for measurement.

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

As is clear from the results in Table 4 and FIG. 9, the extracellular vesicles (exosomes) are captured using the SPFS device, and the inclusions are eluted from the extracellular vesicles captured in the flow path to contain the inclusions. Was able to be measured. Therefore, in the method of the present invention, it is possible to continuously capture extracellular vesicles in a sample, release inclusions from extracellular vesicles, and measure inclusions.

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

Except for using the above sample, exosomes are supplemented on the measurement chip in the same manner as in Example 3, inclusions are eluted from the supplemented exosomes, and an eluate containing the inclusions of exosomes in the flow path (control is Diluted solution) was collected to obtain a sample for measurement.

For the measurement sample obtained above, the signal value that correlates with the amount of HSP70 was measured by SPFS in the same manner as in Example 1. The results are shown in Table 5 and FIG. 10 below.

As is clear from the results in Table 5 and FIG. 10, the measurement method of the present invention selectively measures the substance contained in the extracellular vesicle even if the free inclusions are present in the sample. Was made.

By using the method for measuring extracellular vesicle inclusions and the measurement kit according to the present embodiment, the inclusions are highly sensitive while suppressing the influence of contaminants without extracting extracellular vesicles from the sample. Can be measured. In addition, even if a series of operations such as release of inclusions from extracellular vesicles and dilution of specimens are automatically performed in the device, inclusions can be easily measured without causing insufficient sensitivity. Become. Therefore, the method and kit for measuring extracellular vesicle inclusions according to the present invention are useful for developing new disease markers and diagnostic kits.

100, 200, 300, 400, 500 Measuring chip 110 Prism 111 Incident surface 112 Film formation surface 113 Exit surface 120 Metal film 130 Anti-inclusion antibody (first binding substance)
131 Anti-inclusion antibody (second binding substance)
140 Inclusions 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 layer 421 Adhesive layer 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 (14)

A step of preparing a measuring chip containing a metal membrane and a first binding substance that binds to the inclusions of extracellular vesicles immobilized on the metal membrane.
The process of releasing inclusions from the extracellular vesicles contained in the sample,
A step of providing the inclusions on the metal film and binding the inclusions contained in the sample to the first binding substance.
The step of labeling the inclusions before or after binding to the first binding substance with a fluorescent substance via the second binding substance that binds to the inclusions.
In a state where the inclusions labeled with the fluorescent substance are bound to the first binding substance, the metal film is irradiated with excitation light so that surface plasmon resonance occurs in the metal film, and the fluorescent substance is used. The process of detecting the emitted fluorescence and
A method for measuring the inclusions of extracellular vesicles, including. The measuring 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 measuring method according to claim 1 or 2, wherein the inclusion is at least one selected from nucleic acids, proteins, or fragments thereof. The measuring method according to any one of claims 1 to 3, wherein the release of the inclusions is carried out using an eluate. The measuring method according to claim 4, wherein the eluate contains a non-denatured surfactant. The measuring method according to claim 5, wherein the non-denatured surfactant is at least one selected from the group consisting of Triton® X-100, Tween 20 and sodium deoxycholate. The first binding substance binds to the first binding determinant of the inclusion, and the second binding substance binds to the second binding determinant of the inclusion. The measuring method according to any one of claims 1 to 6, wherein the first binding determinant and the second binding determinant are different from each other. The measuring 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 arranged on the prism, and the metal film is arranged on the prism.
The excitation light irradiates the metal film via a prism.
The measuring method according to any one of claims 1 to 8. The metal film contains a diffraction grating and contains a diffraction grating.
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 measuring method according to any one of claims 1 to 8. An eluate for elution of the inclusions of extracellular vesicles from the extracellular vesicles,
A measuring chip containing a metal film and a first binding substance immobilized on the metal film and bound to the inclusions.
A labeling reagent for labeling the inclusions with a fluorescent substance, and
including,
Measurement kit for extracellular vesicle inclusions. The measurement kit according to claim 11, wherein the inclusion is at least one selected from nucleic acids, proteins, or fragments thereof. The measurement kit according to claim 11 or 12, wherein the eluate is a non-denatured surfactant. The measurement kit according to claim 13, wherein the non-denatured surfactant is at least one selected from the group consisting of Triton® X-100, sodium deoxycholate, and Tween 20.

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