WO2023058624A1

WO2023058624A1 - Dyeing method, evaluation method and sample

Info

Publication number: WO2023058624A1
Application number: PCT/JP2022/037046
Authority: WO
Inventors: 敦郎巽; 恵祐森近; 博之横田
Original assignee: コニカミノルタ株式会社
Priority date: 2021-10-08
Filing date: 2022-10-04
Publication date: 2023-04-13

Abstract

The present invention relates to a dyeing method that involves the use of fluorescent nanoparticles and makes it possible to suppress the separation of fluorescent nanoparticles. The dyeing method according to the present invention includes a step for immobilizing a first binding substance on the periphery of a sample substance by covalent bonding, and a step for binding a second binding substance that modifies the surfaces of the fluorescent nanoparticles to the first binding substance and immobilizing the fluorescent nanoparticles.

Description

Staining method, evaluation method and specimen

The present invention relates to staining methods, evaluation methods and specimens.

Immunohistochemistry (IHC) using antibodies labeled with enzymes or fluorescent dyes is widely used as a staining method for detecting target substances (e.g., specific proteins, drugs, etc.) in tissue specimens. . When an enzyme-labeled antibody is used, the target substance is detected by observing a chromogenic reaction product generated by the action of the enzyme on a chromogenic substrate such as 3,3'-diaminobenzidine (DAB). On the other hand, when a fluorochrome-labeled antibody is used, the target substance is detected by observing the fluorescence emitted from the fluorochrome. However, all of these conventional detection methods using immunohistochemical staining lack sensitivity and quantification.

In order to solve the above problems, it has been proposed to use antibodies labeled with fluorescent nanoparticles containing multiple fluorescent dyes instead of enzymes or fluorescent dyes. For example, US Pat. No. 6,200,000 discloses immunohistochemical staining using antibodies conjugated with biotin via a spacer of predetermined length and fluorescent nanoparticles modified with streptavidin. The fluorescent nanoparticles are indirectly bound to the target substance by binding the antibody to the target substance in the tissue sample and further binding the biotin that labels the antibody to streptavidin that modifies the fluorescent nanoparticles. By measuring the high-intensity fluorescence emitted from the fluorescent nanoparticles, the target substance can be detected with high sensitivity and quantitatively.

WO2015/133523

As described above, immunohistochemical staining using fluorescent nanoparticles can detect target substances more sensitively and quantitatively than when using enzymes or fluorescent dyes. However, the present inventors have found that in immunohistochemical staining using fluorescent nanoparticles, depending on the type of target substance, the fluorescent nanoparticles rarely produce large bright spots. As a result of further studies by the present inventors, this is because some of the plurality of fluorescent nanoparticles bound to the target substance in the tissue specimen were released from the tissue specimen and aggregated with other fluorescent nanoparticles. It was speculated that Aggregation of the fluorescent nanoparticles in this way is not preferable from the viewpoint of quantitative detection of the target substance.

Therefore, an object of the present invention is to provide a staining method using fluorescent nanoparticles, which is capable of suppressing release of the fluorescent nanoparticles. Another object of the present invention is to provide an evaluation method including the staining method, and a specimen prepared by the staining method.

A staining method according to one embodiment of the present invention comprises the steps of: immobilizing a first binding substance around a target substance by covalent bonding; binding to immobilize the fluorescent nanoparticles.

An evaluation method according to one embodiment of the present invention includes the steps of: immobilizing a first binding substance around a target substance by covalent bonding; binding and immobilizing the fluorescent nanoparticles; obtaining information about the immobilized fluorescent nanoparticles; and evaluating the target substance based on the information about the fluorescent nanoparticles. .

A specimen according to one embodiment of the present invention is modified with a target substance, a first binding substance covalently immobilized around the target substance, and a second binding substance capable of binding to the first binding substance. and the second binding substance that modifies the fluorescent nanoparticles is bound to the first binding substance.

According to the present invention, by retaining the fluorescent nanoparticles with the first binding substance that is covalently immobilized around the target substance, it is possible to suppress the release of the fluorescent nanoparticles from the surroundings of the target substance. Therefore, according to the present invention, aggregation of fluorescent nanoparticles can be prevented, and target substances can be detected more quantitatively.

FIG. 1 is a schematic diagram showing the surroundings of a target substance in a specimen stained by a conventional staining method. FIG. 2 is a schematic diagram showing the surroundings of a target substance in a specimen stained by the staining method according to the embodiment. 3A to 3D are schematic diagrams showing an example of the procedure of the staining method according to the embodiment. FIG. 4A is a photograph showing the staining result of a human breast cancer tissue section when the staining method according to the example is used, and FIG. 4B is the staining result of the human breast cancer tissue section when the staining method according to the comparative example is used. is a photograph showing FIG. 5A is a photograph showing the staining result of the HT1080 cell line when the staining method according to the example is used, and FIG. 5B is the photograph showing the staining result of the HT1080 cell line when the staining method according to the comparative example is used. It is a photograph. Figure 6 shows the number of bound anti-HER2 antibodies per cell measured using FACS (FACS value) for each cell line, and per cell stained by the staining method of Example or Comparative Example. is a graph showing the relationship between the fluorescence intensity of . FIG. 7 is a photograph showing the dyeing results of the dyeing method according to the example and the dyeing method according to the comparative example. FIG. 8 is a graph showing the relationship between the tyramide reaction time and the fluorescence intensity per cell stained by the staining method of Example or Comparative Example for each cell line.

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

The staining method according to the present embodiment comprises (1) a step of immobilizing a first binding substance around a target substance by a covalent bond; and immobilizing the fluorescent nanoparticles by binding to a binding substance.

Further, the evaluation method according to the present embodiment includes, in addition to the steps (1) and (2), (3) obtaining information on the immobilized fluorescent nanoparticles; Evaluating the target substance based on particle information.

In the conventional staining method using fluorescent nanoparticles as described in Patent Document 1, for example, as shown in FIG. , and biotin 40 is conjugated with streptavidin 60 , which modifies fluorescent nanoparticles 50 . In this case, one fluorescent nanoparticle 50 is often bound to the specimen (eg, tissue section) at only one point. Also, the primary antibody 20 and the secondary antibody 30 are non-covalently bound to the binding target. Therefore, for example, when the non-covalent bond between the target substance 10 and the primary antibody 20 or the bond between the primary antibody 20 and the secondary antibody 30 is broken, the fluorescent nanoparticles 50 are released from the sample. It will be put away. The released fluorescent nanoparticles 50 may bind to other fluorescent nanoparticles 50 via other biotin 40 or the like bound to the secondary antibody 30 and aggregate.

On the other hand, in the staining method according to the present embodiment, for example, as shown in FIG. A second binding substance 180 that modifies the is bound. In this case, fluorescent nanoparticles 170 are attached to the specimen (eg, tissue section) via strong covalent bonds. Therefore, fluorescent nanoparticles 170 are less likely to be released from the surroundings of target substance 110 . Moreover, in the above case, one fluorescent nanoparticle 170 is often bound to the sample at a plurality of locations. Therefore, even if the binding at one site is broken, the fluorescent nanoparticles 170 will not be released from the surroundings of the target substance 110 .

The steps (1) to (4) above will be described below with reference to FIGS. 3A to 3D.

(1) Immobilization of First Binding Substance First, the first binding substance is immobilized around the target substance in the sample by covalent bonding. Here, the phrase "surrounding the target substance" includes the target substance itself. Thus, the first binding substance may covalently bind to the target substance.

The type of specimen is not particularly limited and can be selected as appropriate according to the purpose. Examples of specimens include human or non-human animal tissues and cells, and cultured cells. For example, to analyze human tumors, human tumor tissue should be prepared as a specimen. Human or non-human animal tissue can be used in the form of tissue sections such as paraffin sections and frozen sections after being fixed. Human or non-human animal cells can also be used in the form of a smear. Cultured cells can also be used in the form of cultured on glass slides and in the form of sections such as paraffin sections and frozen sections.

The type of target substance is not particularly limited and can be appropriately selected according to the purpose. For example, the target substance may be a biological substance such as a protein, a drug, or the like.

The method of immobilizing the first binding substance around the target substance by covalent bonding is not particularly limited. For example, (1-1) a step of directly or indirectly binding a capture substance modified with a catalyst to a target substance (see FIGS. 3A and 3B), and (1-2) a substrate modified with the first binding substance is covalently bound around the target substance in the presence of the catalyst bound to the target substance via the capture substance (see FIG. 3C).

In the step (1-1) above, the catalyst-modified capture substance is directly or indirectly bound to the target substance. At this time, the catalyst-modified capture substance may directly or indirectly bind to the target substance. For example, if the capture agent is an antibody, the catalytically modified capture agent may be a primary antibody that binds to the target agent or a secondary antibody that binds to the primary antibody bound to the target agent. may

The type of capture substance is not particularly limited as long as it can directly or indirectly and specifically bind to the target substance. Capture substances are, for example, antibodies or fragments thereof, aptamers (DNA aptamers, RNA aptamers), and the like. When the catalytically modified capture agent binds directly to the target agent, the capture agent may be an antibody or fragment thereof that specifically binds to the target agent. When the catalytically modified capture agent indirectly binds to the target agent, the capture agent may be a secondary antibody or fragment thereof that specifically binds to the primary antibody specifically bound to the target agent. .

The capture substance is modified with a catalyst. A linker such as polyethylene glycol (PEG) may be inserted between the capture substance and the catalyst. The catalyst promotes the reaction in which the substrate modified with the first binding substance binds around the target substance in the next step (1-2). Therefore, the type of catalyst can be appropriately selected according to the type of substrate. For example, if the substrate is a compound that is radicalized by a peroxidase such as tyramide or styramide, the catalyst can be a peroxidase such as horseradish peroxidase (HRP). Tyramides, styramides, and the like are radicalized by peroxidase in the presence of hydrogen peroxide and bind to electron-rich amino acid residues (eg, tyrosine and tryptophan). Alternatively, when the substrate is a quinone methide precursor containing a fluorine atom, the catalyst can be alkaline phosphatase (ALP). A quinone methide precursor is converted to a quinone methide by hydrolysis of the phosphate group by alkaline phosphatase, and binds to highly nucleophilic functional groups (eg, amino and thiol groups).

For example, in step (1-1), as shown in FIG. combine. After that, the sample is washed to remove the primary antibody 120 that has not bound to the target substance 110 . Then, as shown in FIG. 3B, the specimen is provided with a liquid containing a secondary antibody 130 modified with a catalyst (horseradish peroxidase) 140 to allow the primary antibody 120 bound to the target substance 110 to be modified with the catalyst 140. secondary antibody 130 is bound. After that, the specimen is washed to remove the secondary antibody 130 that has not bound to the primary antibody 120 .

In step (1-2) above, the substrate modified with the first binding substance is covalently bound around the target substance. This reaction proceeds in the presence of a catalyst bound to a target substance via a capture substance. Therefore, the substrate binds only around the target substance.

The substrate is modified with the first binding substance and has the role of immobilizing the first binding substance around the target substance. A linker such as polyethylene glycol (PEG) may be inserted between the substrate and the first binding substance. As described above, the type of substrate can be appropriately selected according to the type of catalyst. Thus, if the catalyst is a peroxidase such as horseradish peroxidase, the substrate can be a compound that is radicalized by the peroxidase such as tyramide or styramide. Alternatively, when the catalyst is alkaline phosphatase, the substrate can be a fluorine atom-containing quinone methide precursor.

The first binding substance is a substance that binds to the second binding substance that modifies the surface of the fluorescent nanoparticles. Therefore, the type of the first binding substance can be appropriately selected according to the type of the second binding substance. For example, if the second binding substance is avidin, streptavidin or neutravidin, the first binding substance can be biotin. Alternatively, the first binding substance may be a small molecule (hapten) recognizable by an antibody, such as fluorescein isothiocyanate (FITC), digoxigenin, 2,4-dinitrophenol, and the like. In this case, the second binding agent can be an antibody or fragment thereof that specifically binds to this small molecule. Alternatively, when the second binding substance is a HaloTag protein that is a mutant of haloalkane dehalogenase from Rhodococcus rhodochrous, the first binding substance can be a HaloTag ligand. From the viewpoint of preventing liberation of the fluorescent nanoparticles, it is preferable that the binding between the first binding substance and the second binding substance be strong. For example, the binding between biotin and avidin is non-covalent but as strong as covalent binding. Also, the binding between the HaloTag protein and the HaloTag ligand is a covalent bond. Therefore, when these bonds are used, the liberation of the fluorescent nanoparticles can be sufficiently prevented even with only one bond. On the other hand, since the binding between a small molecule (hapten) and an antibody is a non-covalent bond, it is preferable to generate multiple binding sites between the specimen and the fluorescent nanoparticles.

For example, in step (1-2), as shown in FIG. 3C, the specimen is provided with a liquid containing a substrate (tyramide) 150 modified with a first binding substance (biotin) 160, and the first binding substance 160 The substrate 150 modified with is covalently bound only around the target substance 110 . After this, the specimen is washed to remove unbound substrate 150 .

(2) Immobilization of Fluorescent Nanoparticles Next, the second binding substance that modifies the surface of the fluorescent nanoparticles is bound to the first binding substance to immobilize the fluorescent nanoparticles. This stains the specimen.

　Fluorescent nanoparticles are nano-sized particles that contain multiple phosphors and are based on particles made of organic or inorganic substances. Although the type of phosphor is not particularly limited, it is, for example, a fluorescent dye or semiconductor nanoparticles. A plurality of phosphors may be present within the particle or may be present on the surface of the particle. The phosphor-accumulating particles are capable of emitting fluorescence of sufficient intensity to show the target substance as a bright spot one molecule at a time.

Examples of organic substances used as base materials include thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin; styrene resin, acrylic resin, acrylonitrile resin, AS resin ( acrylonitrile-styrene copolymer), thermoplastic resins such as ASA resin (acrylonitrile-styrene-methyl acrylate copolymer); other resins such as polylactic acid; and polysaccharides. Examples of inorganic matrix materials include silica and glass. It is preferred that the matrix and the fluorescent substance have substituents or moieties with opposite charges and that electrostatic interaction works.

When the phosphor is a fluorescent dye, the type of fluorescent dye is not particularly limited. Examples of fluorescent dyes include rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, aromatic ring-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, and pyromethene-based dye molecules. Examples of commercially available fluorescent dyes include Alexa Fluor-based dyes (registered trademark, Thermo Fisher Scientific), BODIPY-based dyes (registered trademark, Thermo Fisher Scientific), and Cy-based dyes (registered trademark, Thermo Fisher Scientific). , DY dyes (Dyomics), HiLyte dyes (Anaspec), DyLight dyes (registered trademark, Thermo Fisher Scientific), ATTO dyes (registered trademark, ATTO-TEC), and MFP dyes (MoBiTec) ) is included. These dyes are collectively named based on the main structure (skeleton) or trademark in the compound, and a person skilled in the art can appropriately determine the range of fluorescent dyes belonging to each without excessive trial and error. It is comprehensible.

When the phosphor is a semiconductor nanoparticle, the type of semiconductor that constitutes the semiconductor nanoparticle is not particularly limited as long as it can emit fluorescence. Semiconductors constituting semiconductor nanoparticles are, for example, II-VI group compound semiconductors, III-V group compound semiconductors, or IV group semiconductors. Examples of semiconductors that make up the semiconductor nanoparticles include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si and Ge.

Fluorescent nanoparticles can be produced according to known methods. For example, a liquid containing a phosphor (fluorescent dye or semiconductor nanoparticles) and a silica precursor such as tetraethoxysilane is added dropwise to a solution in which ethanol and ammonia are dissolved to hydrolyze the silica precursor. Fluorescent nanoparticles can be made in which the phosphor is present in a matrix made of silica. In addition, by adding a phosphor (fluorescent dye or semiconductor nanoparticles) to a resin solution or a resin dispersion and stirring, fluorescent nanoparticles in which the phosphor is present on or in the resin matrix are produced. be able to.

In addition, by adding a fluorescent dye to a resin raw material solution and then allowing the polymerization reaction to proceed, fluorescent nanoparticles in which the fluorescent dye is present on the surface or in the resin matrix can be produced. For example, when a thermosetting resin such as a melamine resin is used as a base resin, a raw material for the resin (for example, methylolmelamine, which is a condensate of melamine and formaldehyde), a fluorescent dye, and preferably a surfactant and polymerization. A mixture containing a reaction accelerator (such as an acid) is heated, and a polymerization reaction proceeds by an emulsion polymerization method to produce fluorescent nanoparticles in which a fluorescent dye is present on or in a matrix made of a thermosetting resin. can do. In addition, when using a thermoplastic resin such as a styrene copolymer as the base resin, it contains the raw material of the resin, a fluorescent dye, and a polymerization initiator (benzoyl peroxide, azobisisobutyronitrile, etc.). By heating the mixture and allowing the polymerization reaction to proceed by a radical polymerization method or an ionic polymerization method, fluorescent nanoparticles in which a fluorescent dye is present on or in a matrix made of a thermoplastic resin can be produced.

The average particle diameter of the fluorescent nanoparticles is not particularly limited, but it is usually 10 to 500 nm, preferably 50 to 200 nm, considering the ease of detection as bright spots. Also, the coefficient of variation, which indicates the variation in particle size, is usually 20% or less, preferably 5 to 15%. Fluorescent nanoparticles satisfying such conditions can be produced by adjusting the production conditions. For example, when producing fluorescent nanoparticles by emulsion polymerization, the particle size can be adjusted by the amount of surfactant added. In general, the larger the amount of surfactant relative to the amount of parent material, the smaller the particle size, and the smaller the amount of surfactant relative to the amount of parent material, the larger the particle size. Become.

The particle size of the fluorescent nanoparticles can be measured by measuring the projected area of the fluorescent nanoparticles using a scanning electron microscope (SEM) and converting it into an equivalent circle diameter. The average particle size and coefficient of variation of a population of a plurality of fluorescent nanoparticles are calculated using the particle size (equivalent circle diameter) calculated for a sufficient number (eg, 1000) of fluorescent nanoparticles.

The fluorescent nanoparticles are modified with the second binding substance. A linker such as polyethylene glycol (PEG) may be inserted between the fluorescent nanoparticles and the second binding substance. The second binding substance is a substance that binds to the first binding substance immobilized around the target substance. As described above, the type of the second binding substance can be appropriately selected according to the type of the first binding substance. Thus, if the first binding substance is biotin, the second binding substance can be avidin, streptavidin or neutravidin. Also, if the first binding substance is a small molecule (hapten) recognizable by an antibody, the second binding substance can be an antibody or fragment thereof that specifically binds to this small molecule.

For example, in step (2), as shown in FIG. 3D, the sample is provided with a liquid containing fluorescent nanoparticles 170 modified with a second binding substance (streptavidin) 180, so that the surfaces of the fluorescent nanoparticles 170 are A second binding substance 180 to be modified is bound to the first binding substance 160 . Thereby, the fluorescent nanoparticles 170 are immobilized around the target substance 110 . The specimen is then washed to remove unbound fluorescent nanoparticles 170 .

Through the above steps (1) and (2), the target substance 110, the first binding substance 160 covalently immobilized around the target substance 110, and the second binding substance as shown in FIG. a second binding substance 180 modifying the fluorescent nanoparticles 170 bound to the first binding substance 160 . When the above steps (1-1) and (1-2) were performed, the sample was modified with catalyst 140 directly or indirectly bound to target substance 110, as shown in FIG. It further has a capture substance 130 . In this case, the first binding substance 160 is covalently immobilized around the target substance 110 via the substrate 150 of the catalyst 140 .

As shown in FIG. 2, in the staining method according to the present embodiment, a plurality of first binding substances 160 immobilized around a target substance 110 by covalent bonding are added to a plurality of fluorescent nanoparticles 170 for modifying the surface of the fluorescent nanoparticles 170. of the second binding substance 180 are bound. In this case, fluorescent nanoparticles 170 bind to the specimen via strong covalent bonds. Therefore, fluorescent nanoparticles 170 are less likely to be released from the surroundings of target substance 110 . Also, one fluorescent nanoparticle 170 is often bound to a specimen (eg, tissue section) at multiple locations. Therefore, even if the binding at one site is broken, the fluorescent nanoparticles 170 will not be released from the surroundings of the target substance 110 .

Before and after step (1) or before and after step (2), the specimen may be further stained with another dye or fluorescent dye. For example, when it is desired to stain the nuclei of all cells, the sample may be further stained with Hoechst dye (registered trademark, Thermo Fisher Scientific), DAPI, or the like.

(3) Acquisition of Information on Fluorescent Nanoparticles When performing not only staining of specimens but also evaluation, information on the immobilized fluorescent nanoparticles is further obtained.

The method of obtaining information on immobilized fluorescent nanoparticles is not particularly limited. For example, using a fluorescence microscope, a confocal laser microscope, a whole-slide scanner, etc., the sample is irradiated with excitation light of a wavelength corresponding to the phosphor contained in the fluorescent nanoparticles, and a fluorescence image is obtained from the fluorescence emitted from the fluorescent nanoparticles. should be taken.

(4) Evaluation of Target Substance Finally, the target substance is evaluated based on the information of the fluorescent nanoparticles obtained in step (3).

The method of evaluating the target substance based on the information of the fluorescent nanoparticles is not particularly limited. For example, the captured fluorescence image is image-processed to specify the position and number of the fluorescent nanoparticles. In the fluorescence image, one bright spot is derived from one fluorescent nanoparticle, and thus the size of the bright spot is usually constant. Software used for image processing is not particularly limited, but ImageJ (open source), for example, can be used. By using such image processing software, it is possible to extract luminescent spots of a predetermined wavelength (color) and calculate the sum of their luminances, or to count the number of luminescent spots with a predetermined luminance or more. can be easily done. Then, for example, the distribution and amount of the target substance can be evaluated from the position and number of the fluorescent nanoparticles.

(effect)
As described above, according to the staining method of the present invention, since the fluorescent nanoparticles are retained by the first binding substance immobilized around the target substance by covalent bonding, the fluorescent nanoparticles are released from the surroundings of the target substance. can be suppressed. Therefore, according to the staining method of the present invention, aggregation of the fluorescent nanoparticles can be prevented, and the target substance can be detected more quantitatively.

As shown in the examples below, the staining method according to the present invention can stain specimens more clearly than the conventional staining method using fluorescent nanoparticles.

A staining method including tyramide signal amplification (TSA) (TSA fluorescence method) is also known as immunohistochemical staining using horseradish peroxidase and a substrate (tyramide) as in the present invention. In the TSA fluorescence method, the signal increases as the tyramide reaction time increases. Therefore, in the TSA fluorescence method, the quantification of the signal is impaired due to fluctuations in the reaction time. In contrast, in the staining method according to the present invention, as shown in Examples below, the signal hardly varies even if the reaction time of the substrate modified with the first binding substance varies. Therefore, in the staining method according to the present invention, even if the reaction time fluctuates, the quantification of the signal is less likely to be impaired.

Furthermore, in the TSA fluorescence method, the background tends to be high when there are few target substances. In contrast, as shown in the examples below, the staining method according to the present invention provides a low background even when the amount of the target substance is small.

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

[Experiment 1]
In Experiment 1, the staining method according to the present invention (Example, see FIG. 2) and the conventional staining method using fluorescent nanoparticles (Comparative Example, see FIG. 1) were compared for sections of human breast cancer tissue.

A slide glass on which a paraffin section of human breast cancer tissue was placed was immersed in xylene and ethanol in order to remove paraffin from the section. After washing the section, the section was immersed in the activation solution and heated to activate the antigen. A blocking solution containing bovine serum albumin was applied to the sections to block the tissue surface. A liquid containing anti-HER2 rabbit monoclonal antibody (4B5) (Ventana) was applied to the sections and allowed to stand overnight at 4°C. Sections were then washed.

After that, as an example, a liquid containing a horseradish peroxidase-modified anti-rabbit IgG polyclonal antibody was applied to the sections and allowed to stand at room temperature for 30 minutes. After washing the sections, a biotin-modified tyramide solution diluted in Tris buffer containing hydrogen peroxide was applied to the sections and allowed to stand at room temperature for 10 minutes. Sections were then washed.

On the other hand, as a comparative example, instead of the horseradish peroxidase-modified anti-rabbit IgG polyclonal antibody, the section was provided with a liquid containing biotin-modified anti-rabbit IgG polyclonal antibody, and allowed to stand at room temperature for 30 minutes. Sections were then washed. In a comparative example, no biotin-modified tyramide was applied to the sections.

After that, in both Examples and Comparative Examples, a liquid containing streptavidin-modified fluorescent nanoparticles was applied to the sections and allowed to stand at room temperature. As the fluorescent nanoparticles, fluorescent nanoparticles prepared by the method described in WO2017/109828 were used. After washing, nuclear counterstaining was performed with DAPI and sections were mounted.

FIG. 4A is a photograph showing the staining result of the staining method according to the example, and FIG. 4B is a photograph showing the staining result of the staining method according to the comparative example. The signal due to the fluorescent nanoparticles is red and the signal due to DAPI is blue, but both signals are shown in white (grayscale) in FIGS. 4A and 4B.

By comparing these photographs, it can be seen that the dyeing method (Example) according to the present invention can be dyed more clearly than the conventional dyeing method (Comparative Example). This result suggests that the staining method according to the present invention firmly retains the fluorescent nanoparticles in the vicinity of the target substance and provides a clear stained image.

[Experiment 2]
In Experiment 2, four types of cell lines with different HER2 expression levels were subjected to a staining method according to the present invention (see Example, FIG. 2) and a conventional staining method using fluorescent nanoparticles (Comparative Example, FIG. 1). ) were compared.

In Experiment 2, staining targets were HT1080 (human fibrosarcoma cell line, HER2 score 0), MCF7 (human breast cancer cell line, HER2 score 1+), T47D (human breast cancer cell line, HER2 score 1+), ZR-75-1. (human breast cancer cell line, HER2 score 2+) was used. In Experiment 2, cells were stained in the same manner as in Experiment 1, except that paraffin sections of cultured cells were used instead of paraffin sections of human breast cancer tissue.

FIG. 5A is a photograph showing the staining result of the HT1080 cell line when the staining method according to the example is used, and FIG. 5B is the photograph showing the staining result of the HT1080 cell line when the staining method according to the comparative example is used. It is a photograph. These pictures show only the fluorescence signal from the fluorescent nanoparticles and not the fluorescence signal of the counterstain. A comparison of these photographs shows that the dyeing method according to the present invention (Example) can be dyed more clearly than the conventional dyeing method (Comparative Example).

Figure 6 shows the number of bound anti-HER2 antibodies per cell measured using FACS (FACS value) for each cell line, and per cell stained by the staining method of Example or Comparative Example. is a graph showing the relationship between the fluorescence intensity of . A black circle indicates the result of the dyeing method of the example, and a white circle indicates the result of the dyeing method of the comparative example. From this graph, it can be seen that the dyeing method (Example) according to the present invention can ensure quantitative performance equivalent to or higher than that of the conventional dyeing method (Comparative Example).

[Experiment 3]
In Experiment 3, four types of cell lines with different HER2 expression levels were subjected to a staining method according to the present invention (Example) and a conventional staining method including tyramide signal amplification (TSA) (TSA fluorescence method, Comparative Example). ) were compared.

In Experiment 3, HT1080 (human fibrosarcoma cell line, HER2 score 0), MCF7 (human breast cancer cell line, HER2 score 1+), T47D (human breast cancer cell line, HER2 score 1+), ZR-75-1 were used as staining targets. (human breast cancer cell line, HER2 score 2+) was used.

Examples include the use of paraffin sections of cultured cells instead of paraffin sections of human breast cancer tissue, and the standing time (tyramide Cells were stained in the same procedure as in Experiment 1, except that the reaction time of the reaction) was 6 to 14 minutes.

On the other hand, as a comparative example, streptavidin-modified fluorescent dye (Alexa Fluor 594) was provided to the sections instead of streptavidin-modified fluorescent nanoparticles, and left at room temperature. In a comparative example, sections were not provided with streptavidin-modified fluorescent nanoparticles.

FIG. 7 is a photograph showing the dyeing results of the dyeing method according to the example and the dyeing method according to the comparative example. These pictures show only the fluorescence signal from the fluorescent nanoparticles and not the fluorescence signal of the counterstain. The upper left photograph of FIG. 7 and the photograph of FIG. 5A are both photographs of the HT1080 cell line stained by the staining method according to the present invention, but the gradation (contrast) is different from each other.

FIG. 8 is a graph showing the relationship between the tyramide reaction time and the fluorescence intensity per cell stained by the staining method of Example or Comparative Example for each cell line.

From these results, the staining method (Example) according to the present invention has a low background even if the target substance (HER2) is less than the conventional TSA fluorescence method (Comparative Example), and the expression level of the target substance is low. It can be seen that the staining intensity (fluorescence intensity) changes accordingly. That is, by staining with the staining method according to the present invention, four types of cell lines with different expression levels of target substances can be identified. In addition, in the conventional TSA fluorescence method (comparative example), the signal intensity tends to increase as the reaction time of tyramide increases, whereas in the staining method according to the present invention (example), the same as the TSA fluorescence method It can also be seen that the signal intensity does not depend on the reaction time of tyramide, despite the use of HRP and tyramide in . That is, the staining method according to the present invention has higher stability with respect to reaction time than the TSA fluorescence method.

This application claims priority based on Japanese Patent Application No. 2021-166285 filed on October 8, 2021. All contents described in the specification and drawings are incorporated herein by reference.

The staining method, evaluation method and specimen according to the present invention are useful, for example, for pathological diagnosis.

10 target substance 20 primary antibody 30 secondary antibody 40 biotin 50 fluorescent nanoparticles 60 streptavidin 110 target substance 120 primary antibody 130 secondary antibody 140 catalyst 150 substrate 160 first binding substance 170 fluorescent nanoparticles 180 second binding substance

Claims

covalently immobilizing the first binding substance around the target substance;
binding a second binding substance that modifies the surface of the fluorescent nanoparticles to the first binding substance to immobilize the fluorescent nanoparticles;
staining method, including
The step of immobilizing the first binding substance comprises:
Directly or indirectly binding a catalytically modified capture agent to the target agent;
covalently binding the substrate modified with the first binding substance around the target substance in the presence of the catalyst bound to the target substance via the capture substance;
including,
The dyeing method according to claim 1.
the catalyst is peroxidase,
The substrate is a compound that is radicalized by the peroxidase,
The dyeing method according to claim 2.
the first binding substance is biotin;
wherein said second binding substance is avidin, streptavidin or neutravidin;
The dyeing method according to any one of claims 1 to 3.
covalently immobilizing the first binding substance around the target substance;
binding a second binding substance that modifies the surface of the fluorescent nanoparticles to the first binding substance to immobilize the fluorescent nanoparticles;
obtaining information about the immobilized fluorescent nanoparticles;
evaluating the target substance based on the information of the fluorescent nanoparticles;
evaluation methods, including;
The step of immobilizing the first binding substance comprises:
Directly or indirectly binding a catalytically modified capture agent to the target agent;
covalently binding the substrate modified with the first binding substance around the target substance in the presence of the catalyst bound to the target substance via the capture substance;
including,
The evaluation method according to claim 5.
the catalyst is peroxidase,
The substrate is a compound that is radicalized by the peroxidase,
The evaluation method according to claim 6.
the first binding substance is biotin;
wherein said second binding substance is avidin, streptavidin or neutravidin;
The evaluation method according to any one of claims 5-7.
a target substance;
a first binding substance covalently immobilized around the target substance;
a fluorescent nanoparticle modified with a second binding substance capable of binding to the first binding substance;
has
the second binding substance that modifies the fluorescent nanoparticles is bound to the first binding substance;
Specimen.
further comprising a catalytically modified capture agent directly or indirectly bound to said target agent;
The first binding substance is covalently immobilized around the target substance via the substrate of the catalyst,
A specimen according to claim 9 .
the catalyst is peroxidase,
The substrate is a compound that is radicalized by the peroxidase,
11. Specimen according to claim 10.
the first binding substance is biotin;
wherein said second binding substance is avidin, streptavidin or neutravidin;
Specimen according to any one of claims 9-11.