WO2016152244A1 - Method and system for detecting target biological substance - Google Patents

Method and system for detecting target biological substance Download PDF

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Publication number
WO2016152244A1
WO2016152244A1 PCT/JP2016/052574 JP2016052574W WO2016152244A1 WO 2016152244 A1 WO2016152244 A1 WO 2016152244A1 JP 2016052574 W JP2016052574 W JP 2016052574W WO 2016152244 A1 WO2016152244 A1 WO 2016152244A1
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fluorescent
target biological
detecting
nanoparticles
biological substance
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PCT/JP2016/052574
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French (fr)
Japanese (ja)
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健作 高梨
秀樹 郷田
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コニカミノルタ株式会社
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Priority to JP2017507547A priority Critical patent/JP6687018B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for detecting a target biological substance from a tissue section and a detection system used therefor.
  • fluorescent nanoparticles In conventional immunostaining, antibody-modified “fluorescent nanoparticles” are bound to an antigen of interest on a tissue section, the antigen is labeled, and this is observed and imaged with a fluorescence microscope ( Patent Document 1). Compared with “fluorescent dyes”, fluorescent nanoparticles have the feature that they emit fluorescent light with high brightness and can measure the number of fluorescent bright spots. And the amount can be confirmed. If various types of antigens are labeled with fluorescent nanoparticles having different emission wavelengths, these can also be distinguished and confirmed.
  • the degree of malignancy may be affected by whether or not the distance between antigens is close, and it is important to obtain distance information between antigens. For example, it is known that when HER2 and HER3, which are known as cancer-related antigens, form a dimer (dimer), the malignancy of cancer increases. Nevertheless, conventionally, only fluorescent nanoparticles are used to obtain antigen distribution information, and attention has not been paid to distance information between antigens.
  • FRET fluorescence energy transfer
  • a main object of the present invention is to provide a method for detecting a target biological material that can easily detect whether or not the distance between target biological materials is short.
  • a method for detecting a target biological material from a tissue section Staining a tissue section with a plurality of immunostaining agents including fluorescent nanoparticles having different emission wavelengths from each other; Irradiating the tissue section after staining with excitation light to cause a fluorescent bright spot to appear, and imaging it with an imaging device; Based on the imaging results of the imaging device, generating a plurality of fluorescent images in units of the immunostaining agent, synthesizing the fluorescent images and detecting the overlap of fluorescent bright spots, A method for detecting a target biological substance is provided.
  • the present invention it is possible to easily detect whether or not the distance between the target biological substances is close, because a fluorescent image is generated in units of immunostaining agents and synthesized to detect the overlap of fluorescent bright spots. be able to.
  • the method for detecting a target biological material is a method for detecting a target biological material from a tissue section, as shown in FIG. (S1) staining a tissue section with a plurality of immunostaining agents containing fluorescent nanoparticles having different emission wavelengths from each other; (S2) irradiating a tissue section after staining with excitation light to cause a fluorescent bright spot to appear, and imaging it with an imaging device; (S3) based on the imaging result of the imaging device, generating a plurality of fluorescent images in units of immunostaining, and combining the fluorescent images to detect the overlap of fluorescent bright spots; It has.
  • the staining step S1 will be described, and then the imaging step S2 and the detection step S3 will be described.
  • the detection system 1 of FIG. 2 is used.
  • the target biological material is a target for immunostaining using a fluorescent label for detection or quantification mainly from the viewpoint of pathological diagnosis.
  • Typical target biological materials include biological materials that are expressed in cell membranes of various cancer tissues and can be used as biomarkers.
  • EGFR HER1 (Epidermal Growth Factor Receptor), HER2 (Human Epidermal Growth Factor Receptor), HER3, HER4, VEGFR (Vasular Endothelial Growth Factor Receptor: Vascular Endothelial Cell) Growth factor receptors (IGF), insulin-like growth factor receptor (IGFR), HGFR (hepatocyte growth factor receptor), PD- Proteins that are receptors of the immune system such as 1 (Programmedcell death 1).
  • IGF Intrasular Endothelial Growth Factor Receptor
  • IGFR insulin-like growth factor receptor
  • HGFR hepatocyte growth factor receptor
  • PD- Proteins that are receptors of the immune system such as 1 (Programmedcell death 1).
  • EGFR / HER includes EGFR / HER1 (also referred to as ErbB1) that is overexpressed in cancer tissues such as colorectal cancer, EGFR2 / HER2 (also referred to as ErbB2, neu) that is overexpressed in cancer tissues such as breast cancer, and EGFR3. / HER3 and EGFR4 / HER4 are included.
  • VEGFR includes VEGFR-1 (also referred to as Flt-1), VEGFR-2 (also referred to as Flt-2, KDR) and lymphatic vessels, which are upregulated in vascular endothelial cells in cancer tissues such as liver cancer and esophageal cancer.
  • VEGFR-3 also called Flt-4) whose expression is upregulated in the skin cells is included.
  • immunostaining agent antibody-fluorescent nanoparticle conjugate
  • primary antibodies and fluorescent nanoparticles are used indirectly, that is, other than covalent bonds using antigen-antibody reactions. It is preferable to use a complex linked by these bonds.
  • a complex in which fluorescent nanoparticles are directly bound to a primary antibody or a secondary antibody can also be used as an immunostaining agent.
  • immunostaining agents include [primary antibody against target biological substance] ... [antibody against primary antibody (secondary antibody)] to [fluorescent nanoparticles].
  • “...” represents binding by an antigen-antibody reaction, and the mode of binding indicated by “ ⁇ ” is not particularly limited.
  • a covalent bond, an ionic bond, a hydrogen bond, a coordinate bond, an antigen-antibody bond Examples include biotin avidin reaction, physical adsorption, chemical adsorption, and the like, and a linker molecule may be used as necessary.
  • the immunostaining agent includes fluorescent nanoparticles that emit fluorescence when irradiated with excitation light.
  • a plurality of immunostaining agents are used, and in particular, a plurality of immunostaining agents containing fluorescent nanoparticles having different emission wavelengths from each other are used. It is preferable to use fluorescent nanoparticles having an emission wavelength of 400 to 700 nm. Such an emission wavelength is in the detection sensitivity region of a fluorescence microscope, and a fluorescent bright spot can be detected even with a general-purpose microscope.
  • fluorescent nanoparticles it is preferable that one fluorescent nanoparticle emits short-wavelength fluorescence and the other fluorescent nanoparticle emits long-wavelength fluorescence.
  • One fluorescent nanoparticle has an emission wavelength of 480 to 550 nm (blue to green), preferably 500 to 520 nm (green), and the other fluorescent nanoparticle has an emission wavelength of 550 to 650 nm (orange to red), Preferably, it is 620 to 650 nm (red).
  • the fluorescent nanoparticles those having an average particle diameter of 20 to 300 nm, preferably 50 to 200 nm are preferably used.
  • the particle size of one fluorescent nanoparticle is calculated by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of the fluorescent nanoparticle, and assuming that the cross-sectional shape is a circle. It can be calculated as the diameter of a circle corresponding to the cross-sectional area.
  • the average particle size of a group of a large number of fluorescent nanoparticles is calculated as the arithmetic average after calculating the particle size as described above for a sufficient number (for example, 1000) of fluorescent nanoparticles.
  • an antibody (IgG) that specifically recognizes and binds a protein as a target biological substance as an antigen can be used.
  • an anti-EGFR antibody can be used when EGFR is the target biological material
  • an anti-HER2 antibody can be used when HER2 is the target biological material
  • an anti-HER3 antibody when HER3 is the target biological material.
  • an antibody (IgG) that specifically recognizes and binds to the primary antibody as an antigen can be used.
  • Both the primary antibody and the secondary antibody may be polyclonal antibodies, but from the viewpoint of quantitative stability, monoclonal antibodies are preferred.
  • 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 past.
  • Fluorescent nanoparticles are nano-sized particles that can emit fluorescence with a sufficient intensity to express the target biological substance as a bright spot one molecule at a time.
  • fluorescent nanoparticles preferably, quantum dots (semiconductor nanoparticles) and fluorescent substance integrated nanoparticles are used.
  • Quantum dots As quantum dots, semiconductor nanoparticles containing a II-VI group compound, a III-V group compound, or a group IV element are used. Examples thereof include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.
  • Fluorescent substance integrated nanoparticles are based on particles made of organic or inorganic substances, and a plurality of fluorescent substances (for example, the above-described quantum dots and fluorescent dyes) are encapsulated therein and Nanoparticles having a structure adsorbed on the surface thereof.
  • fluorescent substance-integrated nanoparticles it is preferable that the matrix and the fluorescent substance have substituents or sites having opposite charges, and an electrostatic interaction works.
  • fluorescent substance integrated nanoparticles quantum dot integrated nanoparticles, fluorescent dye integrated nanoparticles, and the like are used.
  • thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, furan resin; styrene Resins, acrylic resins, acrylonitrile resins, AS resins (acrylonitrile-styrene copolymers), ASA resins (acrylonitrile-styrene-methyl acrylate copolymers), etc., resins generally classified as thermoplastic resins; polylactic acid, etc. Examples of other resins: polysaccharides.
  • examples of the inorganic substance include silica and glass.
  • Quantum dot integrated nanoparticle has a structure in which the quantum dot is included in the matrix and / or adsorbed on the surface thereof.
  • the quantum dots may be dispersed inside the matrix and may or may not be chemically bonded to the matrix itself.
  • Fluorescent dye integrated nanoparticle has a structure in which a fluorescent dye is included in the matrix and / or adsorbed on the surface thereof.
  • fluorescent dyes include rhodamine dye molecules, squarylium dye molecules, cyanine dye molecules, aromatic ring dye molecules, oxazine dye molecules, carbopyronine dye molecules, and pyromesene dye molecules.
  • Alexa Fluor registered trademark, manufactured by Invitrogen
  • BODIPY registered trademark, manufactured by Invitrogen
  • Cy registered trademark, manufactured by GE Healthcare
  • HiLite registered trademark, manufactured by Anaspec
  • DyLight registered trademark, manufactured by Thermo Scientific
  • ATTO registered trademark, manufactured by ATTO-TEC
  • MFP registered trademark, manufactured by Mobitec
  • Dye molecules CF (registered trademark, manufactured by Biotium) dye molecules
  • DY registered trademark, manufactured by DYOMICICS
  • CAL registered trademark, manufactured by BioSearch Technologies
  • the generic name of such a dye molecule is named based on the main structure (skeleton) in the compound or a registered trademark, and the range of fluorescent dyes belonging to each of them must be excessively trial and error by those skilled in the art. It can be grasped appropriately.
  • the fluorescent dye When the fluorescent dye is encapsulated in the matrix, the fluorescent dye may be dispersed inside the matrix and may or may not be chemically bonded to the matrix itself.
  • tissue section staining method An example of the staining method will be described.
  • a method for preparing a tissue section to which this staining method can be applied (also simply referred to as “section”, including a section such as a pathological section) is not particularly limited, and a section prepared by a known procedure can be used.
  • Specimen preparation step (1.1) Deparaffinization treatment
  • the section is immersed in a container containing xylene to remove paraffin.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, xylene may be exchanged during the immersion.
  • the section is immersed in a container containing ethanol to remove xylene.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, ethanol may be exchanged during the immersion.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, water may be exchanged during the immersion.
  • the activation process of the target biological material is performed following a known method.
  • the activation conditions are not particularly defined, but as the activation liquid, 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1 M Tris-HCl buffer A liquid etc. can be used.
  • the pH condition is such that a signal is output from a pH range of 2.0 to 13.0 depending on the tissue slice used, and the roughness of the tissue is such that the signal can be evaluated.
  • the pH is 6.0 to 8.0, but for special tissue sections, for example, pH 3.0 is also used.
  • As the heating device an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used.
  • the temperature is not particularly limited, but can be performed at room temperature. The temperature can be 50 to 130 ° C. and the time can be 5 to 30 minutes.
  • the section after the activation treatment is immersed in a container containing PBS and washed.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, PBS may be replaced during the immersion.
  • an immunostaining agent solution containing fluorescent nanoparticles having sites that can be directly or indirectly bound to the target biological material is cut into sections. Put it on and react with the target biological material.
  • the immunostaining agent solution used in the immunostaining step may be prepared in advance before this step.
  • the conditions for performing the immunostaining process should be adjusted as appropriate in order to obtain an appropriate signal according to the conventional immunostaining method. Can do.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the reaction time is preferably 30 minutes or more and 24 hours or less.
  • a known blocking agent such as BSA-containing PBS or a surfactant such as Tween 20 dropwise.
  • the pathological specimen after the immunostaining process is preferably subjected to treatment such as immobilization / dehydration, penetration, and encapsulation so as to be suitable for observation.
  • the immobilization / dehydration treatment may be performed by immersing the pathological specimen in an immobilization treatment liquid (crosslinking agent such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol).
  • the clearing treatment may be performed by immersing the pathological specimen after the fixation / dehydration treatment in a clearing solution (xylene or the like).
  • the encapsulating process may be performed by immersing the pathological specimen that has undergone the penetration process in the encapsulating liquid. Conditions for performing these treatments, for example, the temperature and immersion time when the pathological specimen is immersed in a predetermined treatment solution, can be appropriately adjusted according to the conventional immunostaining method so as to obtain an appropriate signal. it can.
  • a morphological observation staining step can be included so that the morphology of cells, tissues, organs, etc. can be observed in a bright field.
  • the morphological observation staining step can be performed according to a conventional method. For morphological observation of tissue specimens, staining using eosin, in which cytoplasm, stroma, various fibers, erythrocytes, and keratinocytes are stained from red to dark red, is typically used.
  • hematoxylin in which cell nuclei, lime, cartilage tissue, bacteria, and mucus are stained blue-blue to light blue
  • HE staining eosin staining
  • the detection system 1 includes a fluorescence microscope 10, a detection device 20, and a display device 30.
  • the fluorescence microscope 10 includes a light source 12 and an imaging camera 14.
  • the fluorescence microscope 10 is connected to a detection device 20 that controls them, and the detection device 20 is connected to a display device 30 for displaying a fluorescence image.
  • the fluorescence microscope 10 is controlled by the detection device 20, and in the same field of view of the microscope at a desired magnification, each of the plurality of immunostaining agents (fluorescent nanoparticles) used in the immunostaining step is supplied from the light source 12.
  • each of the plurality of immunostaining agents fluorescent nanoparticles used in the immunostaining step is supplied from the light source 12.
  • Corresponding excitation light is irradiated to the pathological specimen, the fluorescent nanoparticles are fluorescently emitted to cause the appearance of fluorescent luminescent spots, and an immunostained image including the fluorescent luminescent spots is captured by the imaging camera 14.
  • the excitation light can be irradiated by using an excitation light filter that selectively transmits a predetermined wavelength as required.
  • the detection device 20 performs image processing on the imaging result of the imaging camera 14, generates a plurality of fluorescent images for each immunostaining agent, and displays them on the display device 30.
  • An example of software that can be used for image processing is “ImageJ” (open source).
  • image processing software By using such image processing software, a fluorescent luminescent spot having a predetermined wavelength (color) is extracted from the immunostained image and the sum of the luminance is calculated, or a fluorescent luminescent spot having a predetermined luminance or higher (described later). And the like.) Can be performed semi-automatically and quickly.
  • an immunostained image stained with the immunostaining agent B containing nanoparticles is captured, in the detection step S3, a fluorescent image 40 including a green fluorescent luminescent spot, and a fluorescent image 42 including a red fluorescent luminescent spot;
  • the two fluorescent images 40 and 42 can be generated.
  • the detection device 20 combines (superimposes) the generated plurality of fluorescent images 40 and 42 to detect the overlap of the fluorescent luminescent spots, and displays the detection result on the display device 30.
  • the green fluorescent spot and the red fluorescent spot are detected independently in the combined fluorescent image 44.
  • the green fluorescent spot and the red fluorescent spot overlap, and the yellow fluorescence A bright spot is detected.
  • the overlapping bright spot of the green fluorescent spot and the red fluorescent spot is displayed in a color (yellow) different from the color of the fluorescent spot (green, red) caused by a single fluorescent nanoparticle. An example is shown.
  • the fluorescence images 40 and 42 are generated in units of the immunostaining agents A and B, and these are synthesized to fluoresce bright spots. Only the overlap is detected, and it is possible to easily detect whether the distance between the target biological substances is close.
  • the color of the overlapping bright spot is displayed in yellow different from the fluorescent bright spot color (green, red) caused by the fluorescent nanoparticles of the immunostaining agents A and B. Whether the distance is short or not can be confirmed visually.
  • the plurality of fluorescent images generated in units of immunostaining agents may be generated by one fluorescence microscope 10 or may be generated by a plurality of fluorescence microscopes 10 that are different for each immunostaining agent.
  • the fluorescence images 40 and 42 may be generated by one fluorescence microscope 10 or may be separately generated by two fluorescence microscopes 10.
  • the fluorescence microscope 10 is also an example of an imaging device that irradiates a tissue section after staining with excitation light to cause a fluorescent luminescent spot to appear and picks up an image.
  • the imaging device is replaced with the fluorescence microscope 10.
  • An imaging device having a function equivalent to this and capable of imaging at high magnification may be used.
  • the human breast tissue specimen is immunostained (IHC method) using the immunostaining agents A and B prepared in (1) above by the method of the following steps (2-1) to (2-13). It was. Using a tissue array slide (CB-A712) manufactured by Cosmo Bio as a pathological specimen, the HER2 staining concentration and the HER3 staining concentration were observed in advance by DAB staining to confirm that HER2 and HER3 were expressed. went.
  • Step (2-7) PBS containing 1% BSA was placed on the pathological specimen and left for 1 hour.
  • Step (2-8) Immunostaining agent A bound with anti-HER2 antibody diluted to 0.05 nM with PBS containing 1% BSA was placed on a pathological specimen and allowed to stand for 3 hours.
  • Step (2-9) The stained pathological specimen was immersed in a container containing PBS for 30 minutes.
  • Step (2-10) Immunostaining agent B bound with anti-HER3 antibody diluted to 0.05 nM with PBS containing 1% BSA was placed on a pathological specimen and left for 3 hours.
  • Step (2-11) The stained pathological specimen was immersed in a container containing PBS for 30 minutes.
  • Observation and imaging with fluorescent microscope The stained tissue sections were observed and imaged using a fluorescent microscope.
  • a fluorescence microscope two types of fluorescence microscopes, a confocal microscope (FV1000 made by Olympus) and a general purpose microscope (BX53 made by Olympus) were used.
  • the number of bright spots was calculated from two fluorescent images of immunostaining agent A (fluorescent nanoparticles 1 / green quantum dots) and immunostaining agent B (fluorescent nanoparticles 2 / red quantum dots). .
  • immunostaining agent A fluorescent nanoparticles 1 / green quantum dots
  • immunostaining agent B fluorescent nanoparticles 2 / red quantum dots.
  • the sample in which each of the fluorescent nanoparticles 1 and 2 is dispersed on the glass plate is observed in advance, and the brightness and light emission pattern of each particle is determined in advance for each fluorescent nanoparticle 1 and 2.
  • the number of bright spots was calculated using this as a reference.
  • the range to be analyzed is an area where cancer cells are present, and the number of bright spots per 100 ⁇ m 2 is calculated by calculating the number of bright spots surrounding ROI as the target area of interest and dividing by 100 ⁇ m 2 . This is the number of bright spots per area of about one cell. In the calculation of the number of overlapping bright spots below, the number of bright spots per 100 ⁇ m 2 was similarly calculated.
  • the bright spot of the fluorescent image by the immunostaining agent A (fluorescent nanoparticle 1 / green quantum dot) is green.
  • the bright spot of the fluorescent image by the immunostaining agent B (fluorescent nanoparticle 2 / red quantum dot) is set to red and the two fluorescent images are synthesized, the green bright spot and the red bright spot
  • the overlapping bright spots were set to yellow (the bright spots other than the overlapping bright spots remained green or red), and the number of bright spots displayed in yellow was calculated.
  • Example 2-1 (1) Preparation of immunostaining agent (1.1) Synthesis of fluorescent dye-integrated nanoparticles 14.4 mg of Sulforhodamine 101 (manufactured by Sigma Aldrich), which is a red luminescent dye, was added to 22 mL of water and dissolved. Thereafter, 2 mL of a 5% aqueous solution of an emulsion (registered trademark) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation) of an emulsifier for emulsion polymerization was added to this solution. This solution was heated to 70 ° C.
  • emulsion registered trademark
  • the mixture was centrifuged at 20000 G for 15 minutes in a centrifuge (Kubota Micro Cooling Centrifuge 3740), and after removing the supernatant, ultrapure water was added and ultrasonically irradiated to redisperse. Centrifugation, supernatant removal, and washing by redispersion in ultrapure water were repeated 5 times.
  • red fluorescent dye-integrated nanoparticles (excitation wavelength: 590 nm, emission wavelength: 620 nm) were produced.
  • red fluorescent dye-integrated nanoparticles Pyrromethene556 dye was used in place of the Sulforhodamine 101 (Texas Red) dye, and green fluorescent dye-integrated nanoparticles (excitation wavelength 490 nm, emission wavelength 520 nm) were also prepared.
  • Step (1-1) 1 mg of the red fluorescent dye-integrated nanoparticles were dispersed in 5 mL of pure water. Next, 100 ⁇ L of aminopropyltriethoxysilane aqueous dispersion (LS-3150, manufactured by Shin-Etsu Chemical Co., Ltd.) was added and stirred at room temperature for 12 hours. Step (1-2): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
  • LS-3150 aminopropyltriethoxysilane aqueous dispersion
  • Step (1-6) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
  • Step (1-8) When 100 ⁇ g of anti-HER3 antibody was dissolved in 100 ⁇ L of PBS, 1M dithiothreitol (DTT) was added and reacted for 30 minutes.
  • Step (1-9) Excess DTT was removed from the reaction mixture with a gel filtration column to obtain a reduced anti-HER3 antibody solution capable of binding to the red fluorescent dye-aggregated nanoparticles.
  • Step (1-10) The red fluorescent dye-integrated nanoparticles dispersion obtained in step (1-7) using the red fluorescent dye-integrated nanoparticles as a starting material, and the reduction obtained in step (1-9) The anti-HER3 antibody solution was mixed in PBS and allowed to react for 1 hour.
  • Step (1-11) 4 ⁇ L of 10 mM mercaptoethanol was added to stop the reaction.
  • Example 2-2 In Example 2-1, instead of red fluorescent dye Sulforhodamine 101, Alexa Fluor 610 was used to produce red fluorescent dye integrated nanoparticles (excitation wavelength 630 nm, emission wavelength 650 nm). Otherwise, in the same manner as in Example 2-1, preparation of immunostaining, staining, observation / imaging with a fluorescence microscope, calculation of the number of bright spots, calculation of the number of overlapping bright spots were performed.
  • Example 2-3 In Example 2-1, instead of red fluorescent dye Sulforhodamine 101, Alexa Fluor 546 was used to produce orange fluorescent dye-integrated nanoparticles (excitation wavelength 530 nm, emission wavelength 550 nm), and ATTO instead of green fluorescent dye Pyrromethene556. Blue fluorescent dye integrated nanoparticles (excitation wavelength 400 nm, emission wavelength 480 nm) were prepared using 390. Otherwise, in the same manner as in Example 2-1, preparation of immunostaining, staining, observation / imaging with a fluorescence microscope, calculation of the number of bright spots, calculation of the number of overlapping bright spots were performed.
  • Example 1 when a confocal microscope was used, a bright spot-like fluorescent image was obtained. On the other hand, when a general-purpose microscope was used, a fluorescent image capable of calculating a bright spot was not obtained. Also in Examples 2-1 to 2-3, using a confocal microscope, bright spot-like fluorescence images were obtained. Even when a general-purpose fluorescence microscope was used, a fluorescent image capable of calculating a bright spot was easily obtained.
  • Example 1 From the results of Examples 1, 2-1, and 2-3 described above, if a fluorescent image is generated in units of immunostaining agents A and B, and these are synthesized to detect overlapping fluorescent spots, the antigens HER2, HER3 It can be seen that it can be easily detected whether or not the distance between them is short. From a comparison between Example 1 and Examples 2-1 to 2-3, it can be seen that it is more useful to use fluorescent dye-integrated nanoparticles as fluorescent nanoparticles in calculating the bright spot.
  • the present invention relates to a method for detecting a target biological material from a tissue section, and can be suitably used particularly for easily detecting whether or not the distance between target biological materials is short.

Abstract

A method for detecting a target biological substance, said method comprising: step (S1) for staining a tissue section with a plurality of immunostaining agents containing fluorescent nanoparticles having different light emission wavelengths from each other; step (S2) for irradiating the stained tissue section with excitation light and imaging the fluorescent bright spots thus appearing with an imaging device; and step (S3) for generating a plurality of fluorescent images respectively for the individual immunostaining agents on the basis of the imaged results from the imaging device and composing the fluorescent images to thereby detect overlapped fluorescent bright spots.

Description

目的生体物質の検出方法および検出システムMethod and system for detecting target biological material
　本発明は、組織切片から目的生体物質を検出する方法およびこれに用いられる検出システムに関する。 The present invention relates to a method for detecting a target biological substance from a tissue section and a detection system used therefor.
　従来の免疫染色において、組織切片上で興味のある抗原に対し、抗体修飾した「蛍光ナノ粒子」を結合させ、抗原を標識し、これを蛍光顕微鏡で観察・撮像することが行なわれている（特許文献１参照）。蛍光ナノ粒子には、「蛍光色素」と比較して、輝点状に高輝度で蛍光発光し蛍光輝点数の計測が可能といった特徴があり、顕微鏡画像から蛍光輝点を検出すると、抗原の状態や量を確認することができる。多種類の抗原を、互いに発光波長の異なる蛍光ナノ粒子で標識すれば、これらも区別し確認しうる。 In conventional immunostaining, antibody-modified “fluorescent nanoparticles” are bound to an antigen of interest on a tissue section, the antigen is labeled, and this is observed and imaged with a fluorescence microscope ( Patent Document 1). Compared with “fluorescent dyes”, fluorescent nanoparticles have the feature that they emit fluorescent light with high brightness and can measure the number of fluorescent bright spots. And the amount can be confirmed. If various types of antigens are labeled with fluorescent nanoparticles having different emission wavelengths, these can also be distinguished and confirmed.
　癌関連の抗原については、抗原間の距離が近いかどうかで悪性度に影響を与える場合があり、抗原間の距離情報を得ることは重要である。たとえば、癌関連の抗原として知られるＨＥＲ２とＨＥＲ３とがダイマー（２量体）を形成すると、癌の悪性度が増大することが知られている。それにもかかわらず、従来は、蛍光ナノ粒子を単独で使用し、抗原の分布情報を得ているにすぎず、抗原間の距離情報については着目されていなかった。 For cancer-related antigens, the degree of malignancy may be affected by whether or not the distance between antigens is close, and it is important to obtain distance information between antigens. For example, it is known that when HER2 and HER3, which are known as cancer-related antigens, form a dimer (dimer), the malignancy of cancer increases. Nevertheless, conventionally, only fluorescent nanoparticles are used to obtain antigen distribution information, and attention has not been paid to distance information between antigens.
　この点、「蛍光色素」を用いた免疫染色においては、蛍光エネルギー移動（ＦＲＥＴ；Fluorescence Resonance Energy Transfer）を検出し、抗原間（蛍光色素間）の距離を見積もることが行なわれている（非特許文献１、２参照）。
　「ＦＲＥＴ」とは、２個の蛍光色素が近接（距離＜約１０ｎｍ）したときに起こる蛍光色素間の相互作用であり、励起光の照射により第１の蛍光色素（ドナー）が励起され、励起状態のドナー色素から第２の蛍光色素（アクセプター）へ励起エネルギーが移動する現象である。かかるＦＲＥＴが起こると、ドナー色素の蛍光強度が低下し、アクセプター色素の蛍光発光が起こる。 In this regard, in immunostaining using a “fluorescent dye”, fluorescence energy transfer (FRET) is detected and the distance between antigens (between fluorescent dyes) is estimated (non-patent). References 1 and 2).
“FRET” is an interaction between fluorescent dyes that occurs when two fluorescent dyes come close to each other (distance <about 10 nm), and the first fluorescent dye (donor) is excited by irradiation with excitation light. This is a phenomenon in which excitation energy is transferred from a donor dye in a state to a second fluorescent dye (acceptor). When such FRET occurs, the fluorescence intensity of the donor dye decreases and the acceptor dye emits fluorescence.
国際公開第２０１２／０２９７５２号International Publication No. 2012/029752
　しかしながら、ＦＲＥＴを検出する方法では、蛍光色素同士が近接しているかどうか（抗原間の距離が近いかどうか）を検出することは可能であるものの、蛍光色素自体の蛍光強度が弱いため高感度な検出が要求されるし、ＦＲＥＴが起こったかどうかを識別するための特殊なフィルターも必要になり、抗原間の距離情報を得るのが煩雑になる。
　したがって本発明の主な目的は、目的生体物質間の距離が近いかどうかを容易に検出することができる目的生体物質の検出方法を提供することにある。 However, in the method for detecting FRET, it is possible to detect whether the fluorescent dyes are close to each other (whether the distance between the antigens is close), but since the fluorescent intensity of the fluorescent dye itself is weak, it is highly sensitive. Detection is required, and a special filter for identifying whether or not FRET has occurred is also required, which makes it difficult to obtain distance information between antigens.
Accordingly, a main object of the present invention is to provide a method for detecting a target biological material that can easily detect whether or not the distance between target biological materials is short.
　上記課題を解決するため、本発明によれば、
　組織切片から目的生体物質を検出する方法であって、
　互いに発光波長が異なる蛍光ナノ粒子を含む複数の免疫染色剤で組織切片を染色する工程と、
　染色後の組織切片に励起光を照射して蛍光輝点を出現させ、それを撮像装置で撮像する工程と、
　前記撮像装置の撮像結果に基づき、前記免疫染色剤単位で複数の蛍光画像を生成し、それら蛍光画像を合成して蛍光輝点の重複を検出する工程と、
　を備えることを特徴とする目的生体物質の検出方法が提供される。 In order to solve the above problems, according to the present invention,
A method for detecting a target biological material from a tissue section,
Staining a tissue section with a plurality of immunostaining agents including fluorescent nanoparticles having different emission wavelengths from each other;
Irradiating the tissue section after staining with excitation light to cause a fluorescent bright spot to appear, and imaging it with an imaging device;
Based on the imaging results of the imaging device, generating a plurality of fluorescent images in units of the immunostaining agent, synthesizing the fluorescent images and detecting the overlap of fluorescent bright spots,
A method for detecting a target biological substance is provided.
　本発明によれば、蛍光画像を免疫染色剤単位で生成し、これを合成して蛍光輝点の重複を検出するだけであるため、目的生体物質間の距離が近いかどうかを容易に検出することができる。 According to the present invention, it is possible to easily detect whether or not the distance between the target biological substances is close, because a fluorescent image is generated in units of immunostaining agents and synthesized to detect the overlap of fluorescent bright spots. be able to.
目的生体物質の検出方法を概略的に示すフローチャートである。It is a flowchart which shows the detection method of the target biological material roughly. 目的生体物質の検出システムの概略構成を示す図である。It is a figure which shows schematic structure of the detection system of the target biological material. 蛍光輝点の重複を検出する際のイメージ図であって、抗原間の距離が離間している場合を示す図である。It is an image figure at the time of detecting duplication of a fluorescent luminescent spot, Comprising: It is a figure which shows the case where the distance between antigens is separated. 蛍光輝点の重複を検出する際のイメージ図であって、抗原間の距離が近接している場合を示す図である。It is an image figure at the time of detecting duplication of a fluorescent luminescent spot, Comprising: It is a figure which shows the case where the distance between antigens is near.
　以下、図面を参照しながら本発明の好ましい実施形態について説明する。
　本明細書中、「～」で示す数値範囲には、その前後に記載される数値が下限値および上限値として含まれる。 Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.
In the present specification, the numerical value range indicated by “to” includes numerical values described before and after the numerical value as a lower limit value and an upper limit value.
　本発明にかかる目的生体物質の検出方法は、組織切片から目的生体物質を検出する方法であって、図１に示すとおり、
（Ｓ１）互いに発光波長が異なる蛍光ナノ粒子を含む複数の免疫染色剤で組織切片を染色する工程と、
（Ｓ２）染色後の組織切片に励起光を照射して蛍光輝点を出現させ、それを撮像装置で撮像する工程と、
（Ｓ３）撮像装置の撮像結果に基づき、免疫染色剤単位で複数の蛍光画像を生成し、それら蛍光画像を合成して蛍光輝点の重複を検出する工程と、
　を備えている。 The method for detecting a target biological material according to the present invention is a method for detecting a target biological material from a tissue section, as shown in FIG.
(S1) staining a tissue section with a plurality of immunostaining agents containing fluorescent nanoparticles having different emission wavelengths from each other;
(S2) irradiating a tissue section after staining with excitation light to cause a fluorescent bright spot to appear, and imaging it with an imaging device;
(S3) based on the imaging result of the imaging device, generating a plurality of fluorescent images in units of immunostaining, and combining the fluorescent images to detect the overlap of fluorescent bright spots;
It has.
　はじめに染色工程Ｓ１について説明し、その後に撮像工程Ｓ２および検出工程Ｓ３について説明する。特に、後者の撮像工程Ｓ２および検出工程Ｓ３では図２の検出システム１を使用する。 First, the staining step S1 will be described, and then the imaging step S2 and the detection step S3 will be described. In particular, in the latter imaging step S2 and detection step S3, the detection system 1 of FIG. 2 is used.
［目的生体物質］
　目的生体物質とは、主に病理診断の観点からの検出または定量のために、蛍光標識体を用いた免疫染色の対象とするものをいい、組織切片に発現している生体物質、特にタンパク質（抗原）である。
　典型的な目的生体物質としては、各種の癌組織の細胞膜で発現しており、バイオマーカーとして利用することができる生体物質が挙げられる。
　たとえば、ＥＧＦＲ（ＨＥＲ１）（Epidermal Growth Factor Receptor：上皮増殖因子受容体）、ＨＥＲ２（Human Epidermal Growth Factor Receptor：ヒト上皮増殖因子受容体）、ＨＥＲ３、ＨＥＲ４、ＶＥＧＦＲ（Vasular Endothelial Growth Factor Receptor：血管内皮細胞増殖因子受容体）、ＩＧＦＲ（Insulin-like Growth Factor Receptor：インスリン様増殖因子受容体）、ＨＧＦＲ（Hepatocyte Growth Factor Receptor：肝細胞増殖因子受容体）といった増殖因子の受容体（レセプター）や、ＰＤ－１（Programmedcell death 1）などの免疫系の受容体であるタンパク質が挙げられる。
　ＥＧＦＲ／ＨＥＲには、大腸癌などの癌組織において過剰発現しているＥＧＦＲ／ＨＥＲ１（ＥｒｂＢ１とも呼ばれる）、乳癌などの癌組織において過剰発現しているＥＧＦＲ２／ＨＥＲ２（ＥｒｂＢ２、ｎｅｕとも呼ばれる）、ＥＧＦＲ３／ＨＥＲ３およびＥＧＦＲ４／ＨＥＲ４が包含される。
　ＶＥＧＦＲには、肝臓癌、食道癌などの癌組織における血管内皮細胞において発現が亢進しているＶＥＧＦＲ－１（Ｆｌｔ－１とも呼ばれる）、ＶＥＧＦＲ－２（Ｆｌｔ－２、ＫＤＲとも呼ばれる）およびリンパ管内皮細胞において発現が亢進しているＶＥＧＦＲ－３（Ｆｌｔ－４とも呼ばれる）が包含される。 [Target biological material]
The target biological material is a target for immunostaining using a fluorescent label for detection or quantification mainly from the viewpoint of pathological diagnosis. The biological material expressed in tissue sections, particularly proteins ( Antigen).
Typical target biological materials include biological materials that are expressed in cell membranes of various cancer tissues and can be used as biomarkers.
For example, EGFR (HER1) (Epidermal Growth Factor Receptor), HER2 (Human Epidermal Growth Factor Receptor), HER3, HER4, VEGFR (Vasular Endothelial Growth Factor Receptor: Vascular Endothelial Cell) Growth factor receptors (IGF), insulin-like growth factor receptor (IGFR), HGFR (hepatocyte growth factor receptor), PD- Proteins that are receptors of the immune system such as 1 (Programmedcell death 1).
EGFR / HER includes EGFR / HER1 (also referred to as ErbB1) that is overexpressed in cancer tissues such as colorectal cancer, EGFR2 / HER2 (also referred to as ErbB2, neu) that is overexpressed in cancer tissues such as breast cancer, and EGFR3. / HER3 and EGFR4 / HER4 are included.
VEGFR includes VEGFR-1 (also referred to as Flt-1), VEGFR-2 (also referred to as Flt-2, KDR) and lymphatic vessels, which are upregulated in vascular endothelial cells in cancer tissues such as liver cancer and esophageal cancer. VEGFR-3 (also called Flt-4) whose expression is upregulated in the skin cells is included.
［免疫染色剤（抗体－蛍光ナノ粒子の結合体）］
　免疫染色剤としては、蛍光標識の効率を向上させて蛍光の劣化につながる時間経過をなるべく抑えるために、一次抗体および蛍光ナノ粒子が間接的に、つまり抗原抗体反応などを利用した、共有結合以外の結合によって連結される複合体を用いることが好ましい。染色操作を簡便にするため、免疫染色剤として、一次抗体または二次抗体に蛍光ナノ粒子が直結している複合体を用いることもできる。 [Immunostaining agent (antibody-fluorescent nanoparticle conjugate)]
As an immunostaining agent, in order to improve the efficiency of fluorescent labeling and suppress the time lapse leading to the deterioration of fluorescence as much as possible, primary antibodies and fluorescent nanoparticles are used indirectly, that is, other than covalent bonds using antigen-antibody reactions. It is preferable to use a complex linked by these bonds. In order to simplify the staining operation, a complex in which fluorescent nanoparticles are directly bound to a primary antibody or a secondary antibody can also be used as an immunostaining agent.
　免疫染色剤の一例として、［目的生体物質に対する一次抗体］…［一次抗体に対する抗体（二次抗体）］～［蛍光ナノ粒子］が挙げられる。
　“…”は抗原抗体反応により結合していることを表し、“～”が示す結合の態様としては特に限定されず、たとえば、共有結合、イオン結合、水素結合、配位結合、抗原抗体結合、ビオチンアビジン反応、物理吸着、化学吸着などが挙げられ、必要に応じてリンカー分子を介していてもよい。 Examples of immunostaining agents include [primary antibody against target biological substance] ... [antibody against primary antibody (secondary antibody)] to [fluorescent nanoparticles].
“...” represents binding by an antigen-antibody reaction, and the mode of binding indicated by “˜” is not particularly limited. For example, a covalent bond, an ionic bond, a hydrogen bond, a coordinate bond, an antigen-antibody bond, Examples include biotin avidin reaction, physical adsorption, chemical adsorption, and the like, and a linker molecule may be used as necessary.
　免疫染色剤には励起光の照射を受けて蛍光発光する蛍光ナノ粒子が含まれる。
　染色工程Ｓ１では複数の免疫染色剤が使用され、特に免疫染色剤同士で互いに発光波長が異なる蛍光ナノ粒子を含む複数の免疫染色剤が使用される。
　蛍光ナノ粒子として発光波長が４００～７００ｎｍのものを用いるのがよい。
　かかる発光波長は蛍光顕微鏡の検出感度領域にあり、汎用顕微鏡でも蛍光輝点を検出することができる。
　２種の蛍光ナノ粒子を使用する場合、一方の蛍光ナノ粒子は短波長の蛍光を発光し、他方の蛍光ナノ粒子は長波長の蛍光を発光するのがよい。一方の蛍光ナノ粒子は発光波長が４８０～５５０ｎｍ（青色～緑色）、好ましくは５００～５２０ｎｍ（緑色）であるのがよく、他方の蛍光ナノ粒子は発光波長が５５０～６５０ｎｍ（橙色～赤色）、好ましくは６２０～６５０ｎｍ（赤色）であるのがよい。 The immunostaining agent includes fluorescent nanoparticles that emit fluorescence when irradiated with excitation light.
In the staining step S1, a plurality of immunostaining agents are used, and in particular, a plurality of immunostaining agents containing fluorescent nanoparticles having different emission wavelengths from each other are used.
It is preferable to use fluorescent nanoparticles having an emission wavelength of 400 to 700 nm.
Such an emission wavelength is in the detection sensitivity region of a fluorescence microscope, and a fluorescent bright spot can be detected even with a general-purpose microscope.
When two types of fluorescent nanoparticles are used, it is preferable that one fluorescent nanoparticle emits short-wavelength fluorescence and the other fluorescent nanoparticle emits long-wavelength fluorescence. One fluorescent nanoparticle has an emission wavelength of 480 to 550 nm (blue to green), preferably 500 to 520 nm (green), and the other fluorescent nanoparticle has an emission wavelength of 550 to 650 nm (orange to red), Preferably, it is 620 to 650 nm (red).
　蛍光ナノ粒子として平均粒径が２０～３００ｎｍ、好ましくは５０～２００ｎｍのものを用いるのがよい。
　蛍光ナノ粒子の１粒子の粒径は、走査型電子顕微鏡（ＳＥＭ）を用いて電子顕微鏡写真を撮影して蛍光ナノ粒子の断面積を計測し、その断面形状を円と仮定したときに、その断面積に相当する円の直径として算出することができる。多数の蛍光ナノ粒子からなる集団の平均粒径は、十分な数（たとえば１０００個）の蛍光ナノ粒子について上記のようにして粒径を算出した後、その算術平均として算出される。 As the fluorescent nanoparticles, those having an average particle diameter of 20 to 300 nm, preferably 50 to 200 nm are preferably used.
The particle size of one fluorescent nanoparticle is calculated by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of the fluorescent nanoparticle, and assuming that the cross-sectional shape is a circle. It can be calculated as the diameter of a circle corresponding to the cross-sectional area. The average particle size of a group of a large number of fluorescent nanoparticles is calculated as the arithmetic average after calculating the particle size as described above for a sufficient number (for example, 1000) of fluorescent nanoparticles.
［抗体］
　一次抗体には、目的生体物質としてのタンパク質を抗原として特異的に認識して結合する抗体（ＩｇＧ）を用いることができる。たとえば、ＥＧＦＲを目的生体物質とする場合は抗ＥＧＦＲ抗体を、ＨＥＲ２を目的生体物質とする場合は抗ＨＥＲ２抗体を、ＨＥＲ３を目的生体物質とする場合は抗ＨＥＲ３抗体を、それぞれ用いることができる。
　二次抗体には、一次抗体を抗原として特異的に認識して結合する抗体（ＩｇＧ）を用いることができる。
　一次抗体および二次抗体はいずれも、ポリクローナル抗体であってもよいが、定量の安定性の観点から、モノクローナル抗体が好ましい。抗体を産生する動物（免疫動物）の種類は特に限定されるものではなく、従来と同様、マウス、ラット、モルモット、ウサギ、ヤギ、ヒツジなどから選択すればよい。 [antibody]
As the primary antibody, an antibody (IgG) that specifically recognizes and binds a protein as a target biological substance as an antigen can be used. For example, an anti-EGFR antibody can be used when EGFR is the target biological material, an anti-HER2 antibody can be used when HER2 is the target biological material, and an anti-HER3 antibody when HER3 is the target biological material.
As the secondary antibody, an antibody (IgG) that specifically recognizes and binds to the primary antibody as an antigen can be used.
Both the primary antibody and the secondary antibody may be polyclonal antibodies, but from the viewpoint of quantitative stability, monoclonal antibodies are preferred. 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 past.
［蛍光ナノ粒子］
　蛍光ナノ粒子とは、目的生体物質を１分子ずつ輝点として表すのに十分な強度の蛍光を発することができるナノサイズの粒子である。
　蛍光ナノ粒子として、好ましくは量子ドット（半導体ナノ粒子）、蛍光物質集積ナノ粒子が使用される。 [Fluorescent nanoparticles]
Fluorescent nanoparticles are nano-sized particles that can emit fluorescence with a sufficient intensity to express the target biological substance as a bright spot one molecule at a time.
As the fluorescent nanoparticles, preferably, quantum dots (semiconductor nanoparticles) and fluorescent substance integrated nanoparticles are used.
（１）量子ドット
　量子ドットとしては、ＩＩ－ＶＩ族化合物、ＩＩＩ－Ｖ族化合物またはＩＶ族元素を含有する半導体ナノ粒子が使用される。たとえば、ＣｄＳｅ、ＣｄＳ、ＣｄＴｅ、ＺｎＳｅ、ＺｎＳ、ＺｎＴｅ、ＩｎＰ、ＩｎＮ、ＩｎＡｓ、ＩｎＧａＰ、ＧａＰ、ＧａＡｓ、Ｓｉ、Ｇｅなどが挙げられる。 (1) Quantum dots As quantum dots, semiconductor nanoparticles containing a II-VI group compound, a III-V group compound, or a group IV element are used. Examples thereof include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.
（２）蛍光物質集積ナノ粒子
　蛍光物質集積ナノ粒子は、有機物または無機物でできた粒子を母体とし、複数の蛍光物質（たとえば、上記量子ドット、蛍光色素など）がその中に内包されているおよび／またはその表面に吸着している構造を有する、ナノサイズの粒子である。
　蛍光物質集積ナノ粒子としては、母体と蛍光物質とが、互いに反対の電荷を有する置換基または部位を有し、静電的相互作用が働くものであることが好適である。
　蛍光物質集積ナノ粒子としては、量子ドット集積ナノ粒子、蛍光色素集積ナノ粒子などが使用される。 (2) Fluorescent substance integrated nanoparticles Fluorescent substance integrated nanoparticles are based on particles made of organic or inorganic substances, and a plurality of fluorescent substances (for example, the above-described quantum dots and fluorescent dyes) are encapsulated therein and Nanoparticles having a structure adsorbed on the surface thereof.
As the fluorescent substance-integrated nanoparticles, it is preferable that the matrix and the fluorescent substance have substituents or sites having opposite charges, and an electrostatic interaction works.
As the fluorescent substance integrated nanoparticles, quantum dot integrated nanoparticles, fluorescent dye integrated nanoparticles, and the like are used.
（２．１）母体
　母体のうち、有機物としては、メラミン樹脂、尿素樹脂、アニリン樹脂、グアナミン樹脂、フェノール樹脂、キシレン樹脂、フラン樹脂など、一般的に熱硬化性樹脂に分類される樹脂；スチレン樹脂、アクリル樹脂、アクリロニトリル樹脂、ＡＳ樹脂（アクリロニトリル－スチレン共重合体）、ＡＳＡ樹脂（アクリロニトリル－スチレン－アクリル酸メチル共重合体）など、一般的に熱可塑性樹脂に分類される樹脂；ポリ乳酸等のその他の樹脂；多糖を例示することができる。
　母体のうち、無機物としては、シリカ、ガラスなどを例示することができる。 (2.1) Base material Among the base materials, as organic substances, resins generally classified as thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, furan resin; styrene Resins, acrylic resins, acrylonitrile resins, AS resins (acrylonitrile-styrene copolymers), ASA resins (acrylonitrile-styrene-methyl acrylate copolymers), etc., resins generally classified as thermoplastic resins; polylactic acid, etc. Examples of other resins: polysaccharides.
Of the matrix, examples of the inorganic substance include silica and glass.
（２．２）量子ドット集積ナノ粒子
　量子ドット集積ナノ粒子とは、上記量子ドットが、上記母体の中に内包されている、および／またはその表面に吸着している構造を有する。
　量子ドットが母体に内包されている場合、量子ドットは母体内部に分散されていればよく、母体自体と化学的に結合していてもよいし、していなくてもよい。 (2.2) Quantum dot integrated nanoparticle The quantum dot integrated nanoparticle has a structure in which the quantum dot is included in the matrix and / or adsorbed on the surface thereof.
When the quantum dots are encapsulated in the matrix, the quantum dots may be dispersed inside the matrix and may or may not be chemically bonded to the matrix itself.
（２．３）蛍光色素集積ナノ粒子
　蛍光色素集積ナノ粒子とは、蛍光色素が、上記母体の中に内包されている、および／またはその表面に吸着している構造を有する。
　蛍光色素としては、ローダミン系色素分子、スクアリリウム系色素分子、シアニン系色素分子、芳香環系色素分子、オキサジン系色素分子、カルボピロニン系色素分子、ピロメセン系色素分子などを例示することができる。
　蛍光色素としては、Ａｌｅｘａ　Ｆｌｕｏｒ（登録商標、インビトロジェン社製）系色素分子、ＢＯＤＩＰＹ（登録商標、インビトロジェン社製）系色素分子、Ｃｙ（登録商標、ＧＥヘルスケア社製）系色素分子、ＨｉＬｙｔｅ（登録商標、アナスペック社製）系色素分子、ＤｙＬｉｇｈｔ（登録商標、サーモサイエンティフィック社製）系色素分子、ＡＴＴＯ（登録商標、ＡＴＴＯ－ＴＥＣ社製）系色素分子、ＭＦＰ（登録商標、Ｍｏｂｉｔｅｃ社製）系色素分子、ＣＦ（登録商標、Ｂｉｏｔｉｕｍ社製）系色素分子、ＤＹ（登録商標、ＤＹＯＭＩＣＳ社製）系色素分子、ＣＡＬ（登録商標、ＢｉｏＳｅａｒｃｈ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）系色素分子などを用いることができる。 (2.3) Fluorescent dye integrated nanoparticle The fluorescent dye integrated nanoparticle has a structure in which a fluorescent dye is included in the matrix and / or adsorbed on the surface thereof.
Examples of fluorescent dyes include rhodamine dye molecules, squarylium dye molecules, cyanine dye molecules, aromatic ring dye molecules, oxazine dye molecules, carbopyronine dye molecules, and pyromesene dye molecules.
As fluorescent dyes, Alexa Fluor (registered trademark, manufactured by Invitrogen) -based dye molecule, BODIPY (registered trademark, manufactured by Invitrogen) -based dye molecule, Cy (registered trademark, manufactured by GE Healthcare) -based dye molecule, HiLite (registered) Trademark, manufactured by Anaspec) dye molecule, DyLight (registered trademark, manufactured by Thermo Scientific) dye molecule, ATTO (registered trademark, manufactured by ATTO-TEC) dye molecule, MFP (registered trademark, manufactured by Mobitec) ) Dye molecules, CF (registered trademark, manufactured by Biotium) dye molecules, DY (registered trademark, manufactured by DYOMICICS) dye molecules, CAL (registered trademark, manufactured by BioSearch Technologies) dye molecules, and the like can be used. .
　具体的には、５－カルボキシ－フルオレセイン、６－カルボキシ－フルオレセイン、５，６－ジカルボキシ－フルオレセイン、６－カルボキシ－２’，４，４’，５’，７，７’－ヘキサクロロフルオレセイン、６－カルボキシ－２’，４，７，７’－テトラクロロフルオレセイン、６－カルボキシ－４’，５’－ジクロロ－２’，７’－ジメトキシフルオレセイン、ナフトフルオレセイン、５－カルボキシ－ローダミン、６－カルボキシ－ローダミン、５，６－ジカルボキシ－ローダミン、ローダミン　６Ｇ、テトラメチルローダミン、Ｘ－ローダミン、スルホローダミンB、スルホローダミン１０１、及びAlexa Fluor350、Alexa Fluor 405、Alexa Fluor 430、Alexa Fluor 488、Alexa Fluor 500、Alexa Fluor 514、Alexa Fluor 532、Alexa Fluor 546、Alexa Fluor 555、Alexa Fluor 568、Alexa Fluor 594、Alexa Fluor 610、Alexa Fluor 633、Alexa Fluor 635、Alexa Fluor 647、Alexa Fluor 660、Alexa Fluor 680、Alexa Fluor 700、Alexa Fluor 750、BODIPY FL、BODIPY TMR、BODIPY 493／503、BODIPY 530／550、BODIPY 558／568、BODIPY 564／570、BODIPY 576／589、BODIPY 581／591、BODIPY 630／650、BODIPY 650／665（以上インビトロジェン社製）、メトキシクマリン、クマリン６、クマリン７、スルホクマリン６、スルホクマリン７、エオジン、ＮＢＤ、ピレン、Cy5、Cy5.5、Cy7、HiLyte Fluor 488、HiLyte Fluor 555、HiLyte Fluor 594、HiLyte Fluor 647、HiLyte Fluor 680、HiLyte Fluor 750（登録商標、アナスペック社製）、DyLight 350、DyLight 405、DyLight 488、DyLight 550、DyLight 594、DyLight 633、DyLight 650、DyLight 680、DyLight 755、DyLight 800（登録商標、サーモサイエンティフィック社製）、ATTO 390、ATTO 425、ATTO 465、ATTO 488、ATTO 495、ATTO 514、ATTO 520、ATTO 532、ATTO Rho6G、ATTO 542、ATTO 550、ATTO 565、ATTO Rho3B、ATTO Rho11、ATTO Rho12、ATTO Thio12、ATTO Rho101、ATTO 590、ATTO 594、ATTO Rho13、ATTO 610、ATTO 620、ATTO Rho14、ATTO 633、ATTO 647、ATTO 647N、ATTO 655、ATTO Oxa12、ATTO 665、ATTO 680、ATTO 700、ATTO 725、ATTO 740（登録商標、ＡＴＴＯ－ＴＥＣ社製）、MFP488、MFP555、MFP590、MFP631（登録商標、Ｍｏｂｉｔｅｃ社製）、CF350、CF405S、CF405M、CF488A、CF514、CF532、CF543、CF555、CF568、CF594、CF620R、CF633、CF640R、CF647、CF660C、CF660R、CF680、CF680R、CF750、CF770、CF790（登録商標、Ｂｉｏｔｉｕｍ社製）、DY-350、DY-405、DY-415、DY-480XL、DY-481XL、DY-485XL、DY-490、DY-495、DY-505、DY-500XL、DY-510XL、DY-520XL、DY-521XL、DY-530、DY-547P1、DY-549P1、DY-550、DY-554、DY-555、DY-556、DY-560、DY-590、DY-591、DY-594、DY-605、DY-610、DY-615、DY-630、DY-631、DY-632、DY-633、DY-634、DY-635、DY-636、DY-647P1、DY-648P1、DY-649P1、DY-650、DY-654、DY-651、DY-652、DY-675、DY-676、DY-677、DY-678、DY-679P1、DY-680、DY-681、DY-682、DY-700、DY-701、DY-703、DY-704、DY-730、DY-731、DY-732、DY-734、DY-749P1、DY-750、DY-751、DY-752、DY-754、DY-776、DY-777、DY-778、DY-780、DY-781、DY-782、DY-800、DY-831（ＤＹＯＭＩＣＳ社製）、CAL Fluor Green 520、CAL Fluor Gold 540、CAL Fluor Orange 560、CAL Fluor Red 590、CAL Fluor Red 610、CAL Fluor Red 615、CAL Fluor Red 635、Pulsar 650（登録商標、ＢｉｏＳｅａｒｃｈ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）、5,10,15,20-テトラフェニルポルフィンテトラスルホン酸、亜鉛5,10,15,20-テトラフェニルポルフィンテトラスルホン酸、フタロシアニンテトラスルホン酸、亜鉛フタロシアニンテトラスルホン酸、Ｎ, Ｎ－Ｂｉｓ－（２，６－ｄｉｉｓｏｐｒｏｐｙｌｐｈｅｎｙｌ）－１，６，７，１２－（４－ｔｅｒｔ－ｂｕｔｙｌｐｈｅｎｏｘｙ）－ｐｅｒｙｌｅｎ－３，４，９，１０－ｔｅｔｒａｃａｒｂｏｎａｃｉｄ ｄｉｉｍｉｄｅ、Ｎ,Ｎ’－Ｂｉｓ（２，６－ｄｉｉｓｏｐｒｏｐｙｌｐｈｅｎｙｌ）－１，６，７，１２－ｔｅｔｒａｐｈｅｎｏｘｙｐｅｒｙｌｅｎｅ－３，４：９，１０－ｔｅｔｒａｃａｒｂｏｘｄｉｉｍｉｄｅ、Benzenesulfonic acid, 4,4',4'',4'''-[(1,3,8,10-tetrahydro-1,3,8,10-tetraoxoperylo[3,4-cd:9,10-c'd']dipyran-5,6,12,13-tetrayl)tetralis(oxy)]tetrakis-などを用いることができる。
　これら蛍光色素は単独で使用されてもよいし、複数種が混合され使用されてもよい。 Specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein, 6 -Carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy Rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, sulforhodamine B, sulforhodamine 101, and Alexa Fluor350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500 , Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/550 568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 (manufactured by Invitrogen), methoxycoumarin, coumarin 6, coumarin 7, sulfocoumarin 6, sulfocoumarin 7, Eodine, NBD, pyrene, Cy5, Cy5.5, Cy7, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 594, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750 (registered trademark, manufactured by Anaspec), DyLight 350, DyLight 405, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 755, DyLight 800 (registered trademark, manufactured by Thermo Scientific), ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N , ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740 (registered trademark, manufactured by ATTO-TEC), MFP488, MFP555, MFP590, MFP631 (registered trademark, manufactured by Mobitec), CF350, CF405S, CF405M, CF488A, CF514, CF532, CF543, CF555, CF568, CF594, CF620R, CF633, CF640R, CF647, CF660C, CF660R, CF680, CF680R, CF750, CF770, CF790 (registered trademark, manufactured by Biotium), DY- 350, DY-405, DY-415, DY-480XL, DY-481XL, DY-485XL, DY-490, DY-495, DY-505, DY-500XL, DY-510XL, DY-520XL, DY-521XL, DY-530, DY-547P1, DY-549P1, DY-550, DY-554, DY-555, DY-556, DY-560, DY-590, DY-591, DY-594, DY-605, DY- 610, DY-615, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, DY-647P1, DY-648P1, DY-649P1, D Y-650, DY-654, DY-651, DY-652, DY-675, DY-676, DY-677, DY-678, DY-679P1, DY-680, DY-681, DY-682, DY- 700, DY-701, DY-703, DY-704, DY-730, DY-731, DY-732, DY-734, DY-749P1, DY-750, DY-751, DY-752, DY-754, DY-776, DY-777, DY-778, DY-780, DY-781, DY-782, DY-800, DY-831 (DYOMICS), CAL Fluor Green 520, CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 615, CAL Fluor Red 635, Pulsar 650 (registered trademark, manufactured by BioSearch Technologies), 5,10,15,20-tetraphenylporphine tetrasulfonic acid, zinc 5,10,15,20-tetraphenylporphine tetrasulfonic acid, phthalocyanine tetrasulfonic acid, zinc phthalocyanine tetrasulfonic acid, N, N-Bis- (2,6-diisopropylphenyl) -1,6,7,12- (4 -Tert-butyl phenoxy) -perylen-3,4,9,10-tetracarbonacid diimide, N, N'-Bis (2,6-diisoprophylphenyl) -1,6,7,12-tetraphenoxyperylene-3, 4: 9,10-tetracarboxidi, Benzenesulfonic acid, 4,4 ', 4``, 4'''-[(1,3,8,10-tetrahydro-1,3,8,10-tetraoxoperylo [3,4-cd: 9,10-c 'd'] dipyran-5,6,12,13-tetrayl) tetralis (oxy)] tetrakis- etc. can be used.
These fluorescent dyes may be used alone or in combination of two or more.
　なお、このような色素分子の総称は、化合物中の主要な構造（骨格）または登録商標に基づき命名されており、それぞれに属する蛍光色素の範囲は当業者であれば過度の試行錯誤を要することなく適切に把握できるものである。
　蛍光色素が母体に内包されている場合、蛍光色素は母体内部に分散されていればよく、母体自体と化学的に結合していてもよいし、していなくてもよい。 In addition, the generic name of such a dye molecule is named based on the main structure (skeleton) in the compound or a registered trademark, and the range of fluorescent dyes belonging to each of them must be excessively trial and error by those skilled in the art. It can be grasped appropriately.
When the fluorescent dye is encapsulated in the matrix, the fluorescent dye may be dispersed inside the matrix and may or may not be chemically bonded to the matrix itself.
［組織切片の染色方法］
　染色方法の一例について説明する。
　この染色方法が適用できる組織切片（単に「切片」ともいい、病理切片などの切片も含まれる。）の作製法は特に限定されず、公知の手順により作製されたものを用いることができる。 [Tissue section staining method]
An example of the staining method will be described.
A method for preparing a tissue section to which this staining method can be applied (also simply referred to as “section”, including a section such as a pathological section) is not particularly limited, and a section prepared by a known procedure can be used.
（１）標本作製工程
（１．１）脱パラフィン処理
　キシレンを入れた容器に、切片を浸漬させ、パラフィン除去する。温度は特に限定されるものではないが、室温で行うことができる。浸漬時間は、３分以上３０分以下であることが好ましい。また必要により浸漬途中でキシレンを交換してもよい。 (1) Specimen preparation step (1.1) Deparaffinization treatment The section is immersed in a container containing xylene to remove paraffin. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, xylene may be exchanged during the immersion.
　次いでエタノールを入れた容器に切片を浸漬させ、キシレン除去する。温度は特に限定されるものではないが、室温で行うことができる。浸漬時間は、３分以上３０分以下であることが好ましい。また必要により浸漬途中でエタノールを交換してもよい。 Next, the section is immersed in a container containing ethanol to remove xylene. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, ethanol may be exchanged during the immersion.
　水を入れた容器に、切片を浸漬させ、エタノール除去する。温度は特に限定されるものではないが、室温で行うことができる。浸漬時間は、３分以上３０分以下であることが好ましい。また必要により浸漬途中で水を交換してもよい。 Immerse the slices in a container containing water and remove the ethanol. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, water may be exchanged during the immersion.
（１．２）賦活化処理
　公知の方法に倣い、目的生体物質の賦活化処理を行う。賦活化条件に特に定めはないが、賦活液としては、０．０１Ｍのクエン酸緩衝液（ｐＨ６．０）、１ｍＭのＥＤＴＡ溶液（ｐＨ８．０）、５％尿素、０．１Ｍのトリス塩酸緩衝液などを用いることができる。
　ｐＨ条件は用いる組織切片に応じてｐＨ２．０～１３．０の範囲から、シグナルが出て、組織の荒れがシグナルを評価できる程度となる条件で行う。通常はｐＨ６．０～８．０で行うが、特殊な組織切片ではたとえばｐＨ３．０でも行う。
　加熱機器はオートクレーブ、マイクロウェーブ、圧力鍋、ウォーターバスなどを用いることができる。温度は特に限定されるものではないが、室温で行うことができる。温度は５０～１３０℃、時間は５～３０分で行うことができる。 (1.2) Activation process The activation process of the target biological material is performed following a known method. The activation conditions are not particularly defined, but as the activation liquid, 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1 M Tris-HCl buffer A liquid etc. can be used.
The pH condition is such that a signal is output from a pH range of 2.0 to 13.0 depending on the tissue slice used, and the roughness of the tissue is such that the signal can be evaluated. Usually, the pH is 6.0 to 8.0, but for special tissue sections, for example, pH 3.0 is also used.
As the heating device, an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used. The temperature is not particularly limited, but can be performed at room temperature. The temperature can be 50 to 130 ° C. and the time can be 5 to 30 minutes.
　次いでＰＢＳを入れた容器に、賦活処理後の切片を浸漬させ、洗浄を行う。温度は特に限定されるものではないが、室温で行うことができる。浸漬時間は、３分以上３０分以下であることが好ましい。また必要により浸漬途中でＰＢＳを交換してもよい。 Next, the section after the activation treatment is immersed in a container containing PBS and washed. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, PBS may be replaced during the immersion.
（２）免疫染色工程
　免疫染色工程では、目的生体物質を染色するために、目的生体物質に直接的または間接的に結合しうる部位を有する蛍光ナノ粒子を含む免疫染色剤の溶液を、切片に乗せ、目的生体物質との反応を行う。免疫染色工程に用いる免疫染色剤の溶液については、この工程の前にあらかじめ調製しておけばよい。 (2) Immunostaining step In the immunostaining step, in order to stain the target biological material, an immunostaining agent solution containing fluorescent nanoparticles having sites that can be directly or indirectly bound to the target biological material is cut into sections. Put it on and react with the target biological material. The immunostaining agent solution used in the immunostaining step may be prepared in advance before this step.
　免疫染色工程を行う上での条件、すなわち免疫染色剤の溶液に病理標本を浸漬する際の温度および浸漬時間は、従来の免疫染色法に準じて、適切なシグナルが得られるよう適宜調整することができる。
　温度は特に限定されるものではないが、室温で行うことができる。反応時間は、３０分以上２４時間以下であることが好ましい。
　上述したような処理を行う前に、ＢＳＡ含有ＰＢＳなど公知のブロッキング剤やＴｗｅｅｎ２０などの界面活性剤を滴下することが好ましい。 The conditions for performing the immunostaining process, that is, the temperature and immersion time when immersing the pathological specimen in the immunostaining solution, should be adjusted as appropriate in order to obtain an appropriate signal according to the conventional immunostaining method. Can do.
The temperature is not particularly limited, but can be performed at room temperature. The reaction time is preferably 30 minutes or more and 24 hours or less.
Before performing the above-described treatment, it is preferable to add a known blocking agent such as BSA-containing PBS or a surfactant such as Tween 20 dropwise.
（３）標本後処理工程
　免疫染色工程を終えた病理標本は、観察に適したものとなるよう、固定化・脱水、透徹、封入などの処理を行うことが好ましい。 (3) Specimen post-treatment process The pathological specimen after the immunostaining process is preferably subjected to treatment such as immobilization / dehydration, penetration, and encapsulation so as to be suitable for observation.
　固定化・脱水処理は、病理標本を固定処理液（ホルマリン、パラホルムアルデヒド、グルタールアルデヒド、アセトン、エタノール、メタノールなどの架橋剤）に浸漬すればよい。透徹処理は、固定化・脱水処理を終えた病理標本を透徹液（キシレンなど）に浸漬すればよい。封入処理は、透徹処理を終えた病理標本を封入液に浸漬すればよい。
　これらの処理を行う上での条件、たとえば病理標本を所定の処理液に浸漬する際の温度および浸漬時間は、従来の免疫染色法に準じて、適切なシグナルが得られるよう適宜調整することができる。 The immobilization / dehydration treatment may be performed by immersing the pathological specimen in an immobilization treatment liquid (crosslinking agent such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol). The clearing treatment may be performed by immersing the pathological specimen after the fixation / dehydration treatment in a clearing solution (xylene or the like). The encapsulating process may be performed by immersing the pathological specimen that has undergone the penetration process in the encapsulating liquid.
Conditions for performing these treatments, for example, the temperature and immersion time when the pathological specimen is immersed in a predetermined treatment solution, can be appropriately adjusted according to the conventional immunostaining method so as to obtain an appropriate signal. it can.
（４）任意工程
　もしも必要であれば、明視野において細胞、組織、臓器などの形態を観察することができるようにするための、形態観察染色工程を含めることができる。形態観察染色工程は、常法に従って行うことができる。組織標本の形態観察に関しては、細胞質・間質・各種線維・赤血球・角化細胞が赤～濃赤色に染色される、エオジンを用いた染色が標準的に用いられている。また、細胞核・石灰部・軟骨組織・細菌・粘液が青藍色～淡青色に染色される、ヘマトキシリンを用いた染色も標準的に用いられている（これら２つの染色を同時に行う方法はヘマトキシリン・エオジン染色（ＨＥ染色）として知られている）。
　形態観察染色工程を含める場合は、免疫染色工程の後に行うようにしてもよいし、免疫染色工程の前に行うようにしてもよい。 (4) Optional step If necessary, a morphological observation staining step can be included so that the morphology of cells, tissues, organs, etc. can be observed in a bright field. The morphological observation staining step can be performed according to a conventional method. For morphological observation of tissue specimens, staining using eosin, in which cytoplasm, stroma, various fibers, erythrocytes, and keratinocytes are stained from red to dark red, is typically used. In addition, staining using hematoxylin, in which cell nuclei, lime, cartilage tissue, bacteria, and mucus are stained blue-blue to light blue, is also used as a standard (the method of performing these two stainings simultaneously is hematoxylin, Known as eosin staining (HE staining)).
When the morphological observation staining step is included, it may be performed after the immunostaining step or may be performed before the immunostaining step.
［検出システム］
　撮像工程Ｓ２および検出工程Ｓ３では図２の検出システム１を使用する。
　検出システム１は蛍光顕微鏡１０、検出装置２０および表示装置３０を備えている。蛍光顕微鏡１０は光源１２および撮像カメラ１４を備えている。蛍光顕微鏡１０にはこれらを制御する検出装置２０が接続され、検出装置２０には蛍光画像を表示するための表示装置３０が接続されている。 [Detection system]
In the imaging step S2 and the detection step S3, the detection system 1 in FIG. 2 is used.
The detection system 1 includes a fluorescence microscope 10, a detection device 20, and a display device 30. The fluorescence microscope 10 includes a light source 12 and an imaging camera 14. The fluorescence microscope 10 is connected to a detection device 20 that controls them, and the detection device 20 is connected to a display device 30 for displaying a fluorescence image.
　撮像工程Ｓ２では、検出装置２０で蛍光顕微鏡１０を制御し、所望の倍率における顕微鏡の同一視野において、光源１２から、免疫染色工程に用いられた複数の免疫染色剤（蛍光ナノ粒子）のそれぞれに対応した励起光を病理標本に照射し、それら蛍光ナノ粒子を蛍光発光させ蛍光輝点を出現させ、その蛍光輝点を含む免疫染色像を撮像カメラ１４で撮像する。
　励起光の照射は、必要に応じて所定の波長を選択的に透過させる励起光用フィルターを用いることで照射することができる。
　免疫染色像の撮像の際には、必要に応じて所定の波長を選択的に透過させる蛍光用フィルターを用いることで、目的とする蛍光のみを含み、目的としない蛍光やノイズとなる励起光およびその他の光を排除した免疫染色像を撮像することができる。 In the imaging step S2, the fluorescence microscope 10 is controlled by the detection device 20, and in the same field of view of the microscope at a desired magnification, each of the plurality of immunostaining agents (fluorescent nanoparticles) used in the immunostaining step is supplied from the light source 12. Corresponding excitation light is irradiated to the pathological specimen, the fluorescent nanoparticles are fluorescently emitted to cause the appearance of fluorescent luminescent spots, and an immunostained image including the fluorescent luminescent spots is captured by the imaging camera 14.
The excitation light can be irradiated by using an excitation light filter that selectively transmits a predetermined wavelength as required.
When imaging an immunostained image, use a fluorescent filter that selectively transmits a predetermined wavelength as necessary, so that only the target fluorescence is included, and excitation light that causes unwanted fluorescence or noise and An immunostained image in which other light is excluded can be taken.
　検出工程Ｓ３では、検出装置２０で、撮像カメラ１４の撮像結果を画像処理し、免疫染色剤単位で複数の蛍光画像を生成し、表示装置３０に表示させる。
　画像処理に用いることができるソフトウェアとしては、たとえば「ＩｍａｇｅＪ」（オープンソース）が挙げられる。このような画像処理ソフトウェアを利用することにより、免疫染色像から、所定の波長（色）の蛍光輝点を抽出してその輝度の総和を算出したり、所定の輝度以上の蛍光輝点（後述の重複輝点を含む。）の数を算出したりする処理を、半自動的に、迅速に行うことができる。 In the detection step S <b> 3, the detection device 20 performs image processing on the imaging result of the imaging camera 14, generates a plurality of fluorescent images for each immunostaining agent, and displays them on the display device 30.
An example of software that can be used for image processing is “ImageJ” (open source). By using such image processing software, a fluorescent luminescent spot having a predetermined wavelength (color) is extracted from the immunostained image and the sum of the luminance is calculated, or a fluorescent luminescent spot having a predetermined luminance or higher (described later). And the like.) Can be performed semi-automatically and quickly.
　たとえば、図３Ａ、図３Ｂに示すとおり、目的生体物質として抗原Ａ、Ｂを含む組織切片の病理標本を、緑色で蛍光発光する蛍光ナノ粒子を含む免疫染色剤Ａと、赤色で蛍光発光する蛍光ナノ粒子を含む免疫染色剤Ｂとで染色した免疫染色像を撮像した場合には、検出工程Ｓ３では、緑色の蛍光輝点を含む蛍光画像４０と、赤色の蛍光輝点を含む蛍光画像４２との２枚の蛍光画像４０、４２を生成しうる。 For example, as shown in FIGS. 3A and 3B, a pathological specimen of a tissue section containing antigens A and B as target biological materials, an immunostaining agent A containing fluorescent nanoparticles that fluoresce in green, and fluorescence that fluoresces in red When an immunostained image stained with the immunostaining agent B containing nanoparticles is captured, in the detection step S3, a fluorescent image 40 including a green fluorescent luminescent spot, and a fluorescent image 42 including a red fluorescent luminescent spot; The two fluorescent images 40 and 42 can be generated.
　その後、検出装置２０で、生成したそれら複数の蛍光画像４０、４２を合成して（重ね合わせて）蛍光輝点の重複を検出し、その検出結果を表示装置３０に表示させる。
　かかる場合、図３Ａに示すとおり、抗原Ａ、Ｂ間の距離が離間している場合、合成後の蛍光画像４４では、緑色の蛍光輝点と赤色の蛍光輝点とがそれぞれ独立に検出される。他方、図３Ｂに示すとおり、抗原Ａ、Ｂ間の距離が近接している場合は、合成後の蛍光画像４６では、緑色の蛍光輝点と赤色の蛍光輝点とが重複し、黄色の蛍光輝点が検出される。ここでは、緑色の蛍光輝点と赤色の蛍光輝点との重複輝点を、単独の蛍光ナノ粒子に起因する蛍光輝点の色（緑色、赤色）とは異なる色（黄色）で表示させた例を示している。 Thereafter, the detection device 20 combines (superimposes) the generated plurality of fluorescent images 40 and 42 to detect the overlap of the fluorescent luminescent spots, and displays the detection result on the display device 30.
In such a case, as shown in FIG. 3A, when the distance between the antigens A and B is separated, the green fluorescent spot and the red fluorescent spot are detected independently in the combined fluorescent image 44. . On the other hand, as shown in FIG. 3B, when the distance between the antigens A and B is close, in the fluorescent image 46 after synthesis, the green fluorescent spot and the red fluorescent spot overlap, and the yellow fluorescence A bright spot is detected. Here, the overlapping bright spot of the green fluorescent spot and the red fluorescent spot is displayed in a color (yellow) different from the color of the fluorescent spot (green, red) caused by a single fluorescent nanoparticle. An example is shown.
　以上の実施形態によれば、抗原Ａ、Ｂ間の距離が近いかどうかを検出するにあたり、蛍光画像４０、４２を免疫染色剤Ａ、Ｂ単位で生成し、これを合成して蛍光輝点の重複を検出しているにすぎず、目的生体物質間の距離が近いかどうかを容易に検出することができる。かかる構成では、重複輝点の色を、免疫染色剤Ａ、Ｂの蛍光ナノ粒子に起因する蛍光輝点の色（緑色、赤色）とは異なる黄色で表示しているので、目的生体物質間の距離が近いかどうかを目視でも確認することができる。 According to the above embodiment, when detecting whether the distance between the antigens A and B is short, the fluorescence images 40 and 42 are generated in units of the immunostaining agents A and B, and these are synthesized to fluoresce bright spots. Only the overlap is detected, and it is possible to easily detect whether the distance between the target biological substances is close. In such a configuration, the color of the overlapping bright spot is displayed in yellow different from the fluorescent bright spot color (green, red) caused by the fluorescent nanoparticles of the immunostaining agents A and B. Whether the distance is short or not can be confirmed visually.
　なお、免疫染色剤単位で生成される複数の蛍光画像は、１台の蛍光顕微鏡１０で生成されてもよいし、免疫染色剤ごとに異なる複数台の蛍光顕微鏡１０で生成されてもよい。ここでは、蛍光画像４０、４２は１台の蛍光顕微鏡１０で生成されてもよいし、２台の蛍光顕微鏡１０でそれぞれ別個に生成されてもよい。
　蛍光顕微鏡１０についても、蛍光顕微鏡１０は染色後の組織切片に励起光を照射して蛍光輝点を出現させそれを撮像する撮像装置の一例であり、当該撮像装置として、蛍光顕微鏡１０に代えてこれと同等の機能を有する、高倍率で撮像可能な撮像装置が使用されてもよい。 Note that the plurality of fluorescent images generated in units of immunostaining agents may be generated by one fluorescence microscope 10 or may be generated by a plurality of fluorescence microscopes 10 that are different for each immunostaining agent. Here, the fluorescence images 40 and 42 may be generated by one fluorescence microscope 10 or may be separately generated by two fluorescence microscopes 10.
The fluorescence microscope 10 is also an example of an imaging device that irradiates a tissue section after staining with excitation light to cause a fluorescent luminescent spot to appear and picks up an image. The imaging device is replaced with the fluorescence microscope 10. An imaging device having a function equivalent to this and capable of imaging at high magnification may be used.
（１）免疫染色剤の作製（量子ドットの修飾）
　量子ドットとして市販のアミノ基末端水溶性量子ドット（ライフテクノロジーズ社）を準備し、当該量子ドットの末端にＳＭＣＣ（Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate）を用いてマレイミドを導入し、これにチオール化した抗体（タンパク質）を結合させ、蛍光ナノ粒子の表面を抗体で修飾した。緑色量子ドットには抗ＨＥＲ２抗体を結合させこれを免疫染色剤Ａとし、赤色量子ドットには抗ＨＥＲ３抗体を結合させこれを免疫染色剤Ｂとした。 (1) Preparation of immunostaining agent (modification of quantum dots)
A commercially available amino-terminated water-soluble quantum dot (Life Technologies) was prepared as a quantum dot, and maleimide was introduced into the end of the quantum dot using SMCC (Succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate) Then, a thiolated antibody (protein) was bound thereto, and the surface of the fluorescent nanoparticle was modified with the antibody. An anti-HER2 antibody was bound to the green quantum dot and used as an immunostaining agent A, and an anti-HER3 antibody was bound to the red quantum dot and used as an immunostaining agent B.
（２）染色
　下記工程（２－１）～（２－１３）の方法により、上記（１）で作製した免疫染色剤Ａ、Ｂを用い、ヒト***組織標本の免疫染色（ＩＨＣ法）を行った。
　病理標本としてコスモバイオ社製の組織アレイスライド（ＣＢ－Ａ７１２）を用い、あらかじめＤＡＢ染色によりＨＥＲ２染色濃度およびＨＥＲ３染色濃度を観察し、ＨＥＲ２とＨＥＲ３を発現していることを確認し、それぞれ染色を行った。 (2) Staining The human breast tissue specimen is immunostained (IHC method) using the immunostaining agents A and B prepared in (1) above by the method of the following steps (2-1) to (2-13). It was.
Using a tissue array slide (CB-A712) manufactured by Cosmo Bio as a pathological specimen, the HER2 staining concentration and the HER3 staining concentration were observed in advance by DAB staining to confirm that HER2 and HER3 were expressed. went.
　工程（２－１）：キシレンを入れた容器に病理標本を３０分浸漬させた。途中３回キシレンを交換した。
　工程（２－２）：エタノールを入れた容器に病理標本を３０分浸漬させた。途中３回エタノールを交換した。
　工程（２－３）：水を入れた容器に病理標本を３０分浸漬させた。途中３回水を交換した。
　工程（２－４）：１０ｍＭクエン酸緩衝液（ｐＨ６．０）に病理標本を３０分浸漬させた。
　工程（２－５）：１２１度で１０分オートクレーブ処理を行った。
　工程（２－６）：ＰＢＳを入れた容器に、オートクレーブ処理後の病理標本を３０分浸漬させた。
　工程（２－７）：１％ＢＳＡ含有ＰＢＳを病理標本に載せて、１時間放置した。
　工程（２－８）：１％ＢＳＡ含有ＰＢＳで０．０５ｎＭに希釈した抗ＨＥＲ２抗体が結合された免疫染色剤Ａを、病理標本に載せて３時間放置した。
　工程（２－９）：ＰＢＳを入れた容器に、染色後の病理標本を３０分浸漬させた。
　工程（２－１０）：１％ＢＳＡ含有ＰＢＳで０．０５ｎＭに希釈した抗ＨＥＲ３抗体が結合された免疫染色剤Ｂを、病理標本に載せて３時間放置した。
　工程（２－１１）：ＰＢＳを入れた容器に、染色後の病理標本を３０分浸漬させた。
　工程（２－１２）：病理標本を４％中性パラホルムアルデヒド溶液で１０分間固定処理した後、ＨＥ染色を行った。
　工程（２－１３）：Merck社製Aquatexを滴下後、カバーガラスを載せ、病理標本を封入した。 Step (2-1): The pathological specimen was immersed in a container containing xylene for 30 minutes. The xylene was changed three times during the process.
Step (2-2): The pathological specimen was immersed in a container containing ethanol for 30 minutes. The ethanol was changed three times during the process.
Step (2-3): The pathological specimen was immersed in a container containing water for 30 minutes. The water was changed three times along the way.
Step (2-4): The pathological specimen was immersed in 10 mM citrate buffer (pH 6.0) for 30 minutes.
Step (2-5): Autoclaving was performed at 121 degrees for 10 minutes.
Step (2-6): The autoclaved pathological specimen was immersed in a container containing PBS for 30 minutes.
Step (2-7): PBS containing 1% BSA was placed on the pathological specimen and left for 1 hour.
Step (2-8): Immunostaining agent A bound with anti-HER2 antibody diluted to 0.05 nM with PBS containing 1% BSA was placed on a pathological specimen and allowed to stand for 3 hours.
Step (2-9): The stained pathological specimen was immersed in a container containing PBS for 30 minutes.
Step (2-10): Immunostaining agent B bound with anti-HER3 antibody diluted to 0.05 nM with PBS containing 1% BSA was placed on a pathological specimen and left for 3 hours.
Step (2-11): The stained pathological specimen was immersed in a container containing PBS for 30 minutes.
Step (2-12): The pathological specimen was fixed with a 4% neutral paraformaldehyde solution for 10 minutes and then stained with HE.
Step (2-13): After dropping Aquack made by Merck, a cover glass was placed and a pathological specimen was enclosed.
（３）蛍光顕微鏡による観察・撮像
　蛍光顕微鏡を用いて染色後の組織切片を観察し撮像した。
　蛍光顕微鏡として、共焦点顕微鏡（オリンパス社製FV1000）と汎用顕微鏡（オリンパス社製BX53）との２種の蛍光顕微鏡を用いた。 (3) Observation and imaging with fluorescent microscope The stained tissue sections were observed and imaged using a fluorescent microscope.
As a fluorescence microscope, two types of fluorescence microscopes, a confocal microscope (FV1000 made by Olympus) and a general purpose microscope (BX53 made by Olympus) were used.
（４）輝点数の算出
　免疫染色剤Ａ（蛍光ナノ粒子１／緑色量子ドット）と免疫染色剤Ｂ（蛍光ナノ粒子２／赤色量子ドット）とによる２枚の蛍光画像から、輝点数を算出した。
　輝点数の算出では、蛍光ナノ粒子１、２の各粒子をガラス板上に散布した試料を事前に観察し、蛍光ナノ粒子１、２ごとに１粒子ぶんの輝度・発光パターンを事前に把握しておき、これを基準として用いて輝点数を算出した。
　解析対象とする範囲は癌細胞が存在する領域とし、興味のある対象領域をROIとして囲い輝点数を算出し、100μｍ２で割ることで、100μｍ２あたりの輝点数を算出した。これはおよそ細胞1個程度の面積あたりの輝点数となっている。
　下記の重複輝点数の算出においても同様に100μｍ２あたりの輝点数を算出した。 (4) Calculation of number of bright spots The number of bright spots was calculated from two fluorescent images of immunostaining agent A (fluorescent nanoparticles 1 / green quantum dots) and immunostaining agent B (fluorescent nanoparticles 2 / red quantum dots). .
In the calculation of the number of bright spots, the sample in which each of the fluorescent nanoparticles 1 and 2 is dispersed on the glass plate is observed in advance, and the brightness and light emission pattern of each particle is determined in advance for each fluorescent nanoparticle 1 and 2. The number of bright spots was calculated using this as a reference.
The range to be analyzed is an area where cancer cells are present, and the number of bright spots per 100 μm 2 is calculated by calculating the number of bright spots surrounding ROI as the target area of interest and dividing by 100 μm 2 . This is the number of bright spots per area of about one cell.
In the calculation of the number of overlapping bright spots below, the number of bright spots per 100 μm 2 was similarly calculated.
（５）重複輝点数の算出
　上記（４）の輝点数の算出で用いた２枚の蛍光画像について、免疫染色剤Ａ（蛍光ナノ粒子１／緑色量子ドット）による蛍光画像の輝点を緑色と、免疫染色剤Ｂ（蛍光ナノ粒子２／赤色量子ドット）とによる蛍光画像の輝点を赤色と設定し、２枚の蛍光画像を合成した場合に、緑色の輝点と赤色の輝点との重複輝点を黄色と設定し（重複輝点以外の輝点は緑色または赤色のまま）、黄色に表示される輝点数を算出した。 (5) Calculation of the number of overlapping bright spots For the two fluorescent images used in the calculation of the number of bright spots in (4) above, the bright spot of the fluorescent image by the immunostaining agent A (fluorescent nanoparticle 1 / green quantum dot) is green. When the bright spot of the fluorescent image by the immunostaining agent B (fluorescent nanoparticle 2 / red quantum dot) is set to red and the two fluorescent images are synthesized, the green bright spot and the red bright spot The overlapping bright spots were set to yellow (the bright spots other than the overlapping bright spots remained green or red), and the number of bright spots displayed in yellow was calculated.
［実施例２－１］
（１）免疫染色剤の作製
（１．１）蛍光色素集積ナノ粒子の合成
　蛍光色素として赤色発光色素であるSulforhodamine101（シグマアルドリッチ社製）１４．４ｍｇを水２２ｍＬに加えて溶解させた。その後、この溶液に乳化重合用乳化剤のエマルジョン（登録商標）４３０（ポリオキシエチレンオレイルエーテル、花王社製）の５％水溶液を２ｍＬ加えた。この溶液をホットスターラー上で撹拌しながら７０℃まで昇温させた後、この溶液にメラミン樹脂原料ニカラックＭＸ－０３５（日本カーバイド工業社製）を０．６５ｇ加えた。
　さらに、この溶液に界面活性剤としてドデシルベンゼンスルホン酸（関東化学社製）の１０％水溶液を１０００μＬ加え、７０℃で５０分間加熱撹拌した。その後、９０℃に昇温して２０分間加熱撹拌した。
　得られた色素樹脂粒子の分散液から、余剰の樹脂原料や蛍光色素などの不純物を除くため、純水による洗浄を行った。具体的には、遠心分離機（クボタ社製マイクロ冷却遠心機３７４０）にて２００００Ｇで１５分間、遠心分離し、上澄み除去後、超純水を加えて超音波照射して再分散した。遠心分離、上澄み除去および超純水への再分散による洗浄を５回繰り返した。
　以上の処理により、赤色蛍光色素集積ナノ粒子（励起波長590nm、発光波長620nm）を作製した。さらにかかる赤色蛍光色素集積ナノ粒子の作製において、Sulforhodamine101（Texas Red）色素に代えてPyrromethene556色素を使用し、緑色蛍光色素集積ナノ粒子（励起波長490nm、発光波長520nm）も作製した。 [Example 2-1]
(1) Preparation of immunostaining agent (1.1) Synthesis of fluorescent dye-integrated nanoparticles 14.4 mg of Sulforhodamine 101 (manufactured by Sigma Aldrich), which is a red luminescent dye, was added to 22 mL of water and dissolved. Thereafter, 2 mL of a 5% aqueous solution of an emulsion (registered trademark) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation) of an emulsifier for emulsion polymerization was added to this solution. This solution was heated to 70 ° C. while stirring on a hot stirrer, and then 0.65 g of melamine resin raw material Nicalak MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was added to this solution.
Further, 1000 μL of a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Inc.) as a surfactant was added to this solution, and the mixture was heated and stirred at 70 ° C. for 50 minutes. Then, it heated up at 90 degreeC and heat-stirred for 20 minutes.
In order to remove impurities such as excess resin raw materials and fluorescent dyes from the obtained dispersion of the dye resin particles, washing with pure water was performed. Specifically, the mixture was centrifuged at 20000 G for 15 minutes in a centrifuge (Kubota Micro Cooling Centrifuge 3740), and after removing the supernatant, ultrapure water was added and ultrasonically irradiated to redisperse. Centrifugation, supernatant removal, and washing by redispersion in ultrapure water were repeated 5 times.
Through the above treatment, red fluorescent dye-integrated nanoparticles (excitation wavelength: 590 nm, emission wavelength: 620 nm) were produced. Further, in the production of the red fluorescent dye-integrated nanoparticles, Pyrromethene556 dye was used in place of the Sulforhodamine 101 (Texas Red) dye, and green fluorescent dye-integrated nanoparticles (excitation wavelength 490 nm, emission wavelength 520 nm) were also prepared.
（１．２）蛍光色素集積ナノ粒子の修飾
　下記工程（１－１）～（１－１２）の方法により、上記（１．１）で作製した赤色蛍光色素集積ナノ粒子に対して抗ＨＥＲ３抗体を結合させた。
　工程（１－１）：１ｍｇの赤色蛍光色素集積ナノ粒子を純水５ｍＬに分散させた。次いで、アミノプロピルトリエトキシシラン水分散液（ＬＳ－３１５０、信越化学工業社製）１００μＬを添加し、室温で１２時間撹拌した。
　工程（１－２）：反応混合物を１００００Ｇで６０分遠心分離を行い、上澄みを除去した。
　工程（１－３）：エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を１回ずつ行った。
　工程（１－４）：工程（１－３）で得られたアミノ基修飾した赤色蛍光色素集積ナノ粒子を、ＥＤＴＡ（エチレンジアミン四酢酸）を２ｍＭ含有したＰＢＳを用いて３ｎＭに調製した。
　工程（１－５）：工程（１－４）で調製した溶液に、最終濃度１０ｍＭとなるようＳＭ（ＰＥＧ）１２（サーモサイエンティフィック社製、succinimidyl-［（N-maleomidopropionamid）-dodecaethyleneglycol］ester）を混合し、１時間反応させた。
　工程（１－６）：反応混合液を１００００Ｇで６０分遠心分離を行い、上澄みを除去した。
　工程（１－７）：ＥＤＴＡを２ｍＭ含有したＰＢＳを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順による洗浄を３回行った。最後に５００μＬのＰＢＳを用いて再分散させ、抗体結合用の赤色蛍光色素集積ナノ粒子を得た。 (1.2) Modification of fluorescent dye-integrated nanoparticles Anti-HER3 antibody against the red fluorescent dye-integrated nanoparticles prepared in (1.1) above by the methods of the following steps (1-1) to (1-12) Were combined.
Step (1-1): 1 mg of the red fluorescent dye-integrated nanoparticles were dispersed in 5 mL of pure water. Next, 100 μL of aminopropyltriethoxysilane aqueous dispersion (LS-3150, manufactured by Shin-Etsu Chemical Co., Ltd.) was added and stirred at room temperature for 12 hours.
Step (1-2): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
Step (1-3): Ethanol was added to disperse the precipitate, followed by centrifugation again. Washing with ethanol and pure water was performed once by the same procedure.
Step (1-4): The amino group-modified red fluorescent dye-integrated nanoparticles obtained in Step (1-3) were prepared to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid).
Step (1-5): SM (PEG) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleomidopropionamid) -dodecaethyleneglycol] ester is added to the solution prepared in step (1-4) to a final concentration of 10 mM. ) And reacted for 1 hour.
Step (1-6): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
Step (1-7): PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing | cleaning by the same procedure was performed 3 times. Finally, 500 μL of PBS was redispersed to obtain red fluorescent dye-integrated nanoparticles for antibody binding.
　工程（１－８）：抗ＨＥＲ３抗体１００μｇを１００μＬのＰＢＳに溶解させたところに、１Ｍジチオスレイトール（ＤＴＴ）を添加し、３０分反応させた。
　工程（１－９）：反応混合物についてゲルろ過カラムにより過剰のＤＴＴを除去し、赤色蛍光色素集積ナノ粒子に結合可能な還元化抗ＨＥＲ３抗体溶液を得た。
　工程（１－１０）：赤色蛍光色素集積ナノ粒子を出発原料として工程（１－７）で得られた赤色蛍光色素集積ナノ粒子の分散液と、工程（１－９）で得られた還元化抗ＨＥＲ３抗体溶液とをＰＢＳ中で混合し、１時間反応させた。
　工程（１－１１）：１０ｍＭメルカプトエタノール４μＬを添加し、反応を停止させた。
　工程（１－１２）：反応混合物を１００００Ｇで６０分遠心分離を行い、上澄みを除去した後、ＥＤＴＡを２ｍＭ含有したＰＢＳを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順による洗浄を３回行った。最後に５００μＬのＰＢＳを用いて再分散させ、抗ＨＥＲ３抗体が結合された赤色蛍光色素集積ナノ粒子（免疫染色剤Ｂ）を得た。 Step (1-8): When 100 μg of anti-HER3 antibody was dissolved in 100 μL of PBS, 1M dithiothreitol (DTT) was added and reacted for 30 minutes.
Step (1-9): Excess DTT was removed from the reaction mixture with a gel filtration column to obtain a reduced anti-HER3 antibody solution capable of binding to the red fluorescent dye-aggregated nanoparticles.
Step (1-10): The red fluorescent dye-integrated nanoparticles dispersion obtained in step (1-7) using the red fluorescent dye-integrated nanoparticles as a starting material, and the reduction obtained in step (1-9) The anti-HER3 antibody solution was mixed in PBS and allowed to react for 1 hour.
Step (1-11): 4 μL of 10 mM mercaptoethanol was added to stop the reaction.
Step (1-12): The reaction mixture was centrifuged at 10,000 G for 60 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing | cleaning by the same procedure was performed 3 times. Finally, 500 μL of PBS was redispersed to obtain red fluorescent dye-integrated nanoparticles (immunostaining agent B) to which the anti-HER3 antibody was bound.
　上記（１．１）で作製した緑色蛍光色素集積ナノ粒子の末端にＮＨＳ－ＰＥＧ（polyethylene glycol）－マレイミド試薬を用いてマレイミドを導入し、これにチオール化した抗ＨＥＲ２抗体（タンパク質）を結合させ、これを免疫染色剤Ａとした。 Maleimide was introduced into the end of the green fluorescent dye-integrated nanoparticles prepared in (1.1) above using NHS-PEG (polyethyleneglycol) -maleimide reagent, and thiolated anti-HER2 antibody (protein) was bound thereto. This was designated immunostaining agent A.
（２）染色～蛍光顕微鏡による観察・撮像～輝点数の算出～重複輝点数の算出
　実施例１と同様にして、染色～蛍光顕微鏡による観察・撮像～輝点数の算出～重複輝点数の算出を行った。 (2) Staining-Observation / imaging with a fluorescence microscope-Calculation of the number of bright spots-Calculation of the number of overlapping bright spots In the same manner as in Example 1, staining-Observation / imaging with a fluorescence microscope-Calculation of the number of bright spots-Calculation of the number of bright spots went.
［実施例２－２］
　実施例２－１において、赤色蛍光色素のSulforhodamine101に代えてAlexa Fluor 610を使用し赤色蛍光色素集積ナノ粒子（励起波長630nm、発光波長650nm）を作製した。
　それ以外は実施例２－１と同様にして、免疫染色剤の作製～染色～蛍光顕微鏡による観察・撮像～輝点数の算出～重複輝点数の算出を行った。 [Example 2-2]
In Example 2-1, instead of red fluorescent dye Sulforhodamine 101, Alexa Fluor 610 was used to produce red fluorescent dye integrated nanoparticles (excitation wavelength 630 nm, emission wavelength 650 nm).
Otherwise, in the same manner as in Example 2-1, preparation of immunostaining, staining, observation / imaging with a fluorescence microscope, calculation of the number of bright spots, calculation of the number of overlapping bright spots were performed.
［実施例２－３］
　実施例２－１において、赤色蛍光色素のSulforhodamine101に代えてAlexa Fluor 546を使用し橙色蛍光色素集積ナノ粒子（励起波長530nm、発光波長550nm）を作製するとともに、緑色蛍光色素のPyrromethene556に代えてATTO 390を使用し青色蛍光色素集積ナノ粒子（励起波長400nm、発光波長480nm）を作製した。
　それ以外は実施例２－１と同様にして、免疫染色剤の作製～染色～蛍光顕微鏡による観察・撮像～輝点数の算出～重複輝点数の算出を行った。 [Example 2-3]
In Example 2-1, instead of red fluorescent dye Sulforhodamine 101, Alexa Fluor 546 was used to produce orange fluorescent dye-integrated nanoparticles (excitation wavelength 530 nm, emission wavelength 550 nm), and ATTO instead of green fluorescent dye Pyrromethene556. Blue fluorescent dye integrated nanoparticles (excitation wavelength 400 nm, emission wavelength 480 nm) were prepared using 390.
Otherwise, in the same manner as in Example 2-1, preparation of immunostaining, staining, observation / imaging with a fluorescence microscope, calculation of the number of bright spots, calculation of the number of overlapping bright spots were performed.
Figure JPOXMLDOC01-appb-T000001
  
Figure JPOXMLDOC01-appb-T000001
  
　表１に示すとおり、実施例１では、共焦点顕微鏡を用いると、輝点状の蛍光像を得られた。他方、汎用顕微鏡を用いた場合は、輝点算出が可能な蛍光像は得られなかった。実施例２－１～２－３でも、共焦点顕微鏡を用いると、輝点状の蛍光像を得られた。汎用蛍光顕微鏡を用いた場合も、輝点算出が可能な蛍光像を容易に得られた。
　以上の実施例１、２－１～２－３の結果から、蛍光画像を免疫染色剤Ａ、Ｂ単位で生成し、これを合成して蛍光輝点の重複を検出すれば、抗原ＨＥＲ２、ＨＥＲ３間の距離が近いかどうかを容易に検出しうることがわかる。実施例１と実施例２－１～２－３との比較から、輝点算出するうえでは、蛍光ナノ粒子として蛍光色素集積ナノ粒子を用いるほうが有用であることがわかる。 As shown in Table 1, in Example 1, when a confocal microscope was used, a bright spot-like fluorescent image was obtained. On the other hand, when a general-purpose microscope was used, a fluorescent image capable of calculating a bright spot was not obtained. Also in Examples 2-1 to 2-3, using a confocal microscope, bright spot-like fluorescence images were obtained. Even when a general-purpose fluorescence microscope was used, a fluorescent image capable of calculating a bright spot was easily obtained.
From the results of Examples 1, 2-1, and 2-3 described above, if a fluorescent image is generated in units of immunostaining agents A and B, and these are synthesized to detect overlapping fluorescent spots, the antigens HER2, HER3 It can be seen that it can be easily detected whether or not the distance between them is short. From a comparison between Example 1 and Examples 2-1 to 2-3, it can be seen that it is more useful to use fluorescent dye-integrated nanoparticles as fluorescent nanoparticles in calculating the bright spot.
　本発明は組織切片から目的生体物質を検出する方法に関し、特に目的生体物質間の距離が近いかどうかを容易に検出するのに好適に利用することができる。 The present invention relates to a method for detecting a target biological material from a tissue section, and can be suitably used particularly for easily detecting whether or not the distance between target biological materials is short.
　１　検出システム
　１０　蛍光顕微鏡
　　１２　光源
　　１４　撮像カメラ
　２０　検出装置
　３０　表示装置
　４０、４２、４４、４６　蛍光画像 DESCRIPTION OF SYMBOLS 1 Detection system 10 Fluorescence microscope 12 Light source 14 Imaging camera 20 Detection apparatus 30 Display apparatus 40, 42, 44, 46 Fluorescence image

Claims (8)

  1. 　組織切片から目的生体物質を検出する方法であって、
    　互いに発光波長が異なる蛍光ナノ粒子を含む複数の免疫染色剤で組織切片を染色する工程と、
    　染色後の組織切片に励起光を照射して蛍光輝点を出現させ、それを撮像装置で撮像する工程と、
    　前記撮像装置の撮像結果に基づき、前記免疫染色剤単位で複数の蛍光画像を生成し、それら蛍光画像を合成して蛍光輝点の重複を検出する工程と、
    　を備えることを特徴とする目的生体物質の検出方法。     A method for detecting a target biological material from a tissue section,
    Staining a tissue section with a plurality of immunostaining agents including fluorescent nanoparticles having different emission wavelengths from each other;
    Irradiating the tissue section after staining with excitation light to cause a fluorescent bright spot to appear, and imaging it with an imaging device;
    Based on the imaging results of the imaging device, generating a plurality of fluorescent images in units of the immunostaining agent, synthesizing the fluorescent images and detecting the overlap of fluorescent bright spots,
    A method for detecting a target biological substance, comprising:
  2. 　請求項１に記載の目的生体物質の検出方法において、
    　組織切片を染色する工程では、前記蛍光ナノ粒子として、量子ドット、量子ドット集積ナノ粒子または蛍光色素集積ナノ粒子を用いることを特徴とする目的生体物質の検出方法。     The method for detecting a target biological substance according to claim 1,
    In the step of staining a tissue section, a quantum dot, quantum dot integrated nanoparticle, or fluorescent dye integrated nanoparticle is used as the fluorescent nanoparticle.
  3. 　請求項２に記載の目的生体物質の検出方法において、
    　前記蛍光ナノ粒子として、蛍光色素集積ナノ粒子を用いることを特徴とする目的生体物質の検出方法。     The method for detecting a target biological substance according to claim 2,
    A method for detecting a target biological substance, wherein fluorescent dye-integrated nanoparticles are used as the fluorescent nanoparticles.
  4. 　請求項１～３のいずれか一項に記載の目的生体物質の検出方法において、
    　前記蛍光ナノ粒子として、発光波長が４００～７００ｎｍのものを用いることを特徴とする目的生体物質の検出方法。     In the method for detecting a target biological substance according to any one of claims 1 to 3,
    A method for detecting a target biological substance, wherein the fluorescent nanoparticles have an emission wavelength of 400 to 700 nm.
  5. 　請求項１～４のいずれか一項に記載の目的生体物質の検出方法において、
    　前記蛍光ナノ粒子として、平均粒径が２０～３００ｎｍのものを用いることを特徴とする目的生体物質の検出方法。     The method for detecting a target biological substance according to any one of claims 1 to 4,
    A method for detecting a target biological substance, wherein the fluorescent nanoparticles have an average particle diameter of 20 to 300 nm.
  6. 　互いに発光波長が異なる蛍光ナノ粒子を含む複数の免疫染色剤で染色された組織切片から目的生体物質を検出する検出システムにおいて、
    　染色後の組織切片に励起光を照射して蛍光輝点を出現させ、それを撮像する撮像装置と、
    　前記撮像装置の撮像結果に基づき、前記免疫染色剤単位で複数の蛍光画像を生成し、それら蛍光画像を合成して蛍光輝点の重複を検出する検出装置と、
    　を備えることを特徴とする目的生体物質の検出システム。     In a detection system for detecting a target biological material from a tissue section stained with a plurality of immunostaining agents containing fluorescent nanoparticles having different emission wavelengths,
    An imaging device that irradiates excitation light to a tissue section after staining, causes a fluorescent bright spot to appear, and images it,
    Based on the imaging result of the imaging device, a detection device that generates a plurality of fluorescent images in units of the immunostaining agent, and combines the fluorescent images to detect overlapping fluorescent luminescent spots;
    A system for detecting a target biological substance, comprising:
  7. 　請求項６に記載の目的生体物質の検出システムにおいて、
    　前記蛍光画像を表示する表示装置を備え、
    　前記検出装置が、蛍光輝点の重複輝点を、単独の蛍光ナノ粒子に起因する蛍光輝点とは異なる色で前記表示装置に表示させることを特徴とする目的生体物質の検出システム。     The target biological substance detection system according to claim 6,
    A display device for displaying the fluorescent image;
    A detection system for a target biological material, wherein the detection device displays an overlapping bright spot of fluorescent bright spots on the display device in a color different from a fluorescent bright spot caused by a single fluorescent nanoparticle.
  8. 　請求項６または７に記載の目的生体物質の検出システムにおいて、
    　前記検出装置が、蛍光輝点の重複輝点数を算出することを特徴とする目的生体物質の検出システム。     The system for detecting a target biological material according to claim 6 or 7,
    A detection system for a target biological material, wherein the detection device calculates the number of overlapping bright spots of fluorescent bright spots.
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