WO2020066336A1 - Detection method and assessment method - Google Patents

Abstract

The present invention provides a method for detecting a protein complex by causing blinking of a fluorescent particle, which does not exhibit blinking, due to energy transfer from a fluorescent particle which exhibits excited blinking. The detection method for a protein complex of a protein (A) and a protein (B) according to the present invention, comprises a step (a) for labeling the protein (A) with an antibody to which a fluorescent particle which exhibits blinking is linked, a step (b) for labeling the protein (B) with an antibody to which a fluorescent particle which does not exhibit blinking is linked, and a step (c) for, after steps (a) and (b), irradiating, with light having an excitation wavelength, the fluorescent particle which exhibits blinking, at the same time, detecting bright points of fluorescence of the emission wavelength of the fluorescent particles which do not exhibit blinking, and extracting a blinking bright point from the bright points of fluorescence of the emission wavelength of the fluorescent particles which do not exhibit blinking.

Description

Detection method and evaluation method

The present invention relates to a detection method and an evaluation method using the detection method.

Although low-molecular-weight drugs have been widely used as anticancer drugs, many antibody drugs with higher stability in plasma, higher selectivity, higher therapeutic effect and fewer side effects have been developed in recent years, Used.

抗体 Trastuzumab (Herceptin (registered trademark); Chugai Pharmaceutical Co., Ltd.) is an antibody drug frequently used as an anticancer drug. Trastuzumab is an antibody drug that specifically binds to the HER2 protein, which has gene amplification and overexpression in many types of cancer.Trastuzumab activates antibody-dependent cytotoxicity by binding to the HER2 protein, It is known to exert an antitumor effect by suppressing a cell proliferation signal.

Pertuzumab (Perjeta (registered trademark); Chugai Pharmaceutical Co., Ltd.), which has been recently developed, is also an antibody drug targeting HER2 targeting cancer. It is well known that HER2 forms a heterodimer with the same family of receptors, HER3. Hereinafter, the heterodimer formed from HER2 and HER3 is also referred to as HER2 / HER3 heterodimer. For example, downstream of the HER2 / HER3 heterodimer, the PI3K / Akt pathway that is mainly involved in cell proliferation, cell cycle progression, and maintenance of cell survival is activated (Non-patent Documents 1 and 2), and pertuzumab has HER2 and HER3. Suppresses the progression of cancer by suppressing the PI3K / Akt signal by strongly inhibiting the dimer formation with lactinib (Non-Patent Document 1), lapatinib (Tykerb (registered trademark); GlaxoSmithKline Co., Ltd.) Reported that inhibiting the dimer formation between HER2 and EGFR and inhibiting the tyrosine kinase activity of the two receptors suppresses downstream signal transduction and suppresses cancer progression (Non-Patent Document 3). Have been. Therefore, the detection of heterodimers including HER2 is considered to be greatly related to the proliferation of cancer cells and the progression or exacerbation of cancer, and the detection of such heterodimers is important in the field of drug discovery. It is.

方法 A method using fluorescence resonance energy transfer (FRET) to detect protein binding and interaction is known. FRET is based on the fact that, when different fluorescent chromophores are present at a distance of about 1 to 10 nm and the fluorescent spectrum of one fluorescent dye overlaps the excitation spectrum of another fluorescent dye, the first excited fluorescent dye (donor fluorescence In the biotechnology field, for example, a donor fluorescent substance or an acceptor fluorescent substance is used as a fluorescent substance to emit light by resonating the electrons of the second fluorescent dye (acceptor fluorescent substance) with the electrons of the second fluorescent dye (acceptor fluorescent substance). These phenomena are detected by binding proteins to each other to detect an interaction such as binding of those proteins.

Specifically, when a donor fluorophore-bound protein bound to a donor fluorophore and an acceptor fluorophore-bound protein bound to an acceptor fluorophore form a complex, the two fluorophores exist at extremely short distances. Therefore, when the donor phosphor is irradiated with excitation light, the acceptor phosphor emits fluorescence, so that the binding of each protein can be determined.

Patent Document 1 describes a method for determining whether a heterodimer containing HER2 is formed by measuring FRET between two fluorescent substances using two kinds of antibodies containing different fluorescent substances. (Patent Document 1).

JP 2014-237667 A

(4) The present inventors have attempted to detect a HER2 / HER3 heterodimer by FRET using two types of fluorescent dyes, and found that the color fading occurred within a few seconds in fluorescence observation, which made imaging difficult.

As an alternative, FRET was performed using quantum dots with high light durability and fluorescent dye-integrated particles instead of fluorescent dyes as the fluorescent substance, but the fluorescent dye-integrated particles were excited by excitation light having a wavelength that excites the quantum dots. In some cases, such non-specific fluorescent dye-integrated particles emit fluorescent light, so that it was not possible to reliably determine whether or not the target heterodimer was formed.

In view of such circumstances, an object of the present invention is to provide a detection method capable of detecting a protein complex such as a heterodimer with high sensitivity, and a method for evaluating a drug using the detection method.

The present inventors have conceived to utilize a phenomenon called blinking, which is observed in quantum dots, in order to solve the above-mentioned problem of emission of nonspecific acceptor fluorescent particles.

Blinking is a phenomenon in which a fluorescent substance repeatedly blinks at time intervals of milliseconds to seconds, and is a phenomenon observed in fluorescent molecules used in one molecule such as fluorescent proteins, organic fluorescent dyes, quantum dots, etc. Non-patent document 4), such flickering is not normally seen in fluorescent dye-integrated particles.

However, the present inventors have found that when fluorescent particles that cause blinking and fluorescent particles that do not cause blinking are used, if both are present at extremely close distances, the fluorescent light that causes excited blinking is generated. It has been found that energy transfer from the luminescent particles may cause the fluorescent particles that do not cause blinking to blink. In addition, the present inventors have completed a protein complex detection method using the phenomenon for detection of a protein complex.

That is, the present invention provides, for example, the following [1] to [12].
[1]
A method for detecting a protein complex comprising a protein (A) and a protein (B),
Step (a) of labeling the protein (A) with an antibody bound to fluorescent particles that cause blinking and labeling the protein (B) with an antibody bound to fluorescent particles that do not cause blinking Step (b),
afterwards,
Simultaneously with irradiating the light of the excitation wavelength of the fluorescent particles that cause the blinking, to detect the fluorescent bright spot of the emission wavelength of the fluorescent particles that do not cause the blinking,
Extracting a flickering luminescent spot from the fluorescent luminescent spots having the emission wavelength of the fluorescent particles that do not cause blinking,
A method for detecting a protein complex, wherein the blinking bright spot represents a complex of the protein (A) and the protein (B).
[2]
The fluorescent particles that cause the blinking are quantum dots,
The fluorescent particles that do not cause blinking are fluorescent dye-integrated particles,
Item 10. The method for detecting a protein complex according to Item 1.
[3]
Item 3. The protein according to item 1 or 2, further comprising a step (d) of calculating the number of proteins (A) not forming a protein complex and the number of proteins (B) not forming a protein complex. Complex detection method.
[4]
Item 4. The method for detecting a protein complex according to any one of Items 1 to 3, wherein the protein complex is a heterodimer comprising a protein (A) and a protein (B).
[5]
5. A method for evaluating a drug effect, comprising a step of performing the method for detecting a protein complex according to any one of Items 1 to 4 before and after addition of a drug to a sample.
[6]
Item 6. The evaluation method according to Item 5, wherein the agent is an agent that inhibits formation of a protein complex.
[7]
Item 7. The evaluation method according to Item 5 or 6, wherein the drug is a heterodimer formation inhibitor.
[8]
Item 8. The evaluation method according to any one of Items 5 to 7, wherein the protein complex is a heterodimer composed of HER2 and HER3.
[9]
Item 9. The evaluation method according to any one of Items 5 to 8, wherein the drug is pertuzumab, trastuzumab, or lapatinib.
[10]
Item 10. The evaluation method according to any one of Items 5 to 9, wherein the quantum dots and the fluorescent dye-integrated particles have different excitation wavelengths.
[11]
Item 5. The excitation wavelength band of the quantum dot, wherein the excitation spectrum intensity per fluorescent dye-integrated particle is 1/10 or less of the excitation spectrum intensity per quantum dot particle. Evaluation method described in.
[12]
Item 5. The excitation wavelength band of the quantum dot, wherein the excitation spectrum intensity per fluorescent dye-integrated particle is 1/100 or less of the excitation spectrum intensity per quantum dot particle. Evaluation method described in.

According to the above-mentioned method, non-specific fluorescence emission of the fluorescent particles that do not cause blinking such as fluorescent dye-integrated particles (fluorescence that does not cause blinking and does not depend on the light of the excitation wavelength of the fluorescent particles that cause blinking) Since non-specific fluorescent emission of the luminescent particles does not blink and can be excluded from the detection target, the target protein complex can be detected with high sensitivity.

According to this means, when the fluorescent particles that do not cause blinking such as the fluorescent dye-integrated particles are flickering, the fluorescent light-emitting particles that cause blinking such as quantum dots and the blinking of the fluorescent dye-integrated particles and the like are used. It can be seen that the fluorescent particles that do not cause kinging are located at such a short distance that energy transfer occurs.This indicates that the antibody to which the fluorescent particles that cause blinking binds to the labeled protein. It is possible to detect with high accuracy a protein complex in which an antibody to which fluorescent light-emitting particles are not bound and a labeled protein are bound.

Specifically, for example, HER2 and HER3 are appropriately combined with an antibody bound to a fluorescent particle that causes blinking and an antibody bound to a fluorescent particle that does not cause blinking to cause blinking. The number of HER2 / HER3 heterodimers formed by irradiating light of the excitation wavelength of the fluorescent particles and observing the flashing fluorescent spots of the fluorescent wavelength of the fluorescent particles that do not cause blinking. Can be measured with high accuracy.

Furthermore, in the fluorescence observation as described above, when the cell is irradiated with excitation light, what is called autofluorescence, which is nonspecific fluorescence from a substance present in the cell, may be detected. Also, since it does not flicker, higher detection accuracy of the target protein complex can be expected.

In addition, the effect of the drug can be evaluated by using the method. For example, as described above, the formation of a HER2 / HER3 heterodimer is greatly involved in the growth of cancer cells and the like. Therefore, by observing changes in the number of heterodimers before and after a candidate drug for an anticancer drug using a tumor-derived cultured cell or an experimental animal such as a tumor-bearing animal, the candidate drug becomes HER2 / HER3. It is possible to determine whether or not the formation of heterodimers can be inhibited, and thus it is possible to predict and evaluate the efficacy of the candidate drug against cancer.

FIG. 1 (A) is a schematic diagram of HER2 labeled in Example 1 with a quantum dot-conjugated anti-HER2 mouse monoclonal antibody.

FIG. 1B is a schematic view of a label which is one embodiment of the present invention. In FIG. 1B, an anti-HER2 mouse antibody (having no quantum dots) is bound to HER2, FIG. 4 is a schematic diagram of HER2 labeled with a quantum dot-conjugated anti-mouse IgG antibody prepared in the same manner as in Preparation Example 1 instead of the anti-HER2 antibody.
FIG. 2 (A) is a schematic diagram of HER3 prepared in Preparation Example 2 and labeled with a fluorescent dye-integrated particle-conjugated anti-HER3 mouse monoclonal antibody. FIG. 2 (B) is a schematic diagram of a label which is one embodiment of the present invention. An anti-HER3 mouse monoclonal antibody (without fluorescent dye-incorporated particles) was bound to HER3, and further produced in Preparation Example 2. FIG. 7 is a schematic diagram of HER3 labeled with a fluorescent dye-integrated particle-conjugated anti-mouse IgG antibody prepared in the same manner as in Preparation Example 2 instead of the fluorescent dye-incorporated particle-bound anti-HER3 mouse monoclonal antibody. FIG. 3 is a schematic diagram of a heterodimer comprising HER2 labeled with a quantum dot-conjugated anti-HER2 mouse monoclonal antibody and HER3 labeled with a fluorescent dye-collected particle-conjugated anti-HER3 mouse monoclonal antibody, and detection of the heterodimer.

In the “detection method” of the present invention, a protein complex consisting of the protein (A) and the protein (B) is targeted for detection.
<Detection method>
(Steps (a) and (b))
In the detection method according to the present invention, the step (a) of labeling the protein (A) with an antibody bound to fluorescent particles that cause blinking, and the step of labeling the protein (B) with fluorescent particles that do not cause blinking (B) labeling with the bound antibody. In the steps (a) and (b), the step (a) may be performed first, the step (b) may be performed first, or the steps (a) and (b) may be performed simultaneously. Is also good.

In the steps (a) and (b), specifically, for example, an antibody bound to fluorescent particles that cause blinking and an antibody bound to fluorescent particles that do not cause blinking are dispersed in a diluent. It can be performed by adding a staining solution to a specimen (cultured cells, tissue sections, and the like).

量子 Typically, quantum dots are selected as the fluorescent particles that cause blinking, and fluorescent dye-integrated particles are typically selected as the fluorescent particles that do not cause blinking.

Therefore, preferably, in the detection method of the present invention, the step (a ′) of labeling the protein (A) with a quantum dot-bound antibody and the step (b ′) of labeling the protein (B) with a fluorescent dye-incorporated particle-bound antibody are preferred. Including. In the following description, the case will be mainly described where the fluorescent particles that cause blinking are quantum dots, and the fluorescent particles that do not cause blinking are fluorescent dye-integrated particles.

The quantum dot-bound antibody is obtained by directly or indirectly binding an antibody that specifically recognizes the protein (A) to a quantum dot having a desired excitation wavelength. An antibody in which an antibody that specifically recognizes the protein (B) is directly or indirectly bound to a fluorescent dye-integrated particle having a desired excitation wavelength.

In the present invention, the reason why the non-blinking fluorescent particles flicker is not clear, but the present inventors have found that the non-blinking fluorescent particles do not cause the excited blinking fluorescent particles. It was speculated that energy transfer to the particles was the main cause.

That is, in the present invention, the fluorescent particles that cause blinking such as quantum dots, together with the blinking phenomenon that is caused by being excited by irradiating light of the excitation wavelength of the particles, together with the fluorescent light that does not cause blinking It is considered that since energy transfer occurs to the particles, flickering of the fluorescent particles that does not cause blinking is detected.

Therefore, it is necessary to select a combination in which the emission wavelength of the quantum dot is a wavelength that can excite the fluorescent dye-integrated particles.

量子 Examples of the quantum dots and fluorescent dye-integrated particles that can be used in the present invention include those described in the section of <quantum dots> and <fluorescent dye-integrated particles> below.

As a combination of the quantum dot and the fluorescent dye-integrated particles suitable for the present invention, (particle dot-fluorescent dye-integrated particle), specifically, Qdot (registered trademark) 585 (ThemoFisher @ Scientific) -Texas Red, Qdot @ 585 -Perylenediimide, Qdot @ 525-AlexaFluor532, Qdot @ 545-Cy3, Qdot @ 565-AlexaFluor568, Qdot @ 625-Cy5, Qdot@655-Cy5.5 and the like.

量子 The quantum dots and the fluorescent dye-integrated particles preferably have different excitation wavelengths. The excitation wavelength band of the quantum dot is preferably an excitation wavelength band in which the excitation spectrum intensity per fluorescent dye-integrated particle is 1/10 or less of the excitation spectrum intensity per quantum dot particle. An excitation wavelength band of 1 or less is particularly preferred.

The excitation wavelength band of the fluorescent dye-integrated particles is preferably an excitation wavelength band in which the excitation spectrum intensity per quantum dot particle is one-tenth or less of the excitation spectrum intensity per the fluorescent dye-integrated particle. , And an excitation wavelength band that is 1/100 or less is particularly preferable. Further, the quantum dots and the fluorescent dye-integrated particles preferably have different emission wavelengths, respectively, and the difference between the emission wavelengths is preferably from 10 nm to 40 nm, particularly preferably from 30 nm to 40 nm. .

抗体 An antibody that specifically recognizes the protein (A) and an antibody that specifically recognizes the protein (B) are not particularly limited, but it is preferable to select an antibody that does not affect the formation of the protein complex. When the protein (A) and the protein (B) are the same protein, the antibody that specifically recognizes the protein (A) and the antibody that specifically recognizes the protein (B) are different from each other. An antibody that recognizes an epitope is preferred. In addition, the antibody used for each is preferably a monoclonal antibody. The type of animal that produces the antibody (immunized animal) is not particularly limited, and may be selected from mice, rats, guinea pigs, rabbits, goats, sheep, and the like, as in the related art. For example, it is preferable to use an anti-HER2 mouse monoclonal antibody when HER2 is selected as the protein (A) and an anti-HER3 mouse monoclonal antibody when HER3 is selected as the protein (B).

Furthermore, antibody fragments or antibody derivatives such as chimeric antibodies (humanized antibodies, etc.) and multifunctional antibodies are used, as long as they have the ability to specifically recognize and bind to the target protein, instead of natural full-length antibodies. You can also.

In the quantum dot-bound antibody and the fluorescent dye-bound particle-bound antibody, the mode of binding between the quantum dot or the fluorescent dye-bound particle and the antibody is not particularly limited, and the quantum dot or the fluorescent dye-bound particle and the antibody are directly Examples of the mode of bonding to a compound include a covalent bond, an ionic bond, a hydrogen bond, a coordination bond, physical adsorption, and chemical adsorption. Further, if a product in which a desired quantum dot or a fluorescent dye-integrated particle is bound to a desired antibody in advance is commercially available, it may be used, or may be prepared based on a known method. Good.

The mode in which the quantum dot or the fluorescent dye-integrated particle and the antibody are indirectly bound may be, for example, bound in a known manner via avidins and biotin, or bound via a linker molecule. May be.

The quantum dot-bound antibody is obtained by binding a secondary antibody that specifically recognizes the primary antibody to an antibody (so-called primary antibody) that specifically recognizes the protein (A), and further binding the quantum dot. There may be.

Further, the fluorescent dye-aggregated particle-bound antibody is obtained by binding a secondary antibody specifically recognizing the primary antibody to an antibody (so-called primary antibody) that specifically recognizes the protein (B). May be combined.

When the quantum dot-bound antibody is in a form in which the secondary antibody is bound to the primary antibody and the quantum dot is further bound, the fluorescent dye-integrated particle-bound antibody is bound to the primary antibody and the secondary antibody, and It is preferable that the fluorescent dye-incorporated particles are bound, but at this time, the primary antibody in the quantum dot-bound antibody and the primary antibody in the fluorescent dye-bound particle-bound antibody are preferably derived from different immunized animals. . When using a primary antibody derived from the same immunized animal, it is necessary to use an antibody that recognizes a different epitope for the quantum dot-bound antibody and the secondary antibody in the fluorescent dye-bound particle-bound antibody, respectively, in order to prevent cross-reactivity. .
(Step (c))
In the present invention, after performing the above-mentioned steps (a) and (b), at the same time as irradiating light of the excitation wavelength of the fluorescent particles which cause blinking of the quantum dots or the like, blinking of the fluorescent dye-integrated particles or the like is performed. The step (c) of detecting, by a microscope, a fluorescent bright point having an emission wavelength of the fluorescent light-emitting particles which does not cause the phenomenon, and extracting a blinking bright point from the fluorescent bright points having the emission wavelength of the fluorescent dye-integrated particles.

The microscope for detecting the fluorescent bright spot is not particularly limited as long as it can detect fluorescence, so-called "fluorescence microscope", for example, epi-fluorescence microscope, confocal laser microscope, total reflection illumination fluorescence microscope, Examples include a transmission fluorescence microscope, a multiphoton excitation microscope, and a structured illumination microscope.

滅 The flickering bright spots extracted in the step (c) represent a protein complex in which the protein (A) and the protein (B) are bound. Therefore, the amount of the protein complex can be quantified by extracting the blinking bright points and measuring the number of the bright points.

The blinking interval at the bright spot of the fluorescent dye-integrated particles depends on the blinking interval of the quantum dot, but is usually 1/1000 sec to 1 sec, and from the viewpoint of easy detection, it is 1/4 sec to 1 sec. An interval of seconds is used.

The excitation light is radiated by, for example, a light source such as an ultra-high pressure mercury lamp, a Xe lamp, an LED (light emitting diode), a laser device, or the like provided in a fluorescence microscope, and an excitation light optic that selectively transmits a predetermined wavelength as necessary. Irradiation can be performed by using a filter.

{The fluorescent bright point} can be acquired as a fluorescent image by, for example, capturing an image with a device capable of capturing a fluorescent image, for example, a digital camera provided in a fluorescent microscope.

At the time of photographing, by using an optical filter for fluorescence that selectively transmits a predetermined wavelength as necessary, excitation light that includes only fluorescence of a target wavelength, and becomes undesired fluorescence or noise, and other light. A fluorescent image excluding the light can be taken.

(4) The fluorescent image may be a moving image or a plurality of fluorescent images acquired successively from a plurality of still images (time-lapse photography). Further, the time-lapsed image may be converted into a moving image by using known software. The number of frames per second when performing moving image shooting and the shooting interval when performing time-lapse shooting can be arbitrarily adjusted in accordance with the flashing interval of the fluorescent dye-integrated particles.

蛍 光 The acquired fluorescent image is preferably converted as a digital image, and may be processed or image-processed by a known means.

Software that can be used for image processing includes, for example, “ImageJ” (open source). By using such image processing software, from a fluorescent image, detection of a luminescent point of a predetermined wavelength, extraction of a blinking luminescent point, measurement of the number of luminescent points of a predetermined luminance or more, and the like, as desired. The process of superimposing a plurality of images, the process of counting the number of cells included in the images, and the like can be performed semi-automatically and quickly.
(Step (d))
Further, the number of quantum dots, that is, the total number of proteins (A) can be measured by irradiating the excitation wavelength of the quantum dots and simultaneously detecting the emission wavelength of the quantum dots. In addition, the total number of proteins (B) can be detected by measuring the emission wavelength of the fluorescent integrated particles while irradiating the excitation wavelength of the fluorescent integrated particles. Therefore, by performing these detections, the number of proteins (A) not forming a protein complex, the number of proteins (B) not forming a protein complex, and the number of proteins (A) and protein (B) Information on the number of protein complexes consisting of

Specifically, by subtracting the number of the protein complexes from the total number of the proteins (A), the number of the proteins (A) not forming the protein complexes can be calculated, and the number of the proteins (B) can be calculated. By subtracting the protein complex from the total number, the number of proteins (B) that do not form a protein complex can be calculated.

By this method, for example, the ratio of the protein (A) forming the protein complex is calculated by dividing the number of proteins (A) forming the protein complex by the total number of proteins (A). The ratio of the protein (B) forming the protein complex can be calculated in the same manner.

When the protein (A) and the protein (B) are the same protein and the protein complex is a homodimer as described later, the protein complex is formed by using the same method as described above. The number of the protein (A) (protein (B)) and the number of unformed proteins can be calculated.

染色 If necessary, staining for bright field observation can be performed. The staining method for bright field observation is not particularly limited, but typically, hematoxylin and eosin staining (HE staining) is performed. When a morphological observation staining step is included, it is preferably performed after the steps (a) and (b). When the morphological observation staining step is performed, for example, information such as the number of protein complexes per cell and the localization of the protein complexes in the specimen tissue can be obtained. The amount of the protein complex quantified by extracting the number of blinking bright spots in the step (c) may be determined as, for example, the average number of protein complexes per cell, or the fluorescence used for the detection. The average number per unit area of the image may be obtained.

If necessary, nuclear staining of cells contained in the tissue section can be performed. The method for staining the nucleus is not particularly limited, but it is preferable to perform fluorescent staining using a fluorescent substance having a wavelength characteristic different from the excitation wavelength and emission wavelength of the quantum dots and fluorescent integrated particles. Although such a fluorescent substance is not particularly limited, DAPI (4 ′) is used in view of easiness of staining treatment and handling, and strong distinction of nucleus by emitting strong fluorescence by strongly binding to nuclear DNA. , 6-diamidino-2-phenylindole) is preferably used.
<Protein complex>
The protein complex detected in the present invention is a protein (A) and a protein (B) which form a complex by an arbitrary bonding mode (hydrogen bond, intermolecular force, etc.). The protein (A) and the protein (B) may be the same or different, but are preferably different proteins. Further, it is more preferable that each is a protein that binds in a cell (including on the cell membrane), and it is more preferable that each is a protein involved in signal transduction by binding in a cell to form a complex. . Typically, the protein complex is a heterodimer consisting of protein (A) and protein (B). The location where the protein (A) and the protein (B) are expressed is not particularly limited, but those that are expressed on the cell membrane of a cancer cell are typically selected.

The protein complex comprising the protein (A) and the protein (B) is, for example, a combination that can form a complex (preferably a dimer) between HER families, more specifically, HER2 / HER3, HER2 / HER2, HER2 / HER4, HER3 / HER4, HER4 / HER4, HER2 / CD74, ERα / ERβ, ERα / ERα, Oct4 / Smad3, Integrin αV / β6, HOX / PBX, EGFR / HER2, EGFR / EGFR, EGFR / HER4, VEGFR-1 It is preferably selected from protein complexes such as / VEGFR-1, VEGFR-2 / VEGFR-2, VEGFR-3 / VEGFR-3, PDGFR / c-kit.

Among them, it is more preferable that the protein (A) and the protein (B) are different heterodimers, and a heterodimer composed of HER2 and HER3 is particularly preferable.
<Quantum dot>
Quantum dots (also referred to as semiconductor nanoparticles) are nano-sized particles made of a semiconductor that emit fluorescence when irradiated with light, and usually have a particle size of 0.5 to 100 nm, preferably 0.5 to 50 nm, more preferably 0.5 to 50 nm. Preferably it is 1 to 10 nm.

Examples of the quantum dots used in the present invention include those containing a II-VI compound, a III-V compound, or a IV element. Specifically, CdSe, CdS, CdTe, ZnSe, ZnS , ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, Ge, and the like.

The quantum dot may be a quantum dot having a core / shell structure, for example, quantum dots such as CdSe / ZnSe (core / shell), InP / ZnS (core / shell), and InGaP / ZnS (core / shell). Dots can be used. Specific examples of such quantum dots include, for example, Qdot (registered trademark) 585, Qdot565, Qdot605, Qdot655, Qdot705, and Qdot800 manufactured by Thermo Fisher Scientific.

The excitation wavelength and emission wavelength of the quantum dot are not particularly limited, but it is preferable to select a quantum dot different from the excitation wavelength and emission wavelength of the fluorescent dye-integrated particles described later.
<Fluorescent dye integrated particles>
The fluorescent dye-accumulated particles used in the present invention have a structure in which a plurality of fluorescent dyes are accumulated (encapsulated) inside and / or adsorbed on the surface of a mother body made of an organic or inorganic substance. Having nano-sized particles. When the fluorescent dye is included in the base, the fluorescent substance may be dispersed in the base, and may or may not be chemically bonded to the base itself. For example, the base (for example, resin) and the fluorescent dye may be bonded by electrostatic interaction or may be covalently bonded.

励 起 The excitation wavelength and emission wavelength of the fluorescent dye-integrated particles are not particularly limited, but it is preferable to select fluorescent dye-integrated particles different from the excitation wavelength and the emission wavelength of the quantum dots.

In the present specification, the “fluorescent dye” is irradiated with an electromagnetic wave (X-ray, ultraviolet light, or visible light) having a predetermined wavelength and absorbs its energy to excite electrons, and returns from the excited state to the ground state. In this case, an organic fluorescent dye that is a substance that emits excess energy as an electromagnetic wave, that is, emits “fluorescence”, and is an organic fluorescent compound having a carbon skeleton is preferable. In addition, “fluorescence” has a broad meaning, and includes phosphorescence having a long luminescence lifetime in which luminescence continues even when irradiation of electromagnetic waves for excitation is stopped, and fluorescence in a narrow sense having a short luminescence lifetime.

Examples of the fluorescent dye include a rhodamine dye, a squarylium dye, a cyanine dye, an aromatic ring dye, an oxazine dye, a carbopironine dye, and a pyromesene dye. Specifically, Alexa Fluor (registered trademark) , (Invitrogen), BODIPY (registered trademark, manufactured by Invitrogen), Cy (registered trademark, manufactured by GE Healthcare), DY (registered trademark, manufactured by DYOMICS), HiLite (registered trademark) , Anaspec), DyLight (registered trademark, manufactured by Thermo Scientific), ATTO (registered trademark, manufactured by ATTO-TEC), MFP (registered trademark, manufactured by Mobitec), etc. Can be used. In addition, such dyes are generically named based on the main structure (skeleton) or registered trademark in the compound, and the range of the fluorescent dye belonging to each dye can be determined by those skilled in the art without undue trial and error. It can be properly grasped.

Among the matrixes that form the fluorescent dye-integrated particles, organic substances include resins generally classified as thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin; styrene. Resins generally classified as thermoplastic resins such as resin, acrylic resin, acrylonitrile resin, AS resin (acrylonitrile-styrene copolymer), ASA resin (acrylonitrile-styrene-methyl acrylate copolymer); polylactic acid, etc. Other resins; polysaccharides can be exemplified, and inorganic substances include silica, glass and the like.

Fluorescent dye-integrated particles can be produced by a known method for producing desired fluorescent dye-integrated particles, for example, according to the method described in JP-A-2013-57937. Specifically, for example, a fluorescent dye-integrated particle having silica as a base, a solution in which a fluorescent dye and a silica precursor are dissolved is dropped into a solution in which ethanol and ammonia are dissolved, and the silica precursor Can be produced by hydrolyzing the resin, and the fluorescent dye-integrated resin particles having a resin as a matrix are prepared in advance by preparing a solution of the resin or a dispersion of fine particles, and adding a fluorescent dye thereto. It can be prepared by stirring.

The average particle size of the fluorescent dye-integrated particles is not particularly limited as long as it is a particle size suitable for fluorescent staining, but is usually 10 to 500 nm, preferably 50 to 200 nm in consideration of easy detection as a bright spot. It is. Further, the coefficient of variation indicating the variation of the particle diameter is usually 20% or less, and preferably 5 to 15%. The fluorescent dye-integrated particles satisfying such conditions can be manufactured by adjusting the manufacturing conditions. For example, when producing fluorescent dye-integrated particles by an emulsion polymerization method, the particle size can be adjusted by the amount of surfactant added.

It is preferable that the particle diameters of the quantum dots and the fluorescent dye-integrated particles be determined by taking an electron micrograph using a scanning electron microscope (SEM) or a transmission electron microscope (TEM). The average particle diameter and the coefficient of variation of a group consisting of a plurality of phosphor-integrated particles are calculated as described above for a sufficient number (for example, 1000) of phosphor-integrated particles. It is calculated as an average, and the coefficient of variation is calculated by the formula: 100 × standard deviation of particle size / average particle size.
<Method of evaluating drug effect>
The method for evaluating a drug effect of the present invention includes a step of performing the above-described method for detecting a protein complex before and after addition of a drug to a specimen (cultured cells, tissue slices, or the like) at each time point. . As the specimen, for example, when the drug is an anticancer agent, a tissue section collected from an experimental animal such as a tumor-bearing animal or a cultured cell derived from a tumor tissue can be used.

By using this method, it is possible to observe the amount of the protein complex before and after the addition of the drug and the change in the ratio of the protein (A) and the protein (B) forming the protein complex. The effect of the drug on the formation can be evaluated.

The agent that affects the formation of the protein complex is preferably an agent that inhibits the formation of the protein complex. When the protein complex is a heterodimer, the agent is preferably a heterodimer formation inhibitor. More preferably, for example, it is particularly preferable to be a heterodimer formation inhibitor composed of HER2 and HER3, and most preferably, the drug is pertuzumab, lapatinib, or afatinib.

When the protein complex is a homodimer, it is preferably a homodimer formation inhibitor, and the drug is trastuzumab (trade name: Herceptin (registered trademark), Chugai Pharmaceutical Co., Ltd.), cetuximab (trade name: Arbitux) (Registered trademark), Bristol-Myers @ Squibb) and panitumumab (trade name: Vectibix (registered trademark), Amgen).

効果 The effect of the drug can be evaluated by calculating the number of proteins (A) (protein (B)) forming a protein complex before and after the addition of the drug and the number of the proteins not forming the protein complex.

Further, by observing a change in the amount of the protein complex before and after administration of the candidate drug for cancer treatment, it can be determined whether or not the candidate drug can inhibit the formation of the protein complex. The efficacy of a candidate drug can be predicted and evaluated.

Next, the present invention will be described in more detail with reference to experimental examples, but the present invention is not limited thereto.

[Production Example 1]
<Quantum dot-conjugated anti-HER2 antibody>
An anti-HER2 mouse monoclonal antibody (Invitrogen; product number 3B5) was labeled with biotin using a biotin labeling kit (Biotin Labeling Kit-NH2; Dojindo Laboratories).

Qdot (registered trademark) 585 @ streptavidin conjugate (Invitrogen) (2 nM) is mixed with the biotin-labeled anti-HER2 antibody (20 μg / mL), and quantum dots (Qdot (registered trademark) 585; excitation wavelength 365 nm, emission wavelength 585 nm) Was bound to obtain an anti-HER2 antibody. In the following experimental examples, the obtained quantum dot-conjugated anti-HER2 antibody was obtained by diluting a diluent Power Block (10 × Concentrated) (BIOGENEX LABORATORIES, INC; Code No.B-HK0855) ten times with pure water. It was used.

[Production Example 2]
<Anti-HER3 antibody conjugated with fluorescent dye particles>
(Biotin-labeled antibody)
An anti-HER3 mouse monoclonal antibody (Santa Cruz Biotechnology, Inc .; product number 5A12) was labeled with biotin using a biotin labeling kit (Biotin Labeling Kit-NH2; Dojindo Laboratories).
(Streptavidin-labeled antibody fluorescent dye integrated particles)
The fluorescent dye-integrated particles were prepared by dissolving 2.5 mg of Texas Red (registered trademark) dye “Sulforhodamine 101: excitation wavelength 595 nm, emission wavelength 615 nm” (Sigma-Aldrich) in 22.5 mL of pure water, and then using a hot stirrer. The solution was stirred for 20 minutes while maintaining the temperature at 70 ° C. To the solution after stirring, 1.5 g of a melamine resin "Nikarac MX-035" (Nippon Carbide Industry Co., Ltd.) was added, and the mixture was further heated and stirred under the same conditions for 5 minutes. 100 μL of formic acid was added to the solution after stirring, and the solution was stirred for 20 minutes while maintaining the temperature of the solution at 60 ° C. Then, the solution was allowed to cool to room temperature. The cooled solution was dispensed into a plurality of centrifuge tubes and centrifuged at 12,000 rpm for 20 minutes to precipitate the Texas Red-integrated melamine resin particles (hereinafter, fluorescent dye-incorporated particles) contained in the solution. . The supernatant was removed, and the precipitated fluorescent dye-collected particles were washed with ethanol and water. SEM observation was performed on 1000 of the obtained fluorescent dye-integrated particles, and the average particle size was measured. The average particle size was 80 nm.

(4) 0.1 mg of the fluorescent dye-integrated particles were dispersed in 1.5 mL of EtOH, and 2 μL of aminopropyltrimethoxysilane “LS-3150” (Shin-Etsu Chemical Co., Ltd.) was added, followed by a reaction for 8 hours to perform a surface amination treatment.

Next, the surface-aminated particles were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM EDTA (ethylenediaminetetraacetic acid), and the solution was adjusted to a final concentration of 10 mM. SM (PEG) ₁₂ (Thermo Scientific, succinimidyl-[(N-maleimidopropionamido) -dodecaethyleneglycol] ester) was mixed and reacted for 1 hour. This mixture was centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. Then, PBS containing 2 mM of EDTA was added to disperse the precipitate, and the mixture was centrifuged again. The same procedure was repeated three times to obtain maleimide-modified fluorescent dye-integrated particles.

On the other hand, streptavidin (Wako Pure Chemical Industries, Ltd.) is subjected to a thiol group addition treatment using N-succinimidyl S-acetylthioacetate (SATA), followed by filtration through a gel filtration column to obtain maleimide-modified fluorescent dye-integrated particles. A bondable streptavidin solution to which a thiol group was added was obtained.

(4) The maleimide-modified fluorescent dye-integrated particles and streptavidin to which a thiol group had been added by the above treatment were mixed in PBS containing 2 mM of EDTA, and reacted at room temperature for 1 hour. Thereafter, 10 mM mercaptoethanol was added to stop the reaction. After concentrating the obtained mixture with a centrifugal filter, unreacted streptavidin and the like were removed using a gel filtration column for purification to obtain streptavidin-modified fluorescent dye-integrated particles.

(4) The biotin-labeled anti-HER3 antibody (20 μg / mL) and the streptavidin-modified fluorescent dye-integrated particles (2 nM) were mixed to obtain an anti-HER3 antibody to which the fluorescent dye-integrated particles were bound. The obtained anti-HER3 antibody bound to the fluorescent dye-aggregated particles was diluted in the same manner as in Preparation Example 1 and used in the following experimental examples.

[Experimental example 1]
1.0 × 10 ⁷ human breast cancer-derived cultured cells (cell name AU565; ATCC (registered trademark) NO. CRL-235; Sumisho Pharma International Co., Ltd.) are suspended in a 4% paraformaldehyde solution and reacted at room temperature for 10 minutes. After immobilization, a cell block embedded in an alginate gel was prepared. AU565 is known to be a cell expressing both HER2 and HER3 (HER2 positive; HER3 positive).

The prepared cell block was cut into a paraffin section having a thickness of 3 μm, and the quantum dot-bound anti-HER2 antibody prepared in Preparation Example 1 and the fluorescent dye-integrated particle-bound anti-HER3 antibody prepared in Preparation Example 2 were brought to a final concentration of 10%. Was added to the cells and left at 4 ° C. for 8 hours for staining.

Using a system biological erecting microscope BX53 (Olympus Corporation) equipped with an incubator (Tokai Hit Co., Ltd.) on the microscope stage, at 37 ° C, 5% CO ₂ environment, a mercury lamp and an optical filter for excitation light (stock Irradiate the cells with ultraviolet light with an excitation wavelength of 370 / DF36 (352 to 388 nm) (exposure time 250 ms, ND25, gain x 8) by the company Optoline "QDLP-C Single Band Fluorescence Filter Set". Then, fluorescence observation was performed.

The excitation wavelength is a wavelength at which Qdot (registered trademark) 585 is excited, and a wavelength at which Texas Red of the fluorescent dye-integrated particles is not excited.

In the monochrome mode of a digital camera for microscope (DP80: Olympus Corporation), using a 100 × oil immersion lens, using a resonant scanner with a resolution of 512 × 512, a pixel size of 64.5 nm / pixel, and a Line Average set to 16, Time-lapse photography was performed by continuously photographing for 60 seconds at an exposure time of 250 msec and a scan speed of 400 msec / 1 frame. When the average blink frequency of Qdot (registered trademark) 585 was measured from the result of the photographing, it was 0.5 seconds / time.

Next, at the same excitation wavelength (352 to 388 nm), the fluorescent luminescent spot derived from the fluorescent dye-integrated particles (Texas Red) was measured under the same conditions as in the imaging of Qdot (registered trademark) 585 (exposure time 250 ms, 400 ms / Time-lapse photography was performed at a scan speed of one frame for 60 seconds (continuous photography), and photography was performed using an optical filter for fluorescence (558 to 584 nm).

Using the image analysis software ImageJ (open source), the number of cells in one screen is visually determined from the fluorescence derived from the autofluorescence of the cells using the image analysis software ImageJ (open source). The number of all bright spots in one screen and the number of only bright spots blinking from the bright spots were measured. Furthermore, the number of bright spots per cell was calculated by dividing the number of blinking bright spots derived from the fluorescent dye-accumulated particles in one screen by the number of cells in one screen. Table 1 shows the results.

In the extraction of the blinking bright points, only the bright points that emit light at the time of blinking and have a luminance of a predetermined value or more are extracted and measured. In addition, a signal whose luminance or magnitude was larger than a certain value (eg, the average value of the observed phosphor-integrated particles) was determined to be an aggregated bright spot. The extraction and measurement of the number of the blinking bright points and the measurement of the number of the bright points included in the aggregated bright points can be performed by using the image analysis software ImageJ.

平均 The average blinking frequency of the fluorescent dye-integrated particles (Texas Red) was measured from the results of the above photographing and found to be 0.5 seconds / time. This coincides with the blink interval of Qdot (registered trademark) 585. From this, the energy of the fluorescent emission of Qdot (registered trademark) 585 is transferred to the fluorescent dye-integrated particles, so that the blinking of the fluorescent dye-integrated particles occurs. Suggests that

[Experimental example 2]
Human ovarian cancer-derived cultured cells (cell name SKOV3; ATCC (registered trademark) NO. HTB-77; Sumisho Pharma International Co., Ltd.) were cultured, and staining and detection of blinking spots were performed in the same manner as in Experimental Example 1. . It is known that SKOV3 expresses HER2 while HER3 is a cell that expresses only a small amount. As in Experimental Example 1, the number of bright spots per cell was determined by dividing the number of blinking bright spots derived from the fluorescent dye-integrated particles in one screen by the number of cells in one screen. Table 1 shows the results.

[Experimental example 3]
Human ovarian cancer-derived cultured cells (cell name: MCF7; ATCC (registered trademark) NO. HTB-22; Sumisho Pharma International Co., Ltd.) were cultured, and staining and detection of blinking spots were performed in the same manner as in Experimental Example 1. . It is known that MCF7 hardly expresses HER2, while HER3 expresses HER2.

As in Experimental Example 1, one cell was obtained by dividing the number of blinking points derived from the fluorescent dye-accumulated particles in one screen by the number of cells in one screen visually determined from the fluorescence derived from the autofluorescence of the cells. The number of bright spots per was obtained. Table 1 shows the results.

*: Fluorescent dye-integrated particles that flicker at excitation wavelengths of 352 to 388 nm (Texas Red)
**: Total number of bright spots = sum of fluorescent dye-integrated particles flickering at excitation wavelength of 352 to 388 nm and fluorescent dye-integrating particles not flickering (discussion)
AU565 cells of Experimental Example 1 express both HER2 and HER3. Among them, those whose bright spots are blinking under the condition of the excitation wavelength of 352 to 388 nm are the fluorescent dye-integrated particles (HER3) blinking under the influence of the fluorescent emission of the quantum dots (HER2), that is, the HER2 / HER3 dimer. Is presumed to be formed.

Therefore, the non-blinking fluorescent dye-integrated particles (HER3; 550-400 = 150) observed in Experimental Example 1 are the fluorescent dye-integrated particles non-specifically excited by the excitation wavelength of the quantum dots. It can be inferred that HER3 of the fluorescent dye-integrated particles does not form a dimer with HER2.

That is, about 27% of the bright spots observed under the condition of 352 to 388 nm, which are not originally the excitation wavelength for the fluorescent dye-integrated particles, are not based on the formation of the HER2 / HER3 dimer.

Next, the SKOV3 cell of Experimental Example 2 has a smaller HER3 expression level than the AU565 cell, so that 45 luminescent spots derived from the fluorescent dye-integrated particles under the conditions of the excitation wavelength of 352 to 388 nm are 45 and the AU565 cell It was small compared to. Note that “45” is the sum of the blinking fluorescent dye-integrated particles and the non-specifically excited fluorescent dye-integrated particles at the wavelength of 370 nm, which is the excitation wavelength of the quantum dots. It was also found that there were 25 blinking bright spots, and at least 20 bright spots were not based on the formation of the HER2 / HER3 dimer. That is, it can be understood that about 44% of the bright spots are non-specific due to the excitation light of the quantum dots.

Similarly, in the MCF7 cells of Experimental Example 3, MCF7 hardly expresses HER2 as described above, but HER3 is expressed. Since HER2 is hardly expressed, the number of bright spots of the fluorescent dye-integrated particles flickering due to the influence of the fluorescent light emission of the quantum dots is very small correspondingly (10 bright spots per cell). . The total number of bright spots was 15, indicating that at least 5 bright spots were not based on the formation of the HER2 / HER3 dimer. In other words, it can be understood that about 33% of the bright spot quantum dots are non-specific due to the excitation light.

From the above results, it can be seen that fluorescent dye-integrated particles that do not blink in any cells are observed. This indicates that HER3 (fluorescent dye-integrated particles) that is not a dimer is also included in the detection target in the conventional technique that does not use blinking, and in the method of the present invention, the dimer is more accurately detected. It can be seen that the protein complex that formed can be detected.

[Experimental example 4]
A paraffin section (AU565: (HER2 positive; HER3 positive) cell) prepared in the same manner as in Experimental Example 1 was subjected to the quantum dot-conjugated anti-HER2 antibody prepared in Preparation Example 1 and the fluorescent dye prepared in Preparation Example 2. Staining was performed under the same conditions as in Experimental Example 1 using the anti-HER3 antibody bound to the accumulated particles.

Under the same conditions as in Experimental Example 1, Qdot (registered trademark) 585 is irradiated with ultraviolet light having an excitation wavelength of 352 to 388 nm, which is a wavelength that is excited but is not excited by Texas Red of the fluorescent dye-integrated particles. Fluorescence observation was performed on Qdot (registered trademark) 585.

Further, using the same method as in Experimental Example 1, an image of a bright spot of Qdot (registered trademark) 585 was taken.

Next, under the same conditions as in Experimental Example 1 (exposure time 250 msec, continuous shooting at a scanning speed of 400 msec / 1 frame for 60 seconds), ultraviolet light of 352 to 388 nm was similarly irradiated as excitation light, and fluorescence Using a filter (558 to 584 nm), time-lapse photography of the fluorescent dye-integrated particles was performed.

Using image processing software “ImageJ” (open source), the number of bright spots derived from Qdot (registered trademark) 585 and fluorescent dye-integrated particles (Texas Red) contained in one captured image is measured, and the bright spots are measured. The number of bright spots per cell was obtained by dividing by the number of cells in one screen.

Table 2 shows the results of Experimental Example 4.

*: Number of fluorescent dye-integrated particles (Texas Red) flickering at excitation wavelengths of 352 to 388 nm **: Total number of bright spots = sum of fluorescent dye-integrating particles flickering at excitation wavelengths of 352 to 388 nm and fluorescent dye-integrating particles that do not blink ( Discussion)
As described above, the number of HER2s can be detected by irradiating the excitation wavelength of the quantum dot and simultaneously detecting the fluorescence of the quantum dot. The number of HER3 can be detected by detecting the emission wavelength of. Furthermore, the number of HER2 / HER3 dimers can also be detected by obtaining the fluorescence of the flickering fluorescent dye-integrated particles together with these detections.

Thus, by using the present invention, HER2 and HER3 can be quantified using the same section as that obtained by quantifying the HER2 / HER3 dimer.

For example, the number of bright spots of HER2 based on the quantum dot stained image is 480, while the bright spot of HER2 / HER3 dimer obtained in Experimental Example 1 is different from 400. That is, it can be determined that 400 ÷ 480 = 83% of HER2 exists as a dimer and the residue exists as a monomer.

[Experimental example 5]
Human breast cancer-derived cultured cells (cell name: AU565; ATCC (registered trademark) NO. CRL-235; Sumisho Pharma International Co., Ltd.) were cultured and seeded on a 35 mm culture dish No. 1.5.

(1) One day after seeding, pertuzumab (Perjeta (registered trademark); Chugai Pharmaceutical Co., Ltd.) diluted with a culture solution to a final concentration of 20 μg / mL was added to the cells.

24 hours after adding pertuzumab to the cells, the cells were collected by trypsin treatment, and a cell block was prepared in the same manner as in Experimental Example 1. Using the quantum dot-conjugated anti-HER2 antibody and the fluorescent dye-integrated particle-conjugated anti-HER3 antibody prepared in Preparation Examples 1 and 2, staining and detection of a blinking spot were performed in the same manner as in Experimental Example 1. As a result, the number of blinking bright spots was 18. In addition, the same operation was performed on cells to which pertuzumab was not acted as a comparative object, and the number of blinking bright spots was measured. As a result, 225 blinking bright spots were confirmed.

*: Number of fluorescent dye-integrated particles (Texas Red) flickering at an excitation wavelength of 352 to 388 nm (discussion)
The addition of pertuzumab significantly reduced the number of blinking bright spots to less than one-tenth. This indicates that the HER2 / HER3 dimer was reduced by pertuzumab, and the effect of pertuzumab on inhibiting the formation of the HER2 / HER3 dimer could be visualized.

1 ... HER2
3 ... Cell membrane 5 ... Anti-HER2 mouse monoclonal antibody 6 ... Anti-mouse IgG antibody 10 ... Quantum dot 20 ... HER3
25 anti-HER3 mouse monoclonal antibody 30 fluorescent dye-integrated particles 40 HER2 / HER3 heterodimer 50 excitation light of quantum dots 60 energy transfer from quantum dots

Claims (12)

1. A method for detecting a protein complex comprising a protein (A) and a protein (B),
   Step (a) of labeling the protein (A) with an antibody bound to fluorescent particles that cause blinking and labeling the protein (B) with an antibody bound to fluorescent particles that do not cause blinking Step (b),
   afterwards,
   Simultaneously with irradiating the light of the excitation wavelength of the fluorescent particles that cause the blinking, to detect the fluorescent bright spot of the emission wavelength of the fluorescent particles that do not cause the blinking,
   Extracting a flickering luminescent spot from the fluorescent luminescent spots having the emission wavelength of the fluorescent particles that do not cause blinking,
   A method for detecting a protein complex, wherein the blinking bright spot represents a complex of the protein (A) and the protein (B).
2. The fluorescent particles that cause the blinking are quantum dots,
   The fluorescent particles that do not cause blinking are fluorescent dye-integrated particles,
   A method for detecting a protein complex according to claim 1.
3. 3. The method according to claim 1, further comprising the step (d) of calculating the number of proteins (A) not forming a protein complex and the number of proteins (B) not forming a protein complex. A method for detecting a protein complex.
4. 方法 The method for detecting a protein complex according to any one of claims 1 to 3, wherein the protein complex is a heterodimer composed of a protein (A) and a protein (B).
5. (5) A method for evaluating a drug effect, comprising a step of performing the method for detecting a protein complex according to any one of (1) to (4) before and after the addition of the drug to the sample.
6. The evaluation method according to claim 5, wherein the drug is a drug that inhibits formation of a protein complex.
7. The evaluation method according to claim 5 or 6, wherein the drug is a heterodimer formation inhibitor.
8. 評 価 The evaluation method according to any one of claims 5 to 7, wherein the protein complex is a heterodimer composed of HER2 and HER3.
9. The evaluation method according to any one of claims 5 to 8, wherein the drug is pertuzumab, trastuzumab, or lapatinib.
10. 10. The evaluation method according to claim 5, wherein the quantum dots and the fluorescent dye-integrated particles have different excitation wavelengths.
11. 11. The excitation wavelength band of the quantum dot, wherein the excitation spectrum intensity per fluorescent dye-integrated particle is one tenth or less of the excitation spectrum intensity per quantum dot particle. Evaluation method described in section.
12. 12. The excitation wavelength band of the quantum dot, wherein the excitation spectrum intensity per fluorescent dye-integrated particle is one hundredth or less of the excitation spectrum intensity per quantum dot particle. Evaluation method described in section.

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