WO2022196630A1

WO2022196630A1 - Method for producing antibody-binding nanoparticles, antibody-binding nanoparticles, kit for producing antibody-binding nanoparticles, and method for measuring analyte using antibody-binding nanoparticles

Info

Publication number: WO2022196630A1
Application number: PCT/JP2022/011293
Authority: WO
Inventors: 雅之小野
Original assignee: 株式会社Ｊｖｃケンウッド
Priority date: 2021-03-19
Filing date: 2022-03-14
Publication date: 2022-09-22
Also published as: JP2022145226A

Abstract

Provided is a method for producing antibody-binding nanoparticles, the method comprising (a) a step for binding an antibody and polyethylene glycol to nanoparticles and (b) a step for binding a blocking agent to each of the nanoparticles to which the antibody and the polyethylene glycol have been bound. Also provided are antibody-binding nanoparticles to each of which an antibody, polyethylene glycol and a blocking agent are bound. Also provided is a kit for producing the antibody-binding nanoparticles, which includes nanoparticles, polyethylene glycol and a blocking gent. Also provided is a method for measuring an analyte using the antibody-binding nanoparticles.

Description

Method for producing antibody-bound nanoparticles, antibody-bound nanoparticles, kit for producing antibody-bound nanoparticles, and method for measuring substance to be detected using antibody-bound nanoparticles

TECHNICAL FIELD The present invention relates to a method for producing antibody-bound nanoparticles, antibody-bound nanoparticles, a kit for producing antibody-bound nanoparticles, and a method for measuring a substance to be detected using antibody-bound nanoparticles.
This application claims priority based on Japanese Patent Application No. 2021-046537 filed in Japan on March 19, 2021, the content of which is incorporated herein.

In a system that immunologically captures and labels a detection target (for example, sandwich immunoassay), it is necessary to reduce non-specific adsorption in order to detect low-concentration detection targets with high accuracy. When antibody-bound microparticles (beads) are used as labels, the factor that antibodies (proteins) are easily adsorbed and the factor that non-specific adsorption is small are contradictory. Therefore, nonspecific adsorption is generally reduced by covering (masking) the bead surface with molecules that do not cause nonspecific adsorption after the antibody is bound to the bead surface. A low-molecular-weight compound such as aminoethanol is generally used for masking the bead surface.

In recent years, due to improvements in analytical instruments and reagents, the accuracy and sensitivity of analysis are increasing. As a result, it has become possible to detect the difference in very small amounts of substances to be detected (Patent Document 1). In the use of such high-precision analytical equipment, the reduction of non-specific adsorption to the surface of beads, which are labels, is a problem.

JP 2017-207289 A

　The conventional masking method using low-molecular-weight compounds could not sufficiently reduce non-specific adsorption to the bead surface. In particular, in order to analyze low-concentration substances to be detected with high accuracy, a technique for further reducing non-specific adsorption to the surface of beads, which are labels, is required.

Therefore, this embodiment provides a method for producing antibody-bound nanoparticles that can reduce nonspecific adsorption, antibody-bound nanoparticles produced by the production method, a kit for producing the antibody-bound nanoparticles, And an object thereof is to provide a method for measuring a substance to be detected using antibody-bound nanoparticles.

This embodiment includes the following aspects.
[1] An antibody comprising the steps of: (a) binding an antibody and polyethylene glycol to a nanoparticle; and (b) binding a blocking agent to the nanoparticle to which the antibody and the polyethylene glycol are bound. A method for producing bound nanoparticles.
[2] The step (a) is a step of simultaneously performing a reaction of binding the antibody to the nanoparticles and a reaction of binding the polyethylene glycol to the nanoparticles, wherein the surface of the nanoparticles and the antibody and have a pH at which they are at opposite potentials, and step (a) comprises binding the antibody and the polyethylene glycol to the nanoparticles in a buffer solution containing a condensing agent, [1 ].
[3] In the step (b), in a buffer solution containing the blocking agent, the nanoparticles to which the antibody and the polyethylene glycol are bound are heated at a temperature of 30 to 45° C. at a temperature of 40 to 100 A method for producing antibody-bound nanoparticles according to [1] or [2], which comprises incubating for a period of time.
[4] Antibody-bound nanoparticles, in which a blocking agent is bound to nanoparticles bound to an antibody and polyethylene glycol.
[5] A kit for producing antibody-bound nanoparticles by the production method according to any one of [1] to [3], comprising nanoparticles, antibodies, polyethylene glycol, and a blocking agent.
[6] A step of contacting the antibody-bound nanoparticles according to [4] with a substance to be detected that specifically binds to the antibody bound to the antibody-bound nanoparticles, and binding to the antibody-bound nanoparticles A method for measuring a substance to be detected using antibody-bound nanoparticles, comprising the step of measuring the substance to be detected.

According to this embodiment, a method for producing antibody-bound nanoparticles that can reduce nonspecific adsorption, antibody-bound nanoparticles produced by the production method, a kit for producing the antibody-bound nanoparticles, and a method for measuring a substance to be detected using antibody-bound nanoparticles.

1 is a schematic diagram showing an example of a method for producing antibody-bound nanoparticles according to one embodiment. FIG. FIG. 2 is a top view showing an example of a solid phase carrier; It is an example of an analytical substrate used in an exosome counting device. FIG. 3 is an enlarged schematic diagram showing a state in which antibody-bound nanoparticles are trapped on the solid-phase carrier of FIG. 2; On the solid-phase carrier of FIG. 2, it is a schematic diagram showing an enlarged state in which antibody-bound nanoparticles are specifically bound to exosomes. Exosomes were counted with an ExoCounter (registered trademark) using the anti-CD9 antibody-bound nanoparticles of Example 1, Comparative Example 1-1, and Comparative Example 2-1 in a dilution series of the culture supernatant of the colon cancer cell line HCT116. The results are shown. Figure 5A shows exosome count values in the blank (Dilution factor = 0). Exosomes were counted with an ExoCounter (registered trademark) using the anti-CD63 antibody-bound nanoparticles of Example 2, Comparative Example 1-2, and Comparative Example 2-2 in a dilution series of the culture supernatant of the colon cancer cell line HCT116. The results are shown. Figure 6A shows exosome count values in the blank (Dilution factor = 0). Exosomes were counted with an ExoCounter (registered trademark) using the anti-Her2 antibody-bound nanoparticles of Example 3, Comparative Example 1-3, and Comparative Example 2-3 in a dilution series of the culture supernatant of the breast cancer cell line KPL4. Show the results. FIG. 7A shows exosome count values in the blank (Dilution factor=0) of FIG. 7A. Exosomes were counted with ExoCounter (registered trademark) using the anti-CD147 antibody-binding nanoparticles of Example 4, Comparative Example 1-4, and Comparative Example 2-4 in dilution series of culture supernatant of pancreatic cancer cell line MiaPaca2. Show the results. Figure 8A shows exosome count values in the blank (Dilution factor = 0). The results of comparing the S/N values in Examples and Comparative Examples are shown. S: Count value at Dilution factor=1. N: Count value at Dilution factor=0.

Hereinafter, the present embodiment will be described in detail with reference to the drawings as the case may be. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and overlapping descriptions are omitted. The dimensional ratios in each drawing are exaggerated for explanation and do not necessarily match the actual dimensional ratios.

By "antibody" is meant an immunoglobulin that has antigen-binding activity. An antibody need not be an intact antibody, and may be an antigen-binding fragment, as long as it has antigen-binding activity. As used herein, the term "antibody" includes antigen-binding fragments. An "antigen-binding fragment" is a polypeptide comprising a portion of an antibody that retains the antigen-binding properties of the original antibody. Antigen-binding fragments preferably include all six complementarity determining regions (CDRs) of the original antibody. That is, it preferably contains all of the CDR1, CDR2 and CDR3 of the heavy chain variable region and the CDR1, CDR2 and CDR3 of the light chain variable region. Antigen-binding fragments include, for example, Fab, Fab', F(ab') ₂ , variable region fragments (Fv), disulfide-bonded Fv, single-chain Fv (scFv), sc(Fv) ₂ and the like.

Antibodies may be derived from any organism. Examples of organisms from which antibodies are derived include, but are not limited to, mammals (humans, mice, rats, rabbits, horses, cows, pigs, monkeys, dogs, etc.), birds (chickens, ostriches), and the like.
Antibodies can be of any class and subclass of immunoglobulin. The antibody may be a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody. Antibodies can be produced by known methods such as the immunization method, hybridoma method, and phage display method.

The term "specifically binds" means that it has a high binding property to the target substance and almost no binding property to other substances.

[Method for producing antibody-bound nanoparticles]
In one embodiment, the present disclosure provides methods of making antibody-conjugated nanoparticles. The production method of the present embodiment includes (a) a step of binding an antibody and polyethylene glycol to a nanoparticle, and (b) a step of binding a blocking agent to the nanoparticle to which the antibody and the polyethylene glycol are bound. ,including.

FIG. 1 is a schematic diagram showing an example of the method for producing antibody-bound nanoparticles of this embodiment. First, antibodies 20 and polyethylene glycol 30 are bound to carboxy group-modified nanoparticles 10 (FIGS. 1(A) and (B); step (a)). Next, a blocking agent 40 is bound (FIG. 1(C); step (b)). In this way, antibody-bound nanoparticles 100 bound with antibody 20, polyethylene glycol 30, and blocking agent 40 can be produced.

<Step (a)>
In step (a), nanoparticles are bound to antibodies and polyethylene glycol.

"Nanoparticles" means particles having an average primary particle diameter of nanometer order (less than 1000 nm). The average primary particle size of the nanoparticles can be appropriately selected according to the application of the antibody-bound nanoparticles produced by the production method of this embodiment. Examples of the average primary particle size of nanoparticles include 10 to 1000 nm, 50 to 500 nm, 100 to 300 nm, or 150 to 200 nm.

The material of the nanoparticles is not particularly limited. Materials for the nanoparticles include, for example, resins such as polystyrene and glycidyl methacrylate; metals such as iron, gold, and silver; metal oxides such as zirconia, titania, and iron oxide; and magnetic materials such as ferrite. is not limited to

The nanoparticles may be surface-modified to facilitate binding of antibodies. For example, nanoparticles 1 shown in FIG. 1(A) are carboxy group-modified nanoparticles 10 whose surfaces are modified with carboxy groups. Carboxy groups react with amino groups contained in antibodies in the presence of a suitable condensing agent to form amide bonds. This allows the antibodies to bind to the nanoparticles.
Functional groups used for surface modification of nanoparticles are not particularly limited as long as they are capable of binding antibodies. Functional groups that can be used for surface modification include, for example, carboxy groups, amino groups, and succinimide groups.
Surface modification of nanoparticles with these functional groups can be performed by known methods. Alternatively, commercially available surface-modified nanoparticles may be used. Commercially available nanoparticles include, for example, Tamagawa Seiki FG beads series (COOH beads, NH ₂ beads, NHS beads) and the like.
Modification functional groups on the nanoparticle surface can be appropriately selected according to the protein to be bound to the nanoparticles. When binding an antibody to the nanoparticle surface, the surface of the nanoparticle is preferably modified with a carboxy group.

The antibody can be appropriately selected according to the use of the antibody-bound nanoparticles produced by the production method of this embodiment. For example, when the antibody-conjugated nanoparticles are for exosome detection or cell detection, antibodies that specifically bind to membrane proteins specifically expressed in exosomes or cells to be detected can be used. Alternatively, when the antibody-conjugated nanoparticles are for exosome detection, an antibody that specifically binds to a membrane protein that exosomes have ubiquitously (hereinafter also referred to as "pan-exosome membrane protein") may be used. "Exosomes have ubiquitous" means that a wide variety of exosomes have. Pan-exosomal membrane proteins include, for example, CD9, CD63, and CD81. When the substance to be detected is exosomes, an antibody that specifically binds to CD9, CD63, or CD81 may be used.

When the antibody-bound nanoparticles are for detection of cancer cells or for detection of exosomes released from cancer cells, membrane proteins specifically expressed in cancer cells or exosomes to be detected (hereinafter also referred to as "cancer antigen") Antibodies that specifically bind to may also be used. Examples of cancer antigens include, but are not limited to, Her2, CD147, MUC1, CEA, mesothelin, EGFR, EGFRvIII, MAGE, NY-ESO-1, PSMA, PSA, CD19, VEGFR1, VEGFR2, and the like.

Although polyethylene glycol is not particularly limited, it is preferable to use one having a molecular weight (Mw: weight average molecular weight) of about 1000 to 5000. Mw of polyethylene glycol is preferably 1,500 to 3,000, more preferably 1,500 to 2,500, and still more preferably 1,800 to 2,200. As an example, the Mw of polyethylene glycol is 2000. When the Mw of polyethylene glycol is within the preferred range, the nanoparticles can be efficiently masked. For the Mw, a weight average molecular weight in terms of polystyrene by GPC (gel permeation chromatography) can be used.

　Polyethylene glycol may be terminally modified in order to increase binding to nanoparticles. The type of terminal modification can be appropriately selected according to the surface modification of the nanoparticles. For example, if the nanoparticles are carboxy group-modified, the polyethylene glycol may be amino group-modified to have an amino group at the end. For example, polyethylene glycol may be modified to have a monoamine or oligoamine at one end.

Binding of antibodies to nanoparticles and binding of polyethylene glycol to nanoparticles may be performed simultaneously or separately. When the reaction for binding antibodies to nanoparticles and the reaction for binding polyethylene glycol to nanoparticles are performed separately, first, the reaction for binding antibodies to nanoparticles is performed, and then polyethylene glycol is bound to nanoparticles. It is preferred to carry out the reaction. First, the binding reaction of the antibody to the nanoparticles is performed, and then polyethylene glycol is bound to bind polyethylene glycol to the modified groups on the surface of the nanoparticles to which the antibodies were not bound, thereby masking the modified groups. can.

The binding reaction of nanoparticles with antibodies and polyethylene glycol can be carried out in the presence of a suitable condensing agent. As the condensing agent, those used for dehydration condensation reaction can be used without particular limitation. Examples of condensing agents include carbodiimide-based condensing agents. Examples of carbodiimide condensing agents include 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC/HCl). , N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC), and the like, but are not limited thereto. Among them, the condensing agent is preferably EDC.HCl.

The binding reaction between nanoparticles and antibodies is preferably carried out under pH conditions in which the potentials of the surfaces of the nanoparticles and the antibodies are opposite to each other, in order to allow the binding reaction to proceed efficiently. For example, since the surfaces of nanoparticles modified with carboxy groups are negatively charged, the reaction is carried out under pH conditions such that the antibody is positively charged. Such pH conditions include pH conditions below the isoelectric point of the antibody. Since the isoelectric point of the antibody is about pH 5 to 6, a pH condition of, for example, pH 5 or less can be used. From the viewpoint of suppressing antibody denaturation, the pH is preferably 3.5 or higher, more preferably 4 or higher. Examples of pH conditions for the binding reaction between nanoparticles and antibodies include pH 3.5 to 5, pH 4 to 5, pH 4.2 to 4.8, and the like. As an example, pH 4.5 can be used.

The reaction for binding antibodies to the nanoparticles and the reaction for binding polyethylene glycol to the nanoparticles are carried out in a buffer solution having a pH such that the surfaces of the nanoparticles and the antibodies have opposite potentials and containing a condensing agent. can be done at the same time. The buffer used for the binding reaction is not particularly limited as long as it can achieve the above pH conditions, but an acetate buffer is preferred. Acetate buffer can be prepared by dissolving sodium acetate in a mixed solvent of acetic acid and pure water.

The reaction for binding antibodies to nanoparticles and the reaction for binding polyethylene glycol to nanoparticles can be carried out under temperature conditions of, for example, 20 to 40°C. The reaction time is not particularly limited as long as the time allows the binding reaction to proceed sufficiently. Examples of reaction time include 30 to 300 minutes, 60 to 250 minutes, or 120 to 200 minutes.

Through this step, nanoparticles to which antibodies and polyethylene glycol are bound (see FIG. 1(B)) can be obtained.

<Step (b)>
In step (b), a blocking agent is bound to the nanoparticles to which the antibody and polyethylene glycol are bound.

"Blocking agent" means a compound that binds to nanoparticles in order to suppress non-specific adsorption to the nanoparticles. As the blocking agent, those commonly used as blocking agents in immunochemical detection methods can be used. When the nanoparticles are surface-modified, the blocking agent is preferably capable of reacting with the modifying groups on the surface of the nanoparticles. For example, when the nanoparticles are modified with a carboxy group, a compound containing a carboxy group-reactive group can be used as the blocking agent. Examples of carboxy group-reactive groups include amino groups and hydroxy groups.
When the nanoparticles are modified with carboxy groups, the blocking agent can be, for example, protein such as casein, albumin (eg, bovine serum albumin (BSA)), collagen, or gelatin. Among them, the blocking agent is preferably casein.
A blocking agent may be used individually by 1 type, and may use 2 or more types together.

The reaction for binding the nanoparticles to the blocking agent can be performed by incubating the nanoparticles after step (a) in a buffer solution containing the blocking agent. The buffer used for incubation is not particularly limited, and buffers commonly used for blocking in immunochemical detection methods can be used. Examples of buffers include, but are not limited to, PBS, PBS-T, Tris buffer, HEPES buffer, and the like. The content of the blocking agent in the buffer solution can be, for example, about 0.01 to 5% (w/v). A surfactant such as Tween-20 or a chelating agent such as EDTA may be added to the buffer.

For the incubation conditions, the conditions normally used for blocking treatment can be used. Temperature conditions for incubation include, for example, 30-45°C, 30-40°C, or 35-40°C. As an example, the incubation temperature is 37°C. The incubation time should be a time during which the binding reaction of the blocking agent to the nanoparticles proceeds sufficiently. Incubation times include, for example, 40-100 hours, 50-90 hours, or 60-80 hours.

Through step (b), antibody-bound nanoparticles (see FIG. 1(C)) to which the antibody, polyethylene glycol, and blocking agent are bound can be obtained. In the antibody-bound nanoparticles, a blocking agent is bound to the modified groups on the surface of the nanoparticles to which the antibody and polyethylene glycol were not bound in step (a). Therefore, unreacted modification groups on the surface of the nanoparticles are reduced compared to the nanoparticles after step (a). Therefore, non-specific adsorption to the nanoparticle surface is reduced.

<Optional process>
The manufacturing method according to this embodiment may include optional steps in addition to the steps (a) and (b). The optional step is not particularly limited, but includes, for example, a step of washing the nanoparticles after step (a) or step (b). For example, after step (a) and before step (b), the nanoparticles may be washed with a washing liquid. The washing liquid is not particularly limited, and washing liquids commonly used in immunochemical detection methods can be used. Examples of washing solutions include, but are not limited to, blocking agent-containing buffers, BS, PBS-T, Tris buffers, HEPES buffers, and pure water used in the blocking treatment in step (b).

The antibody-bound nanoparticles obtained by the production method of the present embodiment are prepared by binding the antibody and polyethylene glycol to the nanoparticles in step (a), thereby masking the modified groups on the surface of the nanoparticles to which the antibody is not bound with polyethylene glycol. can do. Furthermore, in step (b), unbound modifying groups can be reduced by binding a blocking agent to the modifying groups on the nanoparticle surface to which antibodies and polyethylene glycol are not bound. Therefore, antibody-bound nanoparticles with reduced nonspecific adsorption can be obtained.

[Antibody-bound nanoparticles]
In one embodiment, the present disclosure provides antibody-conjugated nanoparticles in which a blocking agent is conjugated to the antibody-polyethylene glycol conjugated nanoparticles.

The antibody-bound nanoparticles according to this embodiment can be obtained by the production method of the above embodiment. FIG. 1(C) is a schematic diagram showing an example of antibody-bound nanoparticles according to this embodiment. Antibody 20, polyethylene glycol 30, and blocking agent 40 are bound to nanoparticle 1 in antibody-bound nanoparticles 100 shown in FIG. 1(C).

As the nanoparticles, antibodies, polyethylene glycol, and blocking agents, the same ones as listed in the above section [Method for producing antibody-bound nanoparticles] can be used.

In the antibody-bound nanoparticles according to this embodiment, the antibody-unbound modification groups are masked with polyethylene glycol and a blocking agent. Therefore, nonspecific adsorption can be reduced. Therefore, even when the concentration of the detection target substance is low, the detection target substance can be detected with high sensitivity.

[kit]
In one embodiment, the present invention provides a kit for producing antibody-conjugated nanoparticles according to the above embodiments. A kit according to this embodiment includes nanoparticles, antibodies, polyethylene glycol, and a blocking agent.

Nanoparticles, antibodies, polyethylene glycol, and blocking agents may be the same as those listed in the section [Method for producing antibody-bound nanoparticles] above. The nanoparticles are preferably surface-modified with carboxy groups or the like.
The kit according to this embodiment can be used for producing antibody-bound nanoparticles by the production method according to the above embodiment.

<Arbitrary configuration>
The kit of this embodiment may contain arbitrary components in addition to nanoparticles, antibodies, polyethylene glycol, and blocking agents. Examples of optional components include washing solutions, various reagents such as various buffer solutions, condensing agents, instructions for use, and the like. Examples of the buffer include the buffer used in the step (a) (eg, acetate buffer), the buffer used in the step (b), and the like.

(Solid phase carrier bound with capture antibody)
The kit of this embodiment may further include a configuration for using the produced antibody-binding nanoparticles. For example, it may further comprise a solid phase carrier to which a capturing antibody is bound.

A "capture antibody" is an antibody used to capture a substance to be detected in a sample on a solid-phase carrier in an immunochemical detection method.

The capture antibody uses an antibody that specifically binds to the substance to be detected. The capturing antibody may be the same as or different from the antibody used for the antibody-bound nanoparticles (hereinafter also referred to as "labeled antibody"). For example, when the substance to be detected is exosomes, the labeling antibody and the capturing antibody may be antibodies that specifically bind to the same pan-exosomal membrane protein (eg, CD9). Alternatively, the labeling antibody and the capturing antibody can be antibodies that bind to different pan-exosomal membrane proteins (eg, CD9 and CD63). Alternatively, the capture antibody is an antibody that specifically binds to the pan-exosomal membrane protein (e.g., CD9), and the labeled antibody is the exosomes to be detected (e.g., cancer cell-specific exosomes) specifically expressed membrane Antibodies that specifically bind to proteins (eg, cancer antigens such as Her2 and CD147) may also be used.

The solid-phase carrier on which the capturing antibody is immobilized is not particularly limited, and any solid-phase carrier commonly used in immunochemical detection methods can be used. Examples of solid-phase carriers include well plates, substrates, membranes, reaction chambers attached to immunochemical detection devices, and the like.

The material of the solid phase carrier is not particularly limited, and can be appropriately selected according to the type of solid phase carrier. Materials for the solid phase carrier include, for example, resins such as polystyrene, polyolefin (polyethylene, polypropylene, etc.), cycloolefin polymer, polycarbonate, etc.; glass; metals such as gold, iron, zirconia, etc.; Not limited.

A known method can be used to immobilize the capture antibody on the solid phase carrier depending on the type of the solid phase carrier. For example, the surface on which the capturing antibody is immobilized may be surface-treated so that proteins are physically adsorbed by surface hydrophobic interaction. Avidin-biotin conjugation may also be utilized. For example, a biotin-modified capture antibody may be bound to an avidin-modified solid support.
The kit of this embodiment may contain a solid phase carrier to which the capturing antibody is bound. In this case, the user of the kit can bind any antibody to the solid phase carrier.

A kit that further includes a solid-phase carrier to which a capturing antibody is bound can be used to detect a substance to which the antibody contained in the antibody-bonded nanoparticles and the capturing antibody specifically bind. Therefore, a kit further comprising a solid phase carrier bound with a capturing antibody can be used as a detection kit for a substance to be detected.

<<Specific examples of solid phase carriers>>
FIG. 2 shows an example of a solid phase carrier. The solid phase carrier 201 shown in FIG. 2 is an example of an analysis substrate used in an exosome counting device such as ExoCounter (registered trademark).

The solid phase carrier 201 has a disc shape similar to optical discs such as Blu-ray Discs (BD), DVDs, Compact Discs (CDs), for example. A positioning hole 202 is formed in the center of the solid phase carrier 201 . The solid phase carrier 201 is made of, for example, a resin material generally used for optical discs, such as polycarbonate resin or cycloolefin polymer. The solid phase carrier 201 may be in another form, and an optical disc complying with a predetermined standard can also be used.

FIG. 3 is an enlarged view of the surface of the solid phase carrier 201. FIG. On the surface of the solid-phase carrier 201, track regions 205 are formed in which convex portions 203 and concave portions 204 are alternately arranged in the radial direction. The protrusions 203 and the recesses 204 are spirally formed from the inner periphery of the solid phase carrier 201 toward the outer periphery thereof. A track pitch W, which is the radial pitch of the concave portions 204 (convex portions 203), is, for example, 320 nm. A reaction region 210 is formed on the track region 205 of the solid phase carrier 201 (see FIG. 2). In FIG. 2, eight reaction regions 210 are formed at equal intervals so that the center of each reaction region 210 is located on the same circumference Cb with respect to the center Ca of the solid phase carrier 201. The number and formation position of are not limited to this.

FIG. 4 is an enlarged view of the reaction area 210 formed in the track area 205. FIG. A capture antibody 212 is immobilized on a predetermined area (area where the reaction area 210 is formed) on the track area 205 .

≪Usage examples of antibody-bound nanoparticles≫
Using the antibody-bound nanoparticles and the solid phase carrier 201, exosome counting device such as ExoCounter (registered trademark), a method for counting exosomes will be described. In the following examples, exosomes E as a substance to be detected shall be counted.

First, a sample is supplied to the reaction area 210 on the track area 205, and binding reaction between the capture antibody 212 immobilized on the reaction area 210 and the exosome E in the sample is performed.
The sample is not particularly limited, and any sample can be used. The sample may be a liquid sample in which exosomes E are to be measured. Samples include, for example, body fluid samples (blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal discharge, semen, pleural effusion, gastrointestinal secretions, cerebrospinal fluid, interstitial fluid, lymphatic fluid, etc.), cell culture Examples include, but are not limited to, supernatants and the like.
The binding reaction can be performed by incubating for a predetermined time while the sample is supplied to the reaction area 210 . The incubation time may be a time sufficient for exosome E in the sample to bind to the capturing antibody. Examples of the incubation time include 30 minutes or more, 1 to 10 hours, 2 to 6 hours, 2 to 4 hours, or 2 to 3 hours. Examples of the incubation temperature include 10 to 40°C, 20 to 40°C, or 30 to 40°C. In order to improve the reaction efficiency between the capturing antibody 212 and exosomes E, the solid phase carrier 201 may be gently shaken during incubation.
When the sample contains exosomes E, the binding reaction causes the capture antibody 212 to capture the exosomes E in the sample (see FIG. 4).

After the binding reaction, the solid phase carrier 201 may be washed with a washing liquid as appropriate. By washing the solid-phase carrier 201, substances not bound to the capturing antibody 212 can be removed. The washing liquid is not particularly limited, and washing liquids commonly used in immunochemical detection methods can be used without particular limitations. Examples of washing solutions include, but are not limited to, buffers such as PBS, Tris buffer, and HEPES buffer; surfactants such as Tween 20 added to the buffer; and pure water.

Before supplying the sample to the reaction area 210, the reaction area 210 may be blocked. The blocking treatment can be performed by supplying a blocking solution to the reaction area 210 and incubating it. The blocking solution is not particularly limited, and those commonly used in immunochemical detection methods can be used without particular limitation. Blocking solutions include, for example, buffers containing about 1 to 5% skim milk, casein, or bovine serum albumin (BSA). The buffer for the blocking solution is not particularly limited, and examples thereof include PBS, PBS-T, Tris buffer, HEPES buffer and the like.
Examples of the incubation temperature include 10 to 40°C, 20 to 40°C, or 30 to 40°C. Incubation times include 10 to 180 minutes, 20 to 120 minutes, 20 to 100 minutes, or 30 to 60 minutes. By blocking the reaction regions 210, non-specific adsorption to the track regions 205 where the reaction regions 210 are formed can be reduced.

Next, a buffer solution containing the antibody-bound nanoparticles 100 is supplied to the reaction area 210 on the track area 205, and the binding reaction between the exosomes E and the antibody-bound nanoparticles 100 is performed. The buffer for suspending the antibody-bound nanoparticles 100 is not particularly limited, and those commonly used in immunochemical detection methods can be used without particular limitations. Examples of buffers include PBS, Tris buffer, HEPES buffer and the like.
The binding reaction can be performed by incubating for a predetermined period of time in a state in which a buffer solution containing the antibody-bound nanoparticles 100 is supplied to the reaction region 210 . Incubation time, exosomes E, as long as the antibody 20 in the antibody-binding nanoparticles 100 is sufficient time to bind. Examples of the incubation time include 30 minutes or more, 1 to 10 hours, 1 to 6 hours, 1 to 4 hours, 1 to 3 hours, or 1 to 2 hours. Examples of the incubation temperature include 10 to 40°C, 20 to 40°C, or 30 to 40°C. In order to improve the reaction efficiency between the antibody 20 and exosomes E, the solid phase carrier 201 may be gently shaken during incubation.
Due to the binding reaction, the antibody-bound nanoparticles 100 bind to the exosomes E captured by the capturing antibody 212 via the antibody 20 (see FIG. 4). Antibody-bound nanoparticles 100 are thereby trapped in recesses 204 of track region 205 .

Antibody-bound nanoparticles 100 captured in reaction region 210 of solid phase carrier 201 can be counted using a counting device (ExoCounter (registered trademark), etc.) equipped with an optical pickup. The optical pickup has an objective lens 241 . The optical pickup irradiates the solid phase carrier 201 with a laser beam 240a. The laser beam 40a is condensed by the objective lens 241 onto the surface on which the reaction region 210 is formed. The wavelength of the laser beam 40a is, for example, approximately 405 nm.
The optical pickup receives reflected light from the solid-phase carrier 201, detects the received light level of the reflected light, generates a received light level signal, and outputs the received light level signal to a control unit including a CPU or the like. The controller extracts the detection signal of the antibody-bound nanoparticles 100 from the received light level signal output from the optical pickup. The solid-phase carrier 201 is driven at a constant linear velocity Lv, and the laser beam 240a is scanned along the concave portion 204. As shown in FIG. The optical pickup counts the antibody-bound nanoparticles 100 captured in the reaction area 210 by extracting the detection signal of the antibody-bound nanoparticles 100 along the recess 204 .

A measuring device having such a mechanism includes, for example, the counting device described in Japanese Patent Application Laid-Open No. 2017-207289.

The method of using the antibody-bound nanoparticles of the above embodiment is not limited to the above, and can be used for various immunological methods such as ELISA, immunoprecipitation, protein purification, cell separation, and cell counting.

[Method for measuring substance to be detected using antibody-bound nanoparticles]
In one embodiment, the present disclosure provides a method for measuring a substance to be detected using the antibody-bound nanoparticles of the embodiment. The measurement method of the present embodiment includes a step of contacting the antibody-bound nanoparticles with a substance to be detected that specifically binds to the antibody bound to the antibody-bound nanoparticles (hereinafter, also referred to as “step (i)” and a step of measuring the substance to be detected bound to the antibody-bound nanoparticles (hereinafter also referred to as “step (ii)”).

<Step (i)>
In step (i), the antibody-bound nanoparticles are brought into contact with a substance to be detected that specifically binds to the antibody bound to the antibody-bound nanoparticles.

The antibody-conjugated nanoparticles are the antibody-conjugated nanoparticles of the previous embodiments.
A substance that specifically binds to a specific antibody preferably has a high binding property to the specific antibody, but little binding property to other biomolecules. A substance that specifically binds to a specific antibody can be said to be an antigen of the specific antibody.

A substance to be detected means a substance to be measured by the measurement method of this embodiment. The substance to be detected is not particularly limited as long as it causes an antigen-antibody reaction. Substances to be detected include, but are not limited to, proteins, peptides, sugar chains, lipids, and cells, viruses, and extracellular vesicles expressing the above molecules. Extracellular vesicles are vesicles released by cells. The size of extracellular vesicles is about 30 nm to 1 μm in diameter. Extracellular vesicles include exosomes, apoptotic bodies, microvesicles and the like.

When the substance to be detected is an exosome, the antibody bound to the antibody-bound nanoparticles may be an antibody that specifically binds to the pan-exosomal membrane protein. Alternatively, it may be a membrane protein that exosomes that are the substance to be detected specifically express.

The contact between the antibody-bound nanoparticles and the substance to be detected can be carried out by a known method. For example, a method of mixing and incubating a sample containing a substance to be detected and antibody-bound nanoparticles can be mentioned. A sample containing a substance to be detected is not particularly limited, and can be appropriately selected according to the type of the substance to be detected. When the substance to be detected is a biological substance, examples of samples containing the substance to be detected include body fluid samples and cell culture supernatants.

Incubation conditions can be set appropriately according to the type of substance to be detected. The incubation conditions include, for example, the same conditions as those listed in the section <<Usage Examples of Antibody-Binding Nanoparticles>> above.

Contact between the antibody-binding substance and the substance to be detected may be carried out while the substance to be detected is captured on a solid phase carrier. Capturing of the substance to be detected on the solid phase carrier can be carried out by bringing the substance to be detected into contact with the solid phase carrier bound with a capturing antibody that specifically binds to the substance to be detected, followed by incubation.
Incubation conditions can be appropriately set according to the type of substance to be detected. The incubation conditions include, for example, the same conditions as those listed in the section <<Usage Examples of Antibody-Binding Nanoparticles>> above.

After contact between the solid phase carrier and the substance to be detected, the solid phase carrier may be washed as appropriate. Before the contact between the solid phase carrier and the substance to be detected, the solid phase carrier may be appropriately blocked. After contacting the antibody-bound nanoparticles with the substance to be detected, the antibody-bound nanoparticles may be washed as appropriate. Examples of these methods include the methods described in the section <<Usage Examples of Antibody-Binding Nanoparticles>> above.

<Step (ii)>
In step (ii), the substance to be detected bound to the antibody-bound nanoparticles is measured.

The method of measuring the detection target substance bound to the antibody-bound nanoparticles is not particularly limited. When the antibody-bound nanoparticles are bound to the substance to be detected captured by the solid-phase carrier, the substances to be detected are bound to the substance to be detected in the same manner as the methods listed in the section <<Usage Examples of Antibody-Binding Nanoparticles>> above. can be measured.

Alternatively, the antibody-bound nanoparticles may be collected and contacted with a detection antibody that specifically binds to the substance to be detected contained in the collected antibody-bound nanoparticles. The detection antibody is labeled with a labeling substance. Therefore, the substance to be detected can be measured indirectly by detecting the signal of the labeling substance. As the labeling substance, those commonly used in ELISA and the like can be used without particular limitation. Examples of labeling substances include enzyme labels such as peroxidase (eg, horseradish peroxidase) and alkaline phosphatase; carboxyfluorescein (FAM), 6-carboxy-4',5'-dichloro2',7'-dimethoxyfluorescein (JOE ), fluorescein isothiocyanate (FITC), tetrachlorofluorescein (TET), 5′-hexachloro-fluorescein-CE phosphoramidite (HEX), fluorescent labels such as Cy3, Cy5, Alexa568, Alexa647; radioisotopes such as iodine-125 Labels; electrochemiluminescent labels such as ruthenium complexes; biotin and the like.

　The signal of the labeling substance can be detected by a known method according to the type of labeling substance. For example, in the case of enzyme labeling, the signal of the labeling substance can be detected by performing a coloring reaction with the enzyme and detecting the coloring. When the labeling substance is a fluorescent dye, a radioactive isotope label, or an electrochemically luminescent label, the signal of the labeling substance can be detected by detecting the fluorescent signal, the radioactive signal, or the electrochemically luminescent signal. .

Since the measurement method of the present embodiment uses the antibody-bound nanoparticles of the above embodiment, it is possible to reduce non-specific adsorption to the antibody-bound nanoparticles during the binding reaction between the antibody-bound nanoparticles and the substance to be detected. . Therefore, even when the concentration of the substance to be detected in the sample is low, highly accurate measurement with reduced noise can be performed.

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

[Example 1]
<Preparation of antibody-bound nanoparticles>
Antibody-bound nanoparticles of Example 1 were prepared by the following procedure.
(1) 5.76 g of acetic acid was added to 4.44 g of sodium acetate, and the mixture was made up to 50 mL with pure water to prepare a 3M acetate buffer (pH 4.5).
(2) The 3 M acetate buffer was diluted 60 times to prepare a 50 mM acetate buffer (pH 4.5).
(3) Amicon filter (cutoff molecular weight 10 KDa) (Amicon Ultra- 0.5 mL centrifugal filter, MERCK) and centrifuged at 4° C. and 14000 G for 15 minutes.
(4) The filtrate was discarded, and 50 mM acetate buffer (pH 4.5) was added to make about 500 μL.
(5) Centrifuged at 4°C and 14000G for 15 minutes.
(6) The anti-CD9 antibody concentrated on the filter was recovered by reverse centrifugation at 1000 G for 2 minutes.
(7) 50 μL (1 mg) of COOH beads (TAS8848N1140, bead diameter 180 nm±30 nm, carboxy group surface modification (surface modification amount: about 250 nmol/mg), Tamagawa Seiki) was added to a 1.5 mL Protein LoBind tube (Eppendorf). . It was centrifuged at 21130G for 5 minutes and the supernatant was removed.
(8) The entire amount (about 50 μL) of the anti-CD9 antibody collected in (6) was added to the Protein LoBind tube of (7).
(9) After the COOH beads in the Protein LoBind tube were dispersed by ultrasonic treatment, they were stirred at 37°C for 60 minutes using a tube mixer.
(10) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC/HCl) 2.88 g / polyethylene glycol (PEG) (Mw = 2,000; CE210, JSR Life Science) to 0.5 mL On the other hand, a mixture of 3M acetate buffer at a ratio of 1/59 (about 8.5 μL) to the total amount of EDC·HCl and PEG was prepared immediately before use in (12) described below (composition 30 mM EDC.HCl+PEG in 50 mM acetate buffer (pH 4.5)).
(11) The Protein LoBind tube of (9) was centrifuged at 4° C. and 21130 G for 5 minutes, and the supernatant was removed.
(12) 400 μL of the solution prepared in (10) was added, and the COOH beads were dispersed by ultrasonic treatment, followed by stirring with a tube mixer at room temperature for 180 minutes.
(13) Centrifuged at 21130G at 4°C for 5 minutes and removed the supernatant.
(14) Wash with 200 μL of storage buffer (10 mM HEPES-NaOH (pH 7.9), 1 mM EDTA, 0.1% Tween-20, 0.1% Casein) (addition of washing solution, dispersion by sonication, centrifugation) , and one wash with supernatant removal) were repeated three times.
(15) 500 μL of Storage buffer was added to prepare an anti-CD9 antibody-bound nanoparticle suspension with a final concentration of 2 mg/mL.
(16) Next, the tube containing the anti-CD9 antibody-bound nanoparticle suspension of (15) was incubated at 37°C for 3 days.
(17) After (16), the tubes were stored at 4°C.

<Immobilization of Capture Antibody to Solid Phase Carrier>
A disk attached to ExoCounter (registered trademark) (JVC Kenwood Co., Ltd.) was used as a solid phase carrier. An anti-CD9 antibody was immobilized on the disc as a capture antibody.
Anti-CD9 antibody was dissolved in phosphate-buffered saline to 5 μg/mL and 70 μL was injected into wells fixed on the disc. After incubation at 37° C. for 30 minutes, the anti-CD9 antibody was fixed to the disc by hydrophobic adsorption. After incubation, the discs and wells were washed with buffer.

<Blocking of solid-phase carrier>
Then, 200 μL of 1% BSA dissolved in phosphate-buffered saline was poured into the wells immobilized on the discs and incubated at 37° C. for 30 minutes to block the discs and wells. After incubation, the discs and wells were washed with buffer.

<Detection of exosomes>
A culture supernatant of colon cancer cell line HCT116 was used as a sample. The culture supernatant is phosphate-buffered saline, 0.8 times (Dilution factor = 0.8), 0.6 times (Dilution factor = 0.6), 0.4 times (Dilution factor = 0.4 ) and 0.2 times (Dilution factor = 0.2) to prepare samples of each dilution factor. The culture supernatant was used as a sample with a dilution factor of 1, and the phosphate buffer solution was used as a sample with a blank (dilution factor=0).

50 μL of each sample was injected into the wells on the disk on which the anti-CD9 antibody was immobilized, and incubated at 37° C. for 2 hours. After incubation, wells were washed with buffer.

Then, 1 µg of anti-CD9 antibody-bound nanoparticles prepared as described above were injected into the wells and incubated at 37°C for 1.5 hours. After incubation, the wells were washed and dried.

Then, the wells were separated from the disc, and the number of anti-CD9 antibody-bound nanoparticles bound to the disc surface was counted with an ExoCounter (registered trademark).

[Comparative Example 1-1]
<Preparation of antibody-bound nanoparticles>
Antibody-bound nanoparticles of Comparative Example 1-1 were prepared by the following procedure, partially modified with reference to the guidelines of the COOH bead manufacturer.
(1) Add 50 μL (1 mg) of COOH beads (TAS8848N1140, bead diameter 180 nm±30 nm, carboxy group surface modification (surface modification amount: about 250 nmol/mg), Tamagawa Seiki) to a 1.5 mL Protein LoBind tube (Eppendorf), Centrifugation was performed at room temperature at 21130 G for 5 minutes, and the supernatant was removed.
(2) Next, 400 mM EDC·HCl in 50 mM acetate buffer and 400 mM N-hydroxysuccinimide (NHS) in 50 mM acetate buffer were prepared. 100 μL of each was added to the COOH beads of (1), ultrasonicated to disperse the COOH beads, and incubated at room temperature for 30 minutes using a microtube mixer.
(3) Then, 50 μg of anti-CD9 antibody was additionally added, and further incubated at room temperature for 2 hours.
(4) Centrifugation (4°C, 21130G, 5 minutes) was performed, and the supernatant was discarded.
(5) Next, 800 μL of polyethylene glycol (PEG) (Mw=2,000; CE210, JSR Life Science) was added and incubated overnight at 4° C. using a microtube mixer.
(6) Centrifugation (4°C, 21130G, 5 minutes) was performed, and the supernatant was discarded.
(7) Washing with 200 μL of washing buffer (10 mM HEPES-NaOH (pH 7.9), 1 mM EDTA, 0.1% Tween-20) (washing solution addition, dispersion by ultrasonication, centrifugation, and supernatant removal) 1 wash) was repeated 3 times.
(8) 500 μL of washing buffer was added to obtain an anti-CD9 antibody-bound nanoparticle suspension with a final concentration of 2 mg/mL.
(9) The tube containing the anti-CD9 antibody-bound nanoparticle suspension of (8) was stored at 4°C.

<Immobilization of Capture Antibody to Solid Phase Carrier, Blocking of Solid Phase Carrier>
Immobilization of the capturing antibody to the solid-phase carrier and blocking of the solid-phase carrier were carried out in the same manner as in Example 1 above.

<Detection of exosomes>
Detection of exosomes was performed in the same manner as in Example 1 above, except that the anti-CD9 antibody-bound nanoparticles of Comparative Example 1-1 prepared above were used.

[Comparative Example 2-1]
The antibody-conjugated nanoparticles of Comparative Example 2-1 were prepared according to the COOH bead manufacturer's guidelines. Specifically, antibody-bound nanoparticles of Comparative Example 2-1 were prepared by the following procedure.
(1) Protein immobilization buffer (25 mM morpholinoethanesulfonic acid (MES)-NaOH (pH 6), wash/storage buffer (10 mM HEPES-NaOH (pH 7.9), 50 mM KCl, 1 mM EDTA, 10% glycerol), and masking A solution (1 M aminoethanol, 0.1% Nonidet P-40, adjusted to pH 8.0 with HCl) was prepared.
(2) 28.8 mg of N-hydroxysuccinimide (NHS) was dissolved in 250 µL of N,N'-dimethylformamide (DMF) to prepare a 1M NHS solution.
(3) The anti-CD9 antibody was diluted with a protein immobilization buffer to prepare 50 µL or more of a 50 µg/50 µL antibody solution.
(4) 1 mg of COOH beads (TAS8848N1140) was placed in a 1.5 mL microtube.
(5) Centrifugation (15,000 rpm, room temperature, 5 minutes) was performed, and the supernatant was discarded.
(6) 100 μL of DMF was added and ultrasonicated to disperse the COOH beads.
(7) Centrifugation (15,000 rpm, room temperature, 5 minutes) was performed, and the supernatant was discarded.
(8) (6) and (7) were repeated twice (beads were washed three times in total).
(9) 7.68 mg (40 micromol) of EDC.HCl was weighed into another 1.5 mL microtube.
(10) 160 µL of DMF was added to the COOH beads, and ultrasonication was performed to disperse the COOH beads.
(11) 40 μL of 1 M NHS solution was added and mixed.
(12) 200 μL of the COOH bead-NHS solution was added to the EDC weighed in (9) and dispersed by ultrasonication.
(13) The mixture was allowed to react with a microtube mixer at room temperature for 2 hours.
(14) Centrifugation (15,000 rpm, room temperature, 5 minutes) was performed, and the supernatant was discarded.
(15) 100 μL of DMF was added and ultrasonicated to disperse the beads (COOH-NHS modified beads: hereinafter “NHS beads”).
(16) Centrifugation (15,000 rpm, room temperature, 5 minutes) was performed, and the supernatant was discarded.
(17) (15) and (16) were repeated four times (washing the beads five times in total).
(18) 100 µL of DMF was added and ultrasonicated to disperse the beads (NHS beads). (NHS bead concentration: 1 mg/100 μL)
(19) 1 mg of NHS beads was placed in a 1.5 mL microtube.
(20) Centrifugation (15,000 rpm, 4°C, 5 minutes) was performed, and the supernatant was discarded.
(21) 50 μL of methanol was added, and ultrasonication was performed to disperse the NHS beads with ultrasonic waves.
(22) Centrifugation (15,000 rpm, 4°C, 5 minutes) was performed, and the supernatant was discarded.
(23) 50 μL of protein immobilization buffer was added and ultrasonicated to disperse the beads.
(24) 50 μL of anti-CD9 antibody solution was added.
(25) The mixture was reacted at 4°C for 30 minutes with a microtube mixer.
(26) Centrifugation (15,000 rpm, 4°C, 5 minutes) was performed to collect the supernatant.
(27) 250 μL of masking solution was added to disperse the beads.
(28) The mixture was allowed to react overnight (16 to 20 hours) at 4°C with a microtube mixer.
(29) Centrifugation (15,000 rpm, 4°C, 5 minutes) was performed, and the supernatant was discarded.
(30) Added 200 μL of wash/storage buffer to disperse the beads.
(31) Centrifugation (15,000 rpm, 4°C, 5 minutes) was performed, and the supernatant was discarded.
(32) (30) and (31) were repeated two more times (total of three bead washes).
(33) Dispersed in 200 μL of washing/storage buffer and stored at 4°C.

<Detection of exosomes>
Detection of exosomes was performed in the same manner as in Example 1 above, except that the anti-CD9 antibody-bound nanoparticles of Comparative Example 2-1 prepared above were used.

(result)
The results of Example 1, Comparative Example 1-1, and Comparative Example 2-1 are shown in FIG. 5A. In addition, the count value in the blank (Dilution factor=0) sample is shown in FIG. 5B.
In Example 1, compared to Comparative Examples 1-1 and 2-1, the sensitivity was improved and the blank count value was reduced.

[Example 2]
<Preparation of antibody-bound nanoparticles>
Anti-CD63 antibody-bound nanoparticles of Example 2 were prepared in the same manner as in Example 1 except that anti-CD63 antibodies (exosome monoclonal antibody Anti CD63, Cosmo Bio) were used instead of anti-CD9 antibodies. did.

<Detection of exosomes>
Detection of exosomes was performed in the same manner as in Example 1 above, except that the anti-63 antibody-binding nanoparticles of Example 2 prepared above were used.

[Comparative Example 1-2]
<Preparation of antibody-bound nanoparticles>
Anti-CD63 antibody-bound nanoparticles of Comparative Example 1-2 were prepared in the same manner as in Comparative Example 1-1, except that an anti-CD63 antibody was used instead of the anti-CD9 antibody.

<Detection of exosomes>
Detection of exosomes was prepared in the same manner as in Example 1 above, except that the anti-CD63 antibody-bound nanoparticles of Comparative Example 1-2 were used.

[Comparative Example 2-2]
<Preparation of antibody-bound nanoparticles>
Anti-CD63 antibody-bound nanoparticles of Comparative Example 2-2 were prepared in the same manner as in Comparative Example 1-2, except that an anti-CD63 antibody was used instead of the anti-CD9 antibody.

<Detection of exosomes>
Detection of exosomes was prepared in the same manner as in Example 1 above, except that the anti-CD63 antibody-bound nanoparticles of Comparative Example 2-2 were used.

(result)
The results of Example 2, Comparative Example 1-2, and Comparative Example 2-2 are shown in FIG. 6A. In addition, the count value in the blank (Dilution factor=0) sample is shown in FIG. 6B.
Comparative Example 1-2 had the lowest count value in the blank, but markedly decreased sensitivity. On the other hand, in Example 2, both the sensitivity and blank count value were good.

[Example 3]
<Preparation of antibody-bound nanoparticles>
Anti-Her2 antibody-bound nanoparticles of Example 3 were prepared in the same manner as in Example 1, except that an anti-Her2 antibody (monoclonal antibody Anti Her2, BioLegend) was used instead of the anti-CD9 antibody.

<Detection of exosomes>
Exosomes were detected in the same manner as in Example 1 above, except that the breast cancer cell line KPL4 was used instead of the colon cancer cell line HCT116, and the anti-Her2 antibody-bound nanoparticles of Example 3 prepared above were used. .

[Comparative Example 1-3]
<Preparation of antibody-bound nanoparticles>
Anti-Her2 antibody-bound nanoparticles of Comparative Example 1-3 were prepared in the same manner as in Comparative Example 1-1, except that an anti-Her2 antibody was used instead of the anti-CD9 antibody.

<Detection of exosomes>
Exosomes are detected in the same manner as in Example 1 above, except that the breast cancer cell line KPL4 is used instead of the colon cancer cell line HCT116 and the anti-Her2 antibody-bound nanoparticles of Comparative Example 1-3 prepared above are used. gone.

[Comparative Example 2-3]
<Preparation of antibody-bound nanoparticles>
Anti-Her2 antibody-bound nanoparticles of Comparative Example 2-3 were prepared in the same manner as in Comparative Example 1-2, except that an anti-Her2 antibody was used instead of the anti-CD9 antibody.

<Detection of exosomes>
Exosomes are detected in the same manner as in Example 1 above, except that the breast cancer cell line KPL4 is used instead of the colon cancer cell line HCT116, and the anti-Her2 antibody-bound nanoparticles of Comparative Example 2-3 prepared above are used. gone.

(result)
The results of Example 3, Comparative Examples 1-3, and Comparative Examples 2-3 are shown in FIG. 7A. In addition, the count value in the blank (Dilution factor=0) sample is shown in FIG. 7B.
Comparative Example 1-3 had the lowest count value in the blank, but markedly decreased sensitivity. On the other hand, in Example 3, both the sensitivity and blank count value were good.

[Example 4]
<Preparation of antibody-bound nanoparticles>
Anti-CD147 antibody-bound nanoparticles of Example 4 were prepared in the same manner as in Example 1, except that an anti-CD147 antibody (monoclonal antibody Anti CD147, BioLegend) was used instead of the anti-CD9 antibody.

<Detection of exosomes>
Exosomes were detected in the same manner as in Example 1 above, except that the pancreatic cancer cell line MiaPaca2 was used instead of the colon cancer cell line HCT116 and the anti-CD147 antibody-binding nanoparticles of Example 4 prepared above were used. .

[Comparative Example 1-4]
<Preparation of antibody-bound nanoparticles>
Anti-CD147 antibody-bound nanoparticles of Comparative Example 1-4 were prepared in the same manner as in Comparative Example 1-1, except that an anti-CD147 antibody was used instead of the anti-CD9 antibody.

<Detection of exosomes>
Exosomes are detected in the same manner as in Example 1 above, except that the pancreatic cancer cell line MiaPaca2 is used instead of the colon cancer cell line HCT116, and the anti-CD147 antibody-bound nanoparticles of Comparative Example 1-4 prepared above are used. gone.

[Comparative Example 2-4]
<Preparation of antibody-bound nanoparticles>
Anti-CD147 antibody-bound nanoparticles of Comparative Example 2-4 were prepared in the same manner as in Comparative Example 1-2, except that an anti-CD147 antibody was used instead of the anti-CD9 antibody.

<Detection of exosomes>
Exosomes are detected in the same manner as in Example 1 above, except that the pancreatic cancer cell line MiaPaca2 is used instead of the colon cancer cell line HCT116, and the anti-CD147 antibody-bound nanoparticles of Comparative Example 2-4 prepared above are used. gone.

(result)
The results of Example 4, Comparative Examples 1-4, and Comparative Examples 2-4 are shown in FIG. 8A. In addition, the count value in the blank (Dilution factor=0) sample is shown in FIG. 8B.
In Example 4, compared to Comparative Examples 1-4 and 2-4, the sensitivity was improved and the blank count value was reduced.

FIG. 9 shows the results of calculating the S/N value in each of the above examples (S: count value at Dilution factor=1, N: count value at Dilution factor=0). A higher S/N value indicates better sensitivity and blank count value.
Among all the antibody-bound nanoparticles, the antibody-bound nanoparticles of Examples had the highest S/N value. This result indicates that the antibody-bound nanoparticles of Examples have reduced non-specific adsorption and good sensitivity.

According to the present invention, a method for producing antibody-bound nanoparticles that can reduce nonspecific adsorption, antibody-bound nanoparticles produced by the production method, a kit for producing the antibody-bound nanoparticles, and A method for measuring a substance to be detected using antibody-bound nanoparticles is provided.

1 Nanoparticle 10 Carboxy Group-Modified Nanoparticle 20 Antibody 30 Polyethylene Glycol 40 Blocking Agent 100 Antibody-Binding Nanoparticle

Claims

(a) binding an antibody and polyethylene glycol to the nanoparticles;
(b) binding a blocking agent to the nanoparticles to which the antibody and the polyethylene glycol are bound;
A method for producing antibody-conjugated nanoparticles, comprising:
The step (a) is a step of simultaneously performing a reaction of binding the antibody to the nanoparticles and a reaction of binding the polyethylene glycol to the nanoparticles,
having a pH at which the surface of the nanoparticles and the antibody have opposite potentials;
The step (a) comprises binding the antibody and the polyethylene glycol to the nanoparticles in a buffer solution containing a condensing agent.
A method for producing antibody-bound nanoparticles according to claim 1 .
In the step (b), the nanoparticles bound to the antibody and the polyethylene glycol are incubated in a buffer solution containing the blocking agent under a temperature condition of 30 to 45° C. for 40 to 100 hours. including
3. A method for producing antibody-bound nanoparticles according to claim 1 or 2.
　Antibody-bound nanoparticles in which a blocking agent is bound to nanoparticles bound to antibodies and polyethylene glycol.
nanoparticles;
an antibody;
polyethylene glycol;
a blocking agent;
A kit for producing antibody-bound nanoparticles by the production method according to any one of claims 1 to 3, comprising:
A step of contacting the antibody-bound nanoparticles according to claim 4 with a substance to be detected that specifically binds to the antibody bound to the antibody-bound nanoparticles;
measuring the substance to be detected bound to the antibody-bound nanoparticles;
A method for measuring a substance to be detected using antibody-bound nanoparticles, comprising: