WO2016072341A1 - Procédé d'immunocoloration, et trousse de réactifs d'immunocoloration pour utilisation dans ledit procédé - Google Patents

Procédé d'immunocoloration, et trousse de réactifs d'immunocoloration pour utilisation dans ledit procédé Download PDF

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WO2016072341A1
WO2016072341A1 PCT/JP2015/080512 JP2015080512W WO2016072341A1 WO 2016072341 A1 WO2016072341 A1 WO 2016072341A1 JP 2015080512 W JP2015080512 W JP 2015080512W WO 2016072341 A1 WO2016072341 A1 WO 2016072341A1
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antibody
phosphor
antigen
immunostaining
carbon
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PCT/JP2015/080512
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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 JP2016557729A priority Critical patent/JP6743703B2/ja
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    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase

Definitions

  • the present invention relates to an immunostaining method and an immunostaining reagent kit used therefor.
  • pathological diagnosis is performed as one of medical diagnosis.
  • a pathologist diagnoses a disease from data indicating the result of a biopsy performed on a tissue piece collected from a human body, and informs a clinician whether treatment or surgery is necessary.
  • the medical doctor decides the drug treatment policy, and the surgical doctor decides whether or not to perform the operation.
  • tissue specimen In order to provide data for the diagnosis, a tissue specimen (tissue specimen) is prepared by slicing a tissue specimen obtained by organ excision or needle biopsy to a thickness of several microns, In order to obtain various findings after dyeing treatment, observation using an optical microscope or a fluorescence microscope is widely performed. In many cases, tissue sections are prepared by dehydrating and fixing paraffin blocks to fix the collected tissues, then slicing them to a thickness of several ⁇ m and removing the paraffin.
  • tissue section hardly absorbs and scatters light and is almost colorless and transparent
  • morphological observation staining for observing the cell morphology of the tissue section hematoxylin using two dyes, hematoxylin and eosin
  • HE staining morphological observation staining for observing the cell morphology of the tissue section
  • other morphological observation staining include Papanicolaou staining (Pap staining) used for cytodiagnosis.
  • immunostaining is performed on the tissue sections and the like of the subject.
  • a fluorescently labeled antibody is specifically bound to an in vivo molecule (antigen) whose expression level increases or decreases depending on the presence or absence of the disease, and the amount of fluorescent signal derived from the fluorescently labeled antibody is related to the disease.
  • the amount of antigen to be quantified is performed.
  • Conventionally known immunostaining methods include methods using fluorescent labels (fluorescent dyes and fluorescent nanoparticles, particles in which fluorescent dyes and fluorescent nanoparticles are integrated with resin, etc.), and share fluorescent integrated nanoparticles.
  • Method of fluorescently staining the antigen by binding the bound primary antibody to the antigen on the tissue section (primary antibody method), phosphor accumulation in a state where the primary antibody is bound to the antigen on the tissue section
  • a method in which a secondary antibody linked to a nanoparticle via a covalent bond is bound to the primary antibody to fluorescently stain an antigen (secondary antibody method), a fluorescent aggregate nanoparticle to which biotin (or avidin) is added; And a secondary antibody added with avidin (or biotin), and after binding the primary antibody to the antigen on the tissue section, the secondary antibody is bound to the primary antibody
  • the secondary antibody Streptavidin - how through specific binding of biotin to bind the fluorescent integrated nanoparticles fluorescently labeling the antigen (biotin - avid
  • the following immunostaining method can be considered as a method related to the primary antibody method or the secondary antibody method described above.
  • a monoclonal antibody (anti-hapten antibody, eg, anti-FITC antibody) against a hapten (low molecular compound, eg, FITC) to be linked to the primary antibody or secondary antibody is prepared.
  • the phosphor-aggregated nanoparticles are linked to the anti-hapten antibody by a covalent bond or the like.
  • the anti-hapten antibody is specifically bound to a hapten linked to a primary antibody bound to an antigen on a tissue section or to a hapten linked to a secondary antibody bound to the primary antibody.
  • a method of fluorescently labeling the antigen is specifically bound to a hapten linked to a primary antibody bound to an antigen on a tissue section or to a hapten linked to a secondary antibody bound to the primary antibody.
  • hapten-anti-hapten antibody method if a hapten that does not exist in the living body is used as the hapten to be used, non-specific binding occurs without reacting with molecules in the living body as in the biotin-avidin method described above. There is no. Therefore, the problem that the signal noise described above increases does not occur. However, since the binding of hapten-anti-hapten is inferior to the binding between biotin and avidin (binding force), phosphor-aggregated nanoparticles can be converted into primary and secondary antibodies via hapten-anti-hapten binding.
  • the above-mentioned immunostaining method is preferred in which the non-specific adsorption described above is performed and the phosphor-aggregated nanoparticles are bound to the primary antibody, the secondary antibody or the like with high binding ability.
  • the present invention has been made in view of the above problems, and can suppress non-specific adsorption of fluorescent dye nanoparticles that cause noise of fluorescent signals in immunostaining, and can be combined with antibodies and fluorescent integrated nanoparticles.
  • the purpose of the present invention is to provide an immunostaining method that can suppress the decrease in the fluorescence signal by increasing the binding force of the protein, and to provide an immunostaining reagent kit that can be used in the immunostaining method and has excellent long-term storage stability. To do.
  • the inventors of the present invention (1) The Husgen cycloaddition reaction occurring between an azide and an alkyne has very high reaction selectivity, and the azide or alkyne hardly reacts with other compounds to form a bond.
  • (2) the presence of a copper catalyst or the structure of the alkyne eg, using an alkyne having an 8-membered ring structure makes it possible to use Husgen even under mild conditions in water, neutrality, and room temperature.
  • the cyclization reaction occurs rapidly, and further, (3) the Husgen cycloaddition reaction can be used to covalently bond molecules such as monomers, and (4) the auside and alkyne Huisgen cyclization reaction Focusing on the fact that covalent bonds can be formed in the same way as the carboxylic acid active ester-amine coupling reaction and the maleimide-thiol coupling reaction, Coupled to azido - and found that the above problems can be solved by utilizing Hyusugen cycloaddition reaction between an alkyne leading to the present invention.
  • an immunostaining method reflecting one aspect of the present invention is an immunostaining in which an antigen of the tissue section is fluorescently labeled with phosphor-integrated nanoparticles on the tissue section.
  • the phosphor-aggregated nanoparticles may have an azide group (—N 3 ) is introduced, and the carbon-carbon triple bond moiety (C ⁇ C) is introduced on the other side, Immobilizing the antibody to the antigen;
  • a bond via a triazole ring is formed between both molecules of the antibody and the phosphor-aggregated nanoparticle, and the antigen is formed by the formation.
  • fluorescent labeling is performed with the phosphor-integrated nanoparticles.
  • an immunostaining reagent kit reflecting one aspect of the present invention is to fluorescently label the antigen of the tissue section with phosphor-integrated nanoparticles on the tissue section.
  • An immunostaining reagent kit comprising: A labeling reagent comprising phosphor-integrated nanoparticles and an antibody reagent comprising an antibody directly immobilized on the antigen by an antigen-antibody reaction, or another antibody indirectly immobilized via the antibody Has An azide group (—N 3 ) is introduced into one of the phosphor-integrated nanoparticles and the antibody, and a carbon-carbon triple bond moiety (C ⁇ C) is introduced into the other; By the Husgen cycloaddition reaction between the azide group and the carbon-carbon triple bond moiety, a bond via a triazole ring is formed between the antibody and the phosphor-aggregated nanoparticle, and the two molecules are formed by the formation. It is an immunostaining reagent kit that
  • an immunostaining method capable of suppressing the above. Furthermore, there is provided an immunostaining reagent kit that is used in the immunostaining method and has excellent long-term storage stability.
  • FIG. 1 is a diagram illustrating an immunostaining method according to the present invention.
  • a primary antibody binds to an antigen presented on a tissue section
  • a secondary antibody binds to the primary antibody
  • the antigen is fluorescently labeled by specifically causing the Huisgen cycloaddition reaction by the azide group derived from the azide linked to the phosphor-integrated nanoparticles.
  • endogenous biotin and other antigens are present in the tissue section.
  • the azide group portion of the phosphor-aggregated nanoparticles does not react with endogenous biotin or other antigens.
  • FIG. 2 is a diagram showing another example of the immunostaining method according to the present invention in which the azide and the alkyne compound shown in FIG. 1 are replaced. Since the carbon-carbon triple bond part of the phosphor-integrated nanoparticles does not react with endogenous biotin or other antigens, non-specific adsorption of the phosphor-accumulated nanoparticles does not occur due to this reaction. Generation of signal noise is suppressed.
  • FIG. 3 is a diagram for explaining an immunostaining method (hapten-anti-hapten antibody method) according to the prior art.
  • the primary antibody binds to the antigen presented on the tissue section
  • the secondary antibody (linked to the hapten) binds to the primary antibody
  • the secondary antibody binds to the secondary antibody.
  • the antigen is fluorescently labeled by specifically binding to the anti-hapten antibody to which the phosphor-integrated nanoparticles are added.
  • the hapten-anti-hapten binding is weaker than the biotin-avidin binding and is likely to decouple.
  • the phosphor-aggregated nanoparticles bind to unintended sites by non-specific adsorption and cause noise in the fluorescence signal.
  • FIG. 4 is a diagram for explaining a conventional immunostaining method (biotin-avidin method).
  • biotin-avidin method as shown in FIG. 4, the primary antibody binds to the antigen presented on the tissue section, the secondary antibody binds to the primary antibody, and is linked to the secondary antibody.
  • Antigen is fluorescently labeled by specifically reacting avidin linked to the phosphor-integrated nanoparticles with biotin.
  • the endogenous biotin present in the tissue section reacts with the streptavidin portion added to the phosphor-integrated nanoparticles, and the phosphor-integrated nanoparticles are non-specifically adsorbed and are not intended. To cause noise in the fluorescence signal.
  • the immunostaining method according to the present invention is an immunostaining method in which an antigen on a tissue section is fluorescently labeled with phosphor-integrated nanoparticles on the tissue section, and is directly fixed to the antigen by an antigen-antibody reaction.
  • An azide group (—N 3 ) is introduced into one of the antibody or another antibody immobilized indirectly through the antibody and the phosphor-integrated nanoparticles, and the carbon-carbon triple bond moiety (C ⁇ C) is introduced, the antibody is immobilized on the antigen, and both the antibody and the phosphor-aggregated nanoparticles are obtained by a Husgen cycloaddition reaction between the azide group and the carbon-carbon triple bond moiety.
  • a bond via a triazole ring is formed between molecules, and the antigen is fluorescently labeled with the phosphor-integrated nanoparticles by the formation.
  • the phosphor-integrated nanoparticles are obtained by accumulating phosphors. By using such phosphor-integrated nanoparticles, it is possible to increase the amount of fluorescence emitted per particle, that is, the brightness of a bright spot marking a predetermined biomolecule, compared to the phosphor itself.
  • the term “phosphor” refers to a general substance that emits light in a process from an excited state to a ground state by being excited by irradiation with external X-rays, ultraviolet rays, or visible rays. Therefore, the “phosphor” in the present invention is not limited to the transition mode when returning from the excited state to the ground state, but is a substance that emits narrowly defined fluorescence that is light emission accompanying deactivation from the excited singlet. It may be a substance that emits phosphorescence, which is light emission accompanying deactivation from a triplet.
  • the “phosphor” referred to in the present invention is not limited by the light emission lifetime after blocking the excitation light. Therefore, it may be a substance known as a phosphorescent substance such as zinc sulfide or strontium aluminate. Such phosphors can be broadly classified into organic phosphors (fluorescent dyes) and inorganic phosphors.
  • organic phosphors examples include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (registered trademark, manufactured by Invitrogen Corporation) dye molecules, BODIPY (registered trademark, manufactured by Invitrogen Corporation) dyes Molecule, cascade (registered trademark, Invitrogen) dye molecule, coumarin dye molecule, NBD (registered trademark) dye molecule, pyrene dye molecule, Texas Red (registered trademark) dye molecule, cyanine dye molecule, perylene dye Examples thereof include substances known as organic fluorescent dyes, such as dye molecules and oxazine dye molecules.
  • inorganic phosphor examples include quantum dots containing II-VI group compounds, III-V group compounds, or group IV elements as components ("II-VI group quantum dots", " Or III-V quantum dots ”or“ IV quantum dots ”). You may use individually or what mixed multiple types.
  • the quantum dots may be commercially available. Specific examples include, but are not limited to, CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.
  • a quantum dot having the above quantum dot as a core and a shell provided thereon.
  • the core is CdSe and the shell is ZnS
  • CdSe / ZnS when the core is CdSe and the shell is ZnS, it is expressed as CdSe / ZnS.
  • CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO 2 , Si / ZnS, Ge / GeO 2 , Ge / ZnS, and the like can be used, but are not limited thereto.
  • Quantum dots may be subjected to surface treatment with an organic polymer or the like as necessary.
  • organic polymer or the like as necessary. Examples thereof include CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like.
  • the method for producing the phosphor-integrated nanoparticles is not particularly limited, and can be produced by a known method. In general, a production method can be used in which phosphors are gathered together using a resin or silica as a base material (the phosphors are immobilized inside or on the surface of the base material).
  • Examples of a method for producing phosphor-integrated nanoparticles using organic phosphors include a method of forming resin particles having a diameter of nanometer order by fixing a fluorescent dye, which is a phosphor, inside or on the surface of a matrix made of resin. Can do.
  • the method for preparing the phosphor-integrated nanoparticles is not particularly limited.
  • a (co) monomer for synthesizing a resin (thermoplastic resin or thermosetting resin) that forms the matrix of the phosphor-integrated nanoparticles While (co) polymerizing the phosphor, a method of adding the phosphor and incorporating the phosphor into the inside or the surface of the (co) polymer can be used.
  • thermoplastic resin for example, polystyrene, polyacrylonitrile, polyfuran, or a similar resin
  • thermosetting resin for example, polyxylene, polylactic acid, glycidyl methacrylate, polymelamine, polyurea, polybenzoguanamine, polyamide, phenol resin, polysaccharide or similar resin
  • Thermosetting resins, particularly melamine resins are preferred in that elution of the dye encapsulated in the dye resin can be suppressed by treatments such as dehydration, penetration, and encapsulation using an organic solvent such as xylene.
  • polystyrene nanoparticles encapsulating an organic fluorescent dye can be obtained by a copolymerization method using an organic dye having a polymerizable functional group described in US Pat. No. 4,326,008 (1982), or US Pat. No. 5,326,692 (1992). ), And can be used as phosphor-integrated nanoparticles.
  • silica nanoparticles in which an organic phosphor is immobilized inside or on the surface of a matrix made of silica can also be produced.
  • the method for synthesizing FITC-encapsulated silica particles described in Langmuir Vol. 8, Vol. 8, page 2921 (1992) can be referred to.
  • silica nanoparticles encapsulating various fluorescent dyes can be synthesized and used as phosphor-integrated nanoparticles.
  • Examples of a method for producing phosphor-integrated nanoparticles using an inorganic phosphor include a method of forming silica nanoparticles in which quantum dots, which are phosphors, are fixed inside or on the surface of a matrix made of silica. This production method can be referred to the synthesis of CdTe-containing silica nanoparticles described in New Journal of Chemistry Vol. 33, p. 561 (2009).
  • the silica beads are treated with a silane coupling agent to aminate the ends, and semiconductor fine particles as phosphors having carboxy group ends are amided on the surface of the silica beads.
  • a method for collecting phosphors to form phosphor-integrated nanoparticles is also exemplified.
  • a reverse micelle method and a mixture of organoalkoxysilane and alkoxide having an organic functional group with good adsorptivity to semiconductor nanoparticles at the molecular end as a glass precursor are used.
  • glass-like particles in which semiconductor nanoparticles are dispersed and fixed are formed to form phosphor-integrated nanoparticles.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • phosphor-integrated nanoparticles can be produced by immobilizing inorganic phosphors inside or on the surface of a matrix made of resin.
  • polymer nanoparticles encapsulating quantum dots can be prepared using the method of impregnating quantum nanoparticles into polystyrene nanoparticles described in Nature Biotechnology Vol. 19, p. 631 (2001).
  • the average particle size of the phosphor-integrated nanoparticles is preferably from 150 nm to 800 nm, more preferably from 150 nm to 500 nm, from the viewpoint of fluorescence signal intensity.
  • the average particle size of the phosphor-integrated nanoparticles can be examined by a known measurement method.
  • the phosphor-integrated nanoparticles are observed with a transmission electron microscope (TEM), and the number-average particle size of the particle size distribution is obtained therefrom.
  • the particle size distribution of the semiconductor nanoparticles is measured by the dynamic light scattering method. And the method etc. which are calculated
  • the average particle diameter can be measured by observing with a gas adsorption method, a light scattering method, an X-ray small angle scattering method (SAXS), or a scanning electron microscope (SEM).
  • SAXS X-ray small angle scattering method
  • SEM scanning electron microscope
  • the surface of the phosphor-integrated nanoparticles may be optionally modified with a hydrophilic polymer.
  • the hydrophilic polymer include polyethylene glycol, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, polyvinyl pyrrolidone, polyvinyl methyl ether, polyvinyl methyl oxazoline, Polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyaspartamide, synthetic polyamino acid, etc. Can be mentioned.
  • the antibody used in the present invention is selected according to the use, for example, an antibody (primary antibody) against an antigen (eg, HER2 etc.) associated with a disease (malignant tumor etc.), or an antigen-antibody reaction with the primary antibody Means a secondary antibody to an n-th antibody that bind to each other (hereinafter also referred to as “predetermined antibody”).
  • predetermined antibody a secondary antibody to an n-th antibody that bind to each other
  • an azide or alkyne compound is bound to any of these antibodies, and has an azide group or a carbon-carbon triple bond moiety.
  • the term “antibody” is used to include any antibody fragment or derivative, and includes, for example, Fab, Fab′2, CDR, humanized antibody, multifunctional antibody, single chain antibody (ScFv) and the like. .
  • antigens include proteins (polypeptides, oligopeptides, etc.) and amino acids (including modified amino acids), and the proteins or amino acids and carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids. Or a complex with these modified molecules.
  • antigens tumor markers, signal transduction substances, hormones, etc.
  • antigens related to the diseases to be pathologically diagnosed are not particularly limited.
  • examples of the antigen include, for example, TNF- ⁇ (Tumor Necrosis Factor ⁇ ), IL, in addition to antigens related to cancer such as cancer growth regulator, metastasis regulator, growth regulator receptor, and metastasis regulator receptor.
  • Inflammatory cytokines such as the ⁇ 6 (Interleukin-6) receptor and virus-related molecules such as RSV F protein are also included in the “antigen”.
  • proteins derived from cancer-related genes include ALK, FLT3, AXL, FLT4 (VEGFR3, DDR1, FMS (CSF1R), DDR2, EGFR (ERBB1), HER4 (ERBB4), EML4.
  • Proteins derived from breast cancer-related genes that can serve as the above antigens are ATM, BRCA1, and BRCA2. , BRCA3, CCND1, E-Cadherin, ERBB2, ETV6, FGFR1, HRAS, KRAS, NRAS, NTRK3, p53, and PTEN Further, as a protein derived from a gene related to a carcinoid tumor, the antigen can be used. Include BCL2, BRD4, CCND1, CDKN1A, CDKN2A, CTNNB1, HES1, MAP2, MEN1, NF1, NOTCH1, NUT, RAF, SDHD, and VEGFA.
  • proteins that can be used as the antigen and are derived from lung cancer-related genes include ALK, PTEN, CCND1, RASSF1A, CDKN2A, RB1, EGFR, RET, EML4, ROS1, KRAS2, TP53, and MYC.
  • proteins derived from liver cancer-related genes that can serve as the antigen include Axin1, MALAT1, b-catenin, p16 INK4A, c-ERBB-2, p53, CTNNB1, RB1, C clin D1, SMAD2, EGFR, SMAD4, IGFR2, TCF1, KRAS and the like.
  • Alpha, PRCC, ASPSCR1, PSF, CLTC, TFE3, p54nrb / NONO, and TFEB are examples of proteins that can be used as the antigen and are derived from kidney cancer-related genes.
  • proteins that can serve as the antigen and are derived from thyroid cancer-related genes include AKAP10, NTRK1, AKAP9, RET, BRAF, TFG, ELE1, TPM3, H4 / D10S170, and TPR.
  • proteins derived from ovarian cancer-related genes that can be used as the antigen include AKT2, MDM2, BCL2, MYC, BRCA1, NCOA4, CDKN2A, p53, ERBB2, PIK3CA, GATA4, RB, HRAS, RET, KRAS, and RNASET2.
  • proteins that can serve as the antigen and are derived from prostate cancer-related genes can be mentioned as proteins that can serve as the antigen and are derived from prostate cancer-related genes.
  • proteins that can be used as the antigen and derived from bone tumor-related genes include CDH11, COL12A1, CNBP, OMD, COL1A1, THRAP3, COL4A5, and USP6.
  • the linker used in the present invention is a molecule for linking the phosphor-integrated nanoparticles and a predetermined antibody. It is desirable that the predetermined antibody (any one of the primary antibody to the n-th antibody) and the phosphor-integrated nanoparticles are bound by a linker. This is because a clearance is formed between the antibody and the phosphor-integrated nanoparticles by the linker portion, and a decrease in the fluorescence signal of the phosphor-integrated nanoparticles due to an insoluble compound (DAB or the like) can be suppressed.
  • DAB insoluble compound
  • an alkyne compound or an azide is bonded to one end of the hydrophilic polymer (eg, PEG), and a functional group (NHS group, thiol group (—SH), etc.) is bonded to the other end.
  • a functional group such as an amino group or a maleimide group
  • a reaction between the functional group of the linker and a functional group such as an NHS group or an SH group introduced on the surface of the phosphor-aggregated nanoparticle or a predetermined antibody is performed.
  • linker can be introduced into the phosphor-integrated nanoparticles and antibody, and can be used for the Husgen cycloaddition reaction.
  • a linker can be purchased from Thermo Scientific or NANOCOS.
  • the linker is preferably a hydrophilic polymer linker, particularly preferably a polyethylene glycol (PEG) linker, from the viewpoint of hardly causing nonspecific adsorption with biomolecules.
  • PEG polyethylene glycol
  • the length of the linker is used to suppress a reduction in the signal intensity of the fluorescence obtained when the target antigen is stained with an insoluble byproduct caused by a staining reagent for morphological observation (eg, DAB). Therefore, it is preferable to set as follows.
  • the linker directly involved in linking the antibody and the phosphor-integrated nanoparticle is present between the phosphor-integrated nanoparticle and the azide-alkyne bond.
  • the number of oxyethylene units (unit number) is preferably 8 or more, more preferably 8 to 70.
  • the length of the linker is preferably a length corresponding to the PEG linker (having oxyethylene units of 8 to 70).
  • One of the predetermined antibody and phosphor-integrated nanoparticles used in the present invention (one having no carbon-carbon triple bond) has an azide group (-N 3 ) that causes a cycloaddition reaction with the carbon-carbon triple bond moiety.
  • ⁇ Azide ⁇ A method for introducing an azide group into an antibody (any of primary to n-order antibodies) or phosphor-integrated nanoparticles is not particularly limited.
  • an antibody or phosphor-integrated nanoparticles together with an azide group A method of reacting a functional group of such an azide with a functional group of an antibody or phosphor-integrated nanoparticle using a compound (azide) having another functional group capable of binding to a functional group present on the surface of the antibody is preferable. .
  • a preferred azide has an azide group at one end of the molecule and reacts with a functional group (eg, —NH 3 , —SH group) present on the surface of the antibody or phosphor-integrated nanoparticle at the other end to form a covalent bond.
  • a functional group eg, —NH 3 , —SH group
  • examples thereof include azides having other functional groups that can be formed (eg, NHS group, maleimide group, etc.).
  • a portion derived from the hydrophilic polymer described above may be included between the azide group and the functional group of such “azide”.
  • azides examples include NHS (N-hydroxysuccinimide) esters having an azide group, other activated esters having an azide group (sulfo-NHS ester, sulfotetrafluorphenyl (STP) ester, etc.). It can be illustrated. Specific examples of these esters include “Azidobutyric acid NHS ester” (Product No. Cat. # 33720, manufactured by Lumi Probe) (see the following formula (1)), “Sulfo-SANPAH (sulfosuccinimidyl-6- ⁇ 4′-azido -2'-nitrophenylamino ⁇ hexanoate) "(product number 22589, manufactured by Thermo Scientific) (see formula (2) below).
  • NHS N-hydroxysuccinimide
  • STP sulfotetrafluorphenyl
  • azides that can be used in the present invention other than those described above have functional groups such as thiol groups (SH groups) and maleimide groups as in the case of the azides, and are derived from a hydrophilic polymer linker (eg, PEG). Mention may be made of azides having a moiety (see formulas (3) and (4) below).
  • the length of the portion derived from the linker of the hydrophilic polymer in the azide molecule is the number of units (as described above). The length is preferably 8 or more (in oxyethylene units), more preferably 8 or more and 70 or less.
  • ⁇ Maleimide ester> A specific example (maleimide ester) of an azide having a linker-derived moiety other than the above is “Azide-PEG-Maleimide” (Product No .: PG2-AZML-400, 600, 1k, 2k, 3k, 5k, NANOCS) Manufactured).
  • PG2-AZML-400” to “PG2-AZML-5K” are obtained by substituting the NHS groups of the above “PG2-AZNS-400” to “PG2-AZNS-5K” with maleimide groups, respectively.
  • the oxyethylene unit and molecular length are almost the same as the corresponding product (see Table 1).
  • it is preferable that m 8 to 70.
  • the binding between the antibody and the azide is not particularly limited as long as the antibody and the azide can be covalently bound so that the immunoreactivity of the azide-derived azide group (—N 3 ) and the antigen of the antibody is not impaired.
  • the reaction can be performed by reacting the amino group of the antibody with an azide compound having an NHS group. For example, it can be carried out by adding 5 to 100 mol of an azide compound having an NHS group to 1 mol of antibody in 0.05 M sodium borate buffer.
  • the antibody and azide can be bound by reacting the antibody thiol group with an azide compound having maleimide.
  • the antibody is previously reduced or thiolated (2-iminothiolane (2-IT), succinimidylacetylthiopropionate (SATP, N-succinimidyl-S-acetylthiopropionate (manufactured by Thermoscience)) ), Thiolated using succinimido 2-pyridyldithiopropionate (SPDP), etc., and then in 0.05 M sodium borate buffer (sodium borate buffer) to 1 mol of antibody.
  • the azide compound having maleimide can be added in an amount of 5 to 100 mol.
  • azide compound having a maleimide group or an NHS group those described above can be used.
  • the pH value of the above binding reaction is preferably adjusted to 6-8, and 8.3-8. It is more preferable to adjust to 5.
  • the pH can be adjusted with sodium borate or potassium hydroxide.
  • the conditions for the binding reaction are not particularly limited.
  • the binding reaction may be performed at room temperature for at least 1 hour, or may be performed overnight on ice.
  • the antibody obtained by the binding reaction and the azide bound to each other can be purified by removing unreacted substances using a spin column for antibody purification containing a resin or the like.
  • confirmation that the azide and the antibody are bound can be confirmed by, for example, ICP-MS (inductively coupled plasma mass spectrometer).
  • the method for binding the azide to the phosphor-aggregated nanoparticles is not particularly limited as long as the azide can be bound to the phosphor-aggregated nanoparticles.
  • azide (mainly commercially available azide)
  • a functional group e.g., NHS group, maleimide group
  • a functional group e.g., NHS group, maleimide group
  • a functional group e.g., amino group, SH group
  • a method comprising a step of reacting the functional group of the azide and the functional group of the phosphor-integrated nanoparticles to bond the azide to the phosphor-integrated nanoparticles (that is, introducing an azide group) is exemplified.
  • each functional group is phosphor-assembled using a coupling agent such as a silane coupling agent. It can be introduced into nanoparticles (having OH groups on the particle surface).
  • a coupling agent such as a silane coupling agent
  • amino groups can be introduced onto the surface of the phosphor-integrated nanoparticles using aminopropylethyl silicate, tris (2-aminoethyl) amine, or the like.
  • the reaction between the coupling agent and the surface of the phosphor-integrated nanoparticles can be carried out by reacting in the presence of the coupling agent under the general use conditions of the coupling agent. Usually, a method of stirring and reacting at room temperature for several tens of minutes to several tens of hours can be used.
  • the ratio of the coupling agent to be used is 300 to 6000 times, preferably 600 to 5400 times, more preferably 2100 to 3000 times in terms of molar ratio with respect to 1 mol of the phosphor-integrated nanoparticles.
  • the functional groups (amino group, SH group, etc.) on the surface of the phosphor-integrated nanoparticles can be appropriately selected and introduced depending on the type of the base material used for producing the phosphor-integrated nanoparticles. For example, if phosphor-integrated nanoparticles are produced using melamine resin, phosphor-integrated nanoparticles having an amino group or a hydroxyl group can be obtained.
  • a melamine resin When a melamine resin is used, the melamine resin has many secondary amine and tertiary amine moieties, and there are few primary amine moieties (-NH 2 groups) and poor reactivity with NHS esters.
  • the primary amine moiety (—NH 2 group) can be introduced to increase the reactivity with the NHS ester. Good.
  • a thiol group (—SH group) can be introduced on the surface of the phosphor-integrated nanoparticles.
  • a method of once introducing an amino group on the surface of the phosphor-integrated nanoparticles and converting the amino group into a thiol group can be mentioned.
  • the thiolation reagent include 2-iminothiolane, N-succinimidyl-S-acetylthioacetate (SATA), and the like.
  • a molar amount of 2-iminothiolane of 5000 to 50000 times is added to 1 mol of particles in a solvent such as water.
  • the amino group can be introduced into the thiol group by reacting at room temperature for about 1 hour.
  • an amino group or SH group is introduced on the surface of the phosphor-integrated nanoparticles is confirmed by whether or not absorption derived from the amino group or SH group can be observed by, for example, FT-IR method and XPS measurement. be able to.
  • step (II) the functional group (NHS group or the like) of the azide is reacted with the functional group (amino group or the like) of the phosphor integrated nanoparticle to bond the azide to the surface of the phosphor integrated nanoparticle.
  • a buffer solution eg, sodium borate buffer, PBS, etc.
  • the phosphor-integrated nanoparticles having the functional group such as amino group are adjusted to 0.3 to 30 nM, and the azide is mixed so that the final concentration is 0.6 to 120 mM. And an example of reacting at room temperature for several hours (eg, 0.5 to 1.5 hours).
  • washing process (III) after process (II).
  • the reaction mixture obtained through the above step (II) is centrifuged, the supernatant is removed, and a buffer solution such as water or PBS containing 2 mM EDTA is further dispersed in the precipitate. It can be carried out by repeating it twice (a few times).
  • One of the predetermined antibody and phosphor-integrated nanoparticles used in the present invention (one not having an azido group) has a carbon-carbon triple bond (C ⁇ C) that causes a cycloaddition reaction with the azide group.
  • the method for introducing the carbon-carbon triple bond moiety into the antibody (any one of the primary to n-order antibodies) or the phosphor-integrated nanoparticles is not particularly limited.
  • a reactive functional group capable of reacting with a functional group present on the surface of the phosphor-integrated nanoparticle to form a covalent bond unless the carbon-carbon triple bond itself is used to form the covalent bond,
  • a method in which a functional group of such a compound is reacted with a functional group of an antibody or phosphor-integrated nanoparticles using a compound having a reactive group itself may be the reactive group is suitable.
  • a resin having a carbon-carbon triple bond moiety in the side chain portion of the monomer that does not participate in the polymerization reaction in the process of polymerizing the resin monomer while incorporating the aforementioned fluorescent dye By using a monomer, a carbon-carbon triple bond portion can be directly introduced into the phosphor-integrated nanoparticles by the polymerization. Further, for example, a resin monomer having an allyl halide in the side chain is reacted with a compound of R—C ⁇ C—H in the side chain portion to form a terminal portion or a center of the raw material monomer for forming silica resin particles. A carbon-carbon triple bond portion may be introduced into the portion, the polymerization may be performed using this monomer, and the carbon-carbon triple bond portion may be directly introduced into the phosphor-integrated nanoparticles.
  • alkyne compounds are collectively referred to as “alkyne compounds”. That is, the term “alkyne compound” in the present invention is not a term indicating only a linear hydrocarbon (strictly alkyne) and cyclic hydrocarbon (strictly cycloalkyne) having one carbon-carbon triple bond, but an azide group. As long as it contains a carbon-carbon triple bond moiety capable of undergoing a cycloaddition reaction with the group, it may be linear or cyclic, and a group containing an atom other than carbon or a carbon-carbon triple bond moiety It is a broad term that refers to all compounds that may further contain a group other than (for example, the reactive functional group). Such alkyne compounds include, of course, the narrowly defined alkynes and cycloalkynes themselves.
  • Type of alkyne Preferred alkyne compounds have a carbon-carbon triple bond moiety at one end of the molecule and react with a functional group (eg, —NH 3 , —SH group) present on the surface of the antibody or phosphor-integrated nanoparticle at the other end.
  • a functional group eg, —NH 3 , —SH group
  • bifunctional alkyne compounds having a reactive functional group capable of forming a covalent bond eg, NHS group, maleimide group, etc.
  • Such an alkyne compound is capable of covalently bonding a compound having a reactive group to a compound having a carbon-carbon triple bond moiety (which may be the above-described alkyne in a narrow sense) via a molecule serving as a linker as necessary.
  • NHS N-hydroxysuccinimide
  • sulfo-NHS-esters compounds obtained by linking with, for example, compounds having an intercarbon triple bond moiety And sulfotetrafluorophenyl (STP) ester).
  • bifunctional alkyne compound examples include “Pentynoic acid STP ester” (product number Cat. # 33720, manufactured by Lumi Probe Co., Ltd.) (see the following formula (5)).
  • alkyne compound having a linker-derived moiety examples include alkyne compounds having the above-described functional groups (eg, NHS group, maleimide group, etc.) and having a portion derived from a hydrophilic polymer linker (eg, PEG, etc.). (Refer to each formula (6) below).
  • AK-PEG-NHS catalog numbers: PG2-AKNS-400, PG2-AKNS-600, PG2-AKNS-800, PG2-AKNS-1k, PG2-AKNS-2k, PG2-AKNS-3k, PG2- AKNS-5k (manufactured by NANOCS) (see the following formula (6) and table).
  • PG2-AKNS-400, PG2-AKNS-600, PG2-AKNS-800, PG2-AKNS-1k, and PG2-AKNS-2k in which the number (n) of oxyethylene units falls within the range of 8 to 70 are preferable.
  • “NHS-PEG (NH-Boc) -alkyne” product number PEG2920; Iris BIOTECH GMBH
  • n 8 to 70 is preferable.
  • an alkyne compound having a cyclic structure having a carbon-carbon triple bond for example, an 8-membered ring structure in the molecule is preferable.
  • a bonding reaction Husgen cycloaddition reaction
  • a metal catalyst eg, copper catalyst
  • alkyne compound having such a cycloalkyl structure and a reactive functional group for binding to the antibody or phosphor-aggregated nanoparticles as described above include the following.
  • the binding between the antibody and the alkyne is not particularly limited as long as the carbon-carbon triple bond portion derived from the alkyne is not impaired, and the antibody and the alkyne can be covalently bound so that the immunoreactivity of the antibody is not impaired.
  • This can be carried out by reacting the amino group of the antibody with an alkyne compound having an NHS group. For example, it can be carried out by adding 5 to 100 mol of an alkyne compound having an NHS group to 1 mol of antibody in 0.05 M sodium borate buffer.
  • the binding between the antibody and the alkyne can also be performed by reacting the thiol group of the antibody with the alkyne compound having maleimide.
  • the antibody is reduced beforehand or a thiolation reagent (2-iminothiolane (2-IT), succinimidylacetylthiopropionate (SATP, N-succinimidyl-S-acetylthiopropionate (manufactured by Thermoscience)) ), N-succinimidyl 3- (2-pyridyldithio) propionate (succinimido3- (2-pyridyldithio) propionate, SPDP), etc., and then 0.05M sodium borate buffer (Sodium borate buffer) ) Can be carried out by adding 5 to 100 mol of an alkyne compound having maleimide to 1 mol of antibody.
  • alkyne compound having a maleimide group or an NHS group those described above can be used.
  • the pH value of the binding reaction is preferably adjusted to 6-8. More preferably, the pH is adjusted to 8.3 to 8.5. In the example using the sodium borate buffer, the pH can be adjusted with sodium borate or potassium hydroxide.
  • the conditions for the binding reaction are not particularly limited.
  • the binding reaction may be performed at room temperature for at least 1 hour, or may be performed overnight on ice.
  • the antibody obtained by the binding reaction and the alkyne compound bound to each other can be purified by removing unreacted substances using a spin column for antibody purification containing a resin or the like.
  • the confirmation that the alkyne compound and the antibody are bound can be confirmed by, for example, ICP-MS (inductively coupled plasma mass spectrometer).
  • the method for binding the alkyne compound to the phosphor-aggregated nanoparticles is not particularly limited as long as the alkyne compound can be bound to the phosphor-aggregated nanoparticles.
  • As a preferable method for bonding the alkyne compound and the phosphor-aggregated nanoparticles it can be achieved by using an alkyne compound instead of the azide in the above-mentioned “Method of binding phosphor-aggregated nanoparticles and azide”. Therefore, the description is omitted.
  • An immunostaining reagent kit is an immunostaining reagent kit for fluorescently labeling an antigen of a tissue section on a tissue section with phosphor-integrated nanoparticles, the labeling reagent containing the phosphor-integrated nanoparticles, An antibody reagent containing an antibody directly immobilized on an antigen by an antigen-antibody reaction, or another antibody indirectly immobilized via the antibody, and the phosphor-integrated nanoparticles and the antibody An azido group (—N 3 ) is introduced into one of them, and a carbon-carbon triple bond moiety (C ⁇ C) is introduced into the other, and the Huisgen cycloaddition of the azide group and the carbon-carbon triple bond moiety By the reaction, a bond via a triazole ring is formed between the antibody and the phosphor-integrated nanoparticles, and the antigen is fluorescently labeled by directly or indirectly binding the both molecules
  • the antibody reagent is obtained by dissolving a predetermined antibody (eg, HER2 antibody) to which alkyne (or alkyne compound) or azide is bound in a predetermined buffer solution, and the antibody is a specific antigen on a tissue section. That can specifically bind to.
  • a predetermined antibody eg, HER2 antibody
  • alkyne or alkyne compound
  • azide is bound in a predetermined buffer solution
  • the antibody is a specific antigen on a tissue section. That can specifically bind to.
  • secondary antibodies to n-order antibodies may be included in the immunostaining reagent kit of the present invention as separate antibody reagents.
  • the antibody concentration in the antibody reagent is preferably adjusted to a concentration range in which a Huisgen cyclization reaction can occur with the alkyne compound or azide of the phosphor-integrated nanoparticles.
  • the molar concentration of the predetermined antibody in the antibody reagent is set to the phosphor in the labeling reagent. It is desirable to set the molar concentration of the integrated nanoparticles to be approximately the same. For example, if the molar concentration of the phosphor-integrated nanoparticles in the labeling reagent is set to 0.005 nM to 0.5 nM, the molar concentration of the antibody in the antibody reagent is also set in the range of 0.005 nM to 0.5 nM. It is desirable to do.
  • buffers examples include phosphate buffers (including PBS), water, and the like.
  • various blocking agents may be included in the antibody reagent, and the concentration of this blocking agent is preferably set to 1% or less in terms of final concentration.
  • blocking agents include biological substances such as bovine serum albumin (BSA), casein ( ⁇ -casein, ⁇ -casein, ⁇ -casein) and gelatin.
  • the labeling reagent is a fluorinated nanoparticle with alkyne (or alkyne compound) or azide bound dispersed in a specified solvent, and Huisgen cyclization between the antigen and the antibody bound to the tissue section It is used for fluorescent labeling of the antigen by an addition reaction.
  • the concentration of the phosphor-aggregated nanoparticles in the labeling reagent only needs to be adjusted to be higher than the concentration causing the Husgen cycloaddition reaction on the tissue section.
  • An example of the concentration of the phosphor-integrated nanoparticles contained in the labeling reagent is set to 0.005 to 0.500 nM.
  • the buffer that can be used for the labeling reagent examples include phosphate buffer (including PBS), water, MES, and the like.
  • the labeling reagent may contain various blocking agents, and the concentration of the blocking agent is preferably set to 1% or less in terms of final concentration.
  • blocking agents include biological substances such as bovine serum albumin (BSA), casein ( ⁇ -casein, ⁇ -casein, ⁇ -casein) and gelatin.
  • the metal catalyst solution optionally contained in the immunostaining reagent kit according to the present invention is a solution containing a metal ion having a catalytic ability for the Husgen cycloaddition reaction between a predetermined antibody and the above-described phosphor-aggregated nanoparticles.
  • a metal ion having a catalytic ability for the Husgen cycloaddition reaction between a predetermined antibody and the above-described phosphor-aggregated nanoparticles As catalysts, Cu, Zr, W, Fe, Ru, Co, Rh, Ir, Ni, Pd, Pt, Ag, Au, Zn, Cd, Hg, and others that can catalyze the Husgen cycloaddition reaction of azide and alkyne Any one or two or more selected from the group consisting of metal ions can be used, and among them, Cu having excellent reaction efficiency is particularly preferable. In addition to this, for example, a particulate metal catalyst capable of generating metal ions in a solution
  • the average particle diameter of the particulate metal catalyst is preferably 10 nm to 1000 ⁇ m, preferably 10 ⁇ m to 200 ⁇ m or 10 nm to 1000 nm.
  • the catalyst may be a porous non-particulate catalyst, for example, a solid substrate in which catalytically active particles are embedded.
  • the concentration of metal ions in the catalyst solution is such that when the catalyst solution is added to the reaction system, the concentration of metal ions in the reaction system can be adjusted to a concentration at which the Husgen cycloaddition reaction can proceed.
  • concentration at which the Husgen cycloaddition reaction can proceed For example, it is preferable to contain 5 to 500 mM in total of any one or more of the metal ions listed above.
  • the immunostaining method according to the present invention includes an immunoreaction step of binding and fixing the above-mentioned antibody conjugated with alkyne (or alkyne compound) or azide to an antigen on a tissue section by an antigen-antibody reaction, and azide or A staining reaction step of binding the above-described phosphor-aggregated nanoparticles, to which alkyne (or alkyne compound) is bound, to the antibody immobilized on the antigen by Huesgen cycloaddition reaction.
  • the immunostaining method is preferably carried out through the following series of steps including the above two steps (immune reaction step and staining reaction step).
  • the tissue section may be purchased from a commercially available one.
  • the tissue of a subject human, dog, cat, etc.
  • suspected of having various cancers as described above for antigen is used for general histopathological diagnosis. It can be prepared by a known method used. In this case, the tissue section of the subject is first fixed with formalin or the like, dehydrated with alcohol, then treated with xylene, and immersed in high temperature paraffin to embed the paraffin into a tissue section.
  • tissue section is immersed in 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 tissue section is immersed in 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. Further, if necessary, ethanol may be exchanged during the immersion.
  • the tissue section is immersed in water (eg, distilled water) to remove 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. Moreover, you may exchange water in the middle of immersion as needed.
  • Activation treatment step When immunohistochemical staining is performed as histochemical staining, it is preferable to perform activation treatment of a target biomolecule according to 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 ethylenediaminetetraacetic acid (EDTA) solution (pH 8.0), 5% urea, 0.1 M Tris
  • EDTA ethylenediaminetetraacetic acid
  • a hydrochloric acid buffer or the like can be used.
  • a heating device an autoclave, a microwave, a pressure cooker, a water bath, etc. can be used.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the heat treatment temperature for the activation treatment can be 50 to 130 ° C., and the heat treatment time can be 5 to 30 minutes.
  • washing is performed by immersing the sections after the activation treatment in PBS placed in a container.
  • 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.
  • the immune reaction step is a step of immobilizing the aforementioned antibody against an antigen on a tissue section. Specifically, a step of directly immobilizing a primary antibody to an antigen on a tissue section by an antigen-antibody reaction, or in addition to the immobilization, the antigen is separated via the primary antibody. This is a step of indirectly fixing the antibodies (secondary to nth antibodies).
  • the above-mentioned primary antibody (or primary to n-th antibody) is in a concentration higher than the final concentration of the reaction system (eg, 0.01 nM to 0.5 nM) in a buffer solution such as PBS containing 1% by weight or less of a blocking agent.
  • a dispersion prepared by dispersing (or each) the antibody reagent of the immunostaining reagent kit in place of the dispersion, and placing the antigen and the primary antibody (and the primary antibody) (Secondary to nth antibody) are bound (sequentially) following the antibody.
  • reaction conditions at this time include an example in which the antibody solution is reacted overnight at 4 ° C. with respect to a tissue section.
  • the unreacted antibody after the reaction, it is preferable to remove the unreacted antibody by washing the tissue section with a buffer solution (PBS or the like).
  • a buffer solution PBS or the like.
  • the washing step include a process of removing unreacted antibody reagent and the like by immersing the stained tissue section in a container containing PBS or the like for 3 to 30 minutes, for example.
  • PBS or the like may be exchanged during the immersion.
  • any one of the primary antibody to the n-th antibody immobilized on the antigen in the above-described immune reaction step is performed by the Huisgen cyclization addition reaction between azide and alkyne, and the above-mentioned fluorescence This is a step of covalently bonding body-integrated nanoparticles.
  • the reaction temperature of the staining reaction is not particularly limited as long as the Huesgen cycloaddition reaction proceeds, but it is preferably about 1 to 30 ° C., for example.
  • the reaction time of the staining reaction is not particularly limited as long as the above-mentioned Huesgen cycloaddition reaction proceeds. For example, an example of setting it to 1 to 10 hours, more preferably about 2 to 4 hours is given.
  • the final antibody concentration in the reaction system of the staining reaction is preferably 0.01 to 0.50 nM.
  • the phosphor-integrated nanoparticles only need to have a sufficient amount to label the antibody part in the reaction system, and the final concentration is preferably 0.01 to 0.50 nM.
  • a sufficient amount of the catalyst is sufficient in the system, and the final concentration is preferably 5 to 500 mM.
  • washing step After the staining reaction step, it is preferable to perform a washing step of washing the tissue section with PBS to remove unreacted phosphor-integrated nanoparticles.
  • a washing step for example, a washing step in which a tissue section is immersed in PBS adjusted to room temperature (1 to 30 ° C.) and left for 0.5 to 1 hour can be performed.
  • PBS or the like may be exchanged during the immersion.
  • the tissue section is stained with hematoxylin / eosin staining (HE staining) to obtain the cell shape of the tissue section and the position information of each part of the cell.
  • the morphological observation processing step can be arbitrarily performed.
  • the tissue section may be subjected to processing such as penetration and encapsulation for observation.
  • HE staining for example, an immunostained section is stained with Mayer's hematoxylin solution for 5 minutes and then stained with hematoxylin, and then the tissue sample is washed with running water at 45 ° C. for 3 minutes, and then 5% with 1% eosin solution. Perform eosin staining with minute staining.
  • Bright field observation is performed in order to acquire distribution information of cells of tissue sections or cell organs to be stained in the tissue.
  • a tissue section that has been subjected to hematoxylin / eosin staining (HE staining) after immunostaining as described above is observed with an optical microscope.
  • HE staining hematoxylin / eosin staining
  • eosin used for morphological observation staining can not only observe in a bright field, but also emits autofluorescence when irradiated with excitation light of a predetermined wavelength, so that an excitation light with an appropriate wavelength and output is applied to a stained tissue sample. Irradiation can be observed with a fluorescence microscope.
  • HER2 protein as an antigen to be detected as another staining
  • a 4 ⁇ objective lens of an optical microscope is used under irradiation with appropriate illumination light.
  • the objective lens is switched to 10 times, it is confirmed whether the positive findings are localized in the cell membrane or the cytoplasm, and if necessary, further searching is performed with the objective lens 20 times.
  • Fluorescence observation Using a fluorescence microscope, the number of fluorescent bright spots or emission luminance is measured from a wide-field microscope image for the stained section. An excitation light source and a fluorescence detection optical filter corresponding to the absorption maximum wavelength and fluorescence wavelength of the fluorescent substance used are selected. The number of bright spots or emission luminance can be measured by using commercially available image analysis software, for example, all bright spot automatic measurement software G-Count manufactured by Zeonstrom Co., Ltd. Note that image analysis itself using a microscope is well known, and for example, a technique disclosed in Japanese Patent Laid-Open No. 9-197290 can be used.
  • the field of view of the microscopic image is preferably 3 mm 2 or more, more preferably 30 mm 2 or more, and further preferably 300 mm 2 or more. Based on the number of bright spots and / or emission luminance measured from the microscopic image, the expression level of the protein (described above) derived from the specific gene of interest is evaluated.
  • the long-term storage stability of the labeling reagent contained in the immunostaining reagent kit according to the present invention can be evaluated as follows, for example. Using the labeling reagent immediately after production and the labeling reagent exposed to the predetermined acceleration conditions (eg, 30 ° C., 1 month), the above-described immunostaining is performed in the same manner, and the resulting fluorescence intensity (specific signal (Strength) can be determined by quantitative and relative evaluation.
  • the predetermined acceleration conditions eg, 30 ° C., 1 month
  • the intensity of the fluorescent signal obtained using a labeling reagent exposed under accelerated conditions is 70% or more compared to the intensity of the fluorescent signal obtained using a labeling reagent immediately after production, a long-term storage It can be evaluated as a labeling reagent having excellent properties.
  • an immunostaining method in which an antigen of a tissue section is fluorescently labeled with phosphor-integrated nanoparticles on the tissue section, and is directly fixed to the antigen by an antigen-antibody reaction.
  • An azide group (—N 3 ) is introduced into one of the antibody or another antibody immobilized indirectly through the antibody and the phosphor-integrated nanoparticles, and the carbon-carbon triple bond moiety (C ⁇ C) is introduced, the antibody is immobilized on the antigen, and both the antibody and the phosphor-aggregated nanoparticles are obtained by a Husgen cycloaddition reaction between the azide group and the carbon-carbon triple bond moiety.
  • a bond via a triazole ring is formed between molecules, and the antigen is fluorescently labeled with the phosphor-integrated nanoparticles by the formation. Since neither an azide group nor a carbon-carbon triple bond is present in the living body, according to the immunostaining method according to the present invention, an endogenous compound (see FIG. 4) that is a problem in the method using the biotin-avidin reaction (see FIG. 4). Noise due to endogenous biotin can be suppressed.
  • the hapten-antihapten Since the binding ability of the antibody and the phosphor-integrated nanoparticles is higher than that due to the antigen-antibody reaction, and the phosphor-aggregated nanoparticles bind strongly to the antibody, the antibody is immobilized on the tissue section via the antibody. Is less likely to be released, and the decrease in the number of bright spots due to the release is suppressed, resulting in an increase in the fluorescence signal from the bright spots detected by fluorescence observation after immunostaining.
  • the average particle diameter of the phosphor-integrated nanoparticles is 40 nm or more and 500 nm or less, in immunostaining using the phosphor-integrated nanoparticles, at least the position of the antibody specifically bound to the antigen is detected.
  • a fluorescent signal having a sufficiently detectable intensity can be obtained from the bright spot.
  • the tissue is comparable to that immediately after production.
  • a fluorescent signal with an intensity sufficient to detect the antigen on the section is obtained.
  • the average particle of the phosphor-integrated nanoparticles is 150 nm to 500 nm, and further 40 nm to 500 nm, the above-described effect on the fluorescence signal can be suitably obtained.
  • the phosphor-integrated nanoparticles are introduced into the phosphor-integrated nanoparticles by the PEG moiety by introducing the azido group or the carbon-carbon triple bond moiety via a hydrophilic polymer linker (eg, PEG linker).
  • a hydrophilic polymer linker eg, PEG linker
  • the phosphors are accumulated at a position spatially separated from the position of the antibody bound to the antigen on the tissue section by at least the length of the PEG linker.
  • nanoparticles can be arranged, the phosphor-aggregated nanoparticles are not easily obscured by insoluble precipitates by-produced by other staining methods combined with the above immunostaining, resulting in a reduction in fluorescence signal.
  • a fluorescent signal from a bright spot can be more suitably obtained by suppressing the above.
  • the effect (3) can be more suitably obtained. If the linkers of other hydrophilic polymers have the same length, the effect (3) can be obtained more suitably.
  • the Husgen cycloaddition reaction is carried out in the presence of a predetermined metal catalyst (eg, copper catalyst), the Husgen cycloaddition reaction is dramatically accelerated by the predetermined metal catalyst (eg, copper catalyst). Because of acceleration, even when a predetermined antibody or phosphor-integrated nanoparticle has a low concentration (eg, around 0.05 nM), both can be reacted and bonded.
  • a predetermined metal catalyst eg, copper catalyst
  • the Husgen cycloaddition reaction proceeds even without the above-mentioned predetermined metal catalyst, so that the labor of immunostaining can be omitted. It is possible to reduce the number of parts for the immunostaining reagent kit.
  • An immunostaining reagent kit is an immunostaining reagent kit for fluorescently labeling an antigen of a tissue section on a tissue section with phosphor-integrated nanoparticles, the labeling reagent containing the phosphor-integrated nanoparticles, An antibody reagent containing an antibody directly immobilized on an antigen by an antigen-antibody reaction, or another antibody indirectly immobilized via the antibody, and the phosphor-integrated nanoparticles and the antibody An azido group (—N 3 ) is introduced into one of them, and a carbon-carbon triple bond moiety (C ⁇ C) is introduced into the other, and the Huisgen cycloaddition of the azide group and the carbon-carbon triple bond moiety By the reaction, a bond via a triazole ring is formed between the antibody and the phosphor-integrated nanoparticles, and the antigen is fluorescently labeled by directly or indirectly binding the both molecules.
  • the immunostaining kit Like From it and is used to, by using the immunostaining kit, it is possible to highly inspection accuracy immunostaining as described above (1). Furthermore, when the immunostaining kit has the same configuration as the above (2) to (6), the effects described in (2) to (6) can be obtained when the kit is used for fluorescent immunostaining. .
  • sulforhodamine 101 (Sulforhodamine 101, Sigma-Aldrich), which is a red luminescent dye, was added as a fluorescent dye to 22 mL of distilled water and dissolved. Thereafter, 2 mL of a 5% by weight aqueous solution of an emulsion of an emulsifier for emulsion polymerization (registered trademark) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation) or “Latemul (registered trademark) PD-430” (Kao Chemical Co., Ltd.) is added to this solution. added. 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.
  • 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.
  • the obtained melamine particles were positively charged because the melamine resin itself contains many amino groups in the skeleton.
  • the charge of the resin particles was evaluated by analyzing resin components by NMR, IR, etc. and measuring zeta potential.
  • the nanoparticles were observed with a scanning electron microscope (SEM; Model S-800 manufactured by Hitachi (registered trademark)), and the average particle size and coefficient of variation were calculated.
  • the average particle diameter of the obtained phosphor-integrated nanoparticles was 150 nm, and the coefficient of variation was 12%.
  • Production Example 1 was the same as Production Example 1 except that 20.9 mg of sulforhodamine 101 (Sulforhodamine 101, Sigma-Aldrich) and 0.95 g of melamine resin raw material Nicalac MX-035 (Nihon Carbide Industries) were used. Phosphor integrated nanoparticles (average particle size 550 nm) were produced.
  • ⁇ Production Example III ⁇ Manufacture of phosphor-integrated nanoparticles (average particle size 800 nm) ⁇
  • Production Example 1 the same procedure as in Production Example 1 was conducted except that 23.1 mg of sulforhodamine 101 (Sulforhodamine 101, Sigma-Aldrich) and 1.05 g of melamine resin raw material Nicalac MX-035 (Nippon Carbide Industries) were used. Phosphor integrated nanoparticles (average particle size 800 nm) were produced.
  • ⁇ Production Example IV ⁇ Production of phosphor-integrated nanoparticles (average particle size 40 nm) ⁇
  • the phosphor integrated nano-particles were manufactured in the same manner as in Production Example 1 except that 9.9 mg of Sulforhodamine 101 (Sigma Aldrich) and 0.45 g of melamine resin raw material Nicalac MX-035 (Nihon Carbide Industries) were used. Particles (average particle size 40 nm) were produced.
  • Example 1-1 In Example 1-1, as described below, an alkyne compound is bound to an antibody, an azide is bound to a phosphor-aggregated nanoparticle, and a copper ion catalyst solution made of 50 mM copper bromide (CuBr).
  • An immunostaining reagent kit was prepared. At this time, copper bromide (CuBr) was put in the kit in a solid state, and a predetermined amount of water was added to obtain a solution having a predetermined concentration. Furthermore, using this, immunostaining was performed on a specimen slide (tissue array slide) on which tissue sections with different expression levels of HER2 antigen were mounted.
  • Step (1) 1 mg of the above phosphor-integrated nanoparticles were dispersed in 5 mL of pure water to prepare a dispersion. Subsequently, 20 ⁇ L of Tris (2-aminoethyl) amine (Tris (2-aminoethyl) amine) was added to the above dispersion, followed by heating and stirring at 70 ° C. for 20 minutes.
  • Tris (2-aminoethyl) amine Tris (2-aminoethyl) amine
  • Step (2) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
  • Step (3) Ethanol was added to disperse the sediment and centrifuged again. Washing with ethanol and pure water was performed once by the same procedure. When the FT-IR measurement and XPS measurement of the resulting amino group-modified nanoparticles were performed, absorption derived from the amino group could be observed, confirming that the amino group was modified.
  • Step (4) The amino group-modified nanoparticles obtained in step (3) were adjusted to 3 nM using PBS.
  • Step (5) ALK-PEG-NHS (PG2-AKNS-2k, NANOCOS) was mixed with the solution prepared in step (4) to a final concentration of 10 mM and reacted at room temperature for 1 hour.
  • PG2-AKNS-2k, NANOCOS PG2-AKNS-2k, NANOCOS
  • Step (6) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
  • Step (7) PBS containing 2 mM of EDTA was added to disperse the sediment of phosphor-aggregated nanoparticles, and then centrifuged again. The washing
  • an anti-rabbit antibody (5196-4504) manufactured by AbD Serotec was dissolved in PBS so as to be 1.0 mg / mL.
  • "NHS-PEG12-Azide” (Catalog No. 26131, manufactured by Thermo Scientific (or Thermo Fisher Scientific, the same shall apply hereinafter)) is added to 100 mol per 1 mol of this anti-rabbit antibody. And mixed. The mixed solution was further allowed to stand at 37 ° C. for 2 hours to carry out a reaction for binding the NHS group of “NHS-PEG12-Azide” to the amino group of the antibody. After the reaction, the reaction solution was subjected to a gel filtration column (Zaba Spin Desaling Columns: Funakoshi) and fractionated to produce an antibody reagent in which the antibody was dissolved in PBS.
  • a gel filtration column Zaba Spin Desaling Columns: Funakoshi
  • Copper bromide (CuBr) (product number 212865, manufactured by Sigma-Aldrich) was purchased as a copper catalyst, and diluted with PBS so that the copper ion concentration was 50 mM to prepare a metal catalyst solution.
  • IHC Immunohistochemistry
  • a dispersion (labeling reagent) of phosphor-aggregated nanoparticles modified with the alkyne compound, an anti-HER2 antibody (from rabbit, 4B5, manufactured by Ventana), a solution of an anti-rabbit antibody modified with the azide (secondary antibody reagent), and Immunostaining was performed using an immunostaining reagent kit comprising the metal catalyst solution as described below.
  • the tissue array slide was immunostained by performing the following steps in order using the above immunostaining reagent kit and the like.
  • a tissue array slide (br243) manufactured by US Biomax was used, and a tissue section of a breast cancer tissue (HER2 (+) positive) and a normal cell tissue section (HER2 ( ⁇ ) negative) were used. Using.
  • HER2 staining concentration of each tissue section described above was observed by DAB staining, and three lots of HER2 high expression (HER2 3+), HER2 low expression (HER2 +), and HER2 negative (HER2 ⁇ ) were selected. Prepared and immunostained each lot. Note that the “HER2 3+”, “HER2 +”, and “HER2 ⁇ ” correspond to the IHC method criteria “3+”, “1+”, and “0” in Table 3 above, respectively.
  • Step (2C) The tissue section of the tissue array slide that had undergone the step (2B) was immersed in PBS for 30 minutes.
  • HE staining immunostained tissue sections were stained with Mayer's hematoxylin solution for 5 minutes to perform hematoxylin staining. The sections were then washed with running water at 45 ° C. for 3 minutes.
  • eosin staining was performed by staining with 1% eosin solution for 5 minutes. Then, the operation which was immersed in pure ethanol for 5 minutes was performed 4 times, and washing
  • the excitation light was set to 575 to 600 nm by passing through an optical filter.
  • the range of the wavelength (nm) of fluorescence to be observed was also set to 612 to 682 nm by passing through an optical filter.
  • the excitation wavelength conditions at the time of microscopic observation and image acquisition were such that the irradiation energy near the center of the field of view was 900 W / cm 2 for excitation at 580 nm.
  • the exposure time at the time of image acquisition was arbitrarily set (for example, set to 4000 ⁇ sec) so as not to saturate the luminance of the image.
  • a microscope image taken with a fluorescence microscope or the like a portion where the luminance exceeds a predetermined threshold is measured as a bright spot, and the number of phosphor-integrated nanoparticles per cell and the intensity of the fluorescence signal are measured. Calculation and accuracy evaluation were performed (see Table 4).
  • evaluation value (Evaluation value) (Evaluation 1) ⁇ Evaluation of low non-specific adsorption ⁇
  • “Evaluation 1” indicates the result of performing a series of steps including the above immunostaining on a tissue section in which HER2 antigen is not present (IHC method score HER2 (0)) and the evaluation result based on the result.
  • the upper is the result of evaluation 1, the lower number is the number of bright spots), the smaller the number of bright spots, the less non-specific adsorption and the higher the accuracy of antigen detection.
  • the number of bright spots resulting from non-specific adsorption per average cell in the observation field of the tissue section where the HER2 antigen is not present, and the results of accuracy evaluation based on the number of bright spots are shown.
  • the evaluation “ ⁇ ” is the case where the number of bright spots per cell is 5 or less in the above observation, and the non-specific adsorption of the phosphor-integrated nanoparticles and the predetermined antibody is small and the detection accuracy is low. Indicates high.
  • the evaluation “x” is a case where the number of bright spots per cell measured in the above observation is 6 or more, and indicates that non-specific adsorption of phosphor-integrated nanoparticles is large and detection accuracy is low.
  • the evaluation “impossible to measure” indicates a case where the bright spot itself cannot be confirmed.
  • Evaluation 2 is a result of performing a series of steps including the above immunostaining on a tissue section highly expressing the HER2 antigen (IHC method score HER2 (3+)) and an evaluation result (fluorescence signal) (Strength evaluation results). The stronger the fluorescent signal from the bright spot, the easier it is to detect the antigen (the higher the detection accuracy of the antigen).
  • the numerical value of the item of evaluation 2 in Table 4 shows the relative value of the intensity of the fluorescence signal. This relative value is a relative value with respect to other examples and comparative examples, and is expressed as “100” of Example 1-1.
  • the evaluation “ ⁇ ” indicates that the relative value is 70 or more, and that the intensity of the fluorescent signal is sufficient for the bright spot measurement.
  • Evaluation “ ⁇ ” indicates a case where the relative value is 50 or more and less than 70, and indicates that the intensity of the fluorescent signal is slightly inferior, but is a level at which a bright spot can be measured.
  • the evaluation “ ⁇ ” indicates a case where the relative value is 0 or more and less than 50, and indicates that the intensity of the fluorescent signal is insufficient for the bright spot measurement.
  • the evaluation “impossible to measure” indicates a case where the bright spot itself cannot be confirmed.
  • Evaluation 3 indicates the intensity of the fluorescence signal obtained as a relative value when a series of steps including the immunostaining is performed using the labeling reagent stored at 30 ° C. for 1 month in the storage evaluation test ( This relative value is shown as a relative value when the intensity of the fluorescent signal in Evaluation 2 of Example 1-1 is set to “100” as a reference).
  • the value (%) in parentheses indicates the following bright spot retention rate (%) (see the following formula 1).
  • Bright spot retention rate (%) number of bright spots (stored at 30 ° C.
  • the evaluation “ ⁇ ” indicates that the bright spot retention rate (%) is 70% or more, and that the storage stability of the labeling reagent is high (as a result, the storage stability of the immunostaining reagent kit is high). Show. Evaluation “x” shows that a bright spot retention rate (%) is less than 70%, and the said preservability is low.
  • Example 1 except that the phosphor integrated nanoparticles (average particle diameter 550 nm) produced in Production Example II were used instead of the phosphor integrated nanoparticles (average particle diameter 150 nm) used in Example 1-1.
  • phosphor-integrated nanoparticles modified with an alkyne compound (having a carbon-carbon triple bond moiety) were produced, and a series of steps including immunostaining and evaluation were performed. The results are shown in Table 4.
  • Example 1 except that the phosphor-integrated nanoparticles (average particle diameter 800 nm) produced in Production Example III were used instead of the phosphor-integrated nanoparticles (average particle diameter 150 nm) used in Example 1-1.
  • phosphor-integrated nanoparticles modified with an alkyne compound (having a carbon-carbon triple bond moiety) were produced, and a series of steps including immunostaining and evaluation were performed. The results are shown in Table 4.
  • Example 1 except that the phosphor integrated nanoparticles (average particle size 40 nm) produced in Production Example IV were used instead of the phosphor integrated nanoparticles (average particle size 150 nm) used in Example 1-1.
  • phosphor-integrated nanoparticles modified with an alkyne compound (having a carbon-carbon triple bond moiety) were produced, and a series of steps including immunostaining and evaluation were performed. The results are shown in Table 4.
  • Example 2-1 ⁇ Manufacture of phosphor-integrated nanoparticles (150nm) modified with azide ⁇
  • alkyne compound “ALK-PEG-NHS” PG2-AKNS-2k, NANOCOS
  • Phosphor integrated nanoparticles modified with azide having an azide group
  • NHS-PEG12-Azide Catalog No. 26131, manufactured by Thermo Scientific
  • Example 2-1 The operations (preparation of copper catalyst, immunostaining, evaluation, etc.) other than the production of the phosphor-integrated nanoparticles and antibody were performed in the same manner as in Example 1-1.
  • the results of Example 2-1 are shown in Table 4.
  • Example 2-2 In Example 2-1, Example 2-1, except that the phosphor integrated nanoparticles (average particle diameter 550 nm) produced in Production Example II were used instead of the phosphor integrated nanoparticles (average particle diameter 150 nm).
  • the phosphor integrated nanoparticles average particle diameter 150 nm.
  • Example 2-3 In Example 2-1, Example 2-1, except that the phosphor integrated nanoparticles (average particle size 800 nm) produced in Production Example III were used instead of the phosphor integrated nanoparticles (average particle size 150 nm).
  • Example 2-4 In Example 2-1, Example 2-1, except that the phosphor integrated nanoparticles (average particle size 40 nm) produced in Production Example IV were used instead of the phosphor integrated nanoparticles (average particle size 150 nm).
  • the phosphor integrated nanoparticles average particle size 150 nm.
  • streptavidin manufactured by Wako Pure Chemical Industries, Ltd.
  • 2-Iminothiolane manufactured by Pierce
  • This streptavidin solution was desalted with a gel filtration column (Zaba Spin Desaling Columns: Funakoshi) to obtain 0.04 mg of streptavidin having an SH group.
  • Step (1) 1 mg of the above phosphor-integrated nanoparticles were dispersed in 5 mL of pure water to prepare a dispersion. Subsequently, 20 ⁇ L of Tris (2-aminoethyl) amine (Tris (2-aminoethyl) amine) was added to the above dispersion, followed by heating and stirring at 70 ° C. for 20 minutes.
  • Tris (2-aminoethyl) amine Tris (2-aminoethyl) amine
  • Step (2) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
  • Step (3) Ethanol was added to disperse the sediment and centrifuged again. Washing with ethanol and pure water was performed once by the same procedure.
  • the obtained amino group-modified nanoparticles were subjected to FT-IR measurement and XPS measurement. As a result, absorption derived from the amino group was observed, and it was confirmed that the amino group was modified.
  • step (A) was performed using the biotinylated anti-HER2 antibody and phosphor-integrated nanoparticles modified with streptavidin. Went. Other than that, immunostaining and evaluation were performed in the same manner as in Example 1-1. The results are shown in Table 4.
  • anti-fluorescein antibody Anti-Fluorescein, Go-Poly, SP-0601, Vector Laboratories
  • Anti-Fluorescein Go-Poly, SP-0601, Vector Laboratories
  • 2-Iminothiolane Pirce
  • 64 mg / mL at room temperature For 1 hour. That is, a thiol group was introduced to the amino group of the anti-fluorescein antibody.
  • the anti-fluorescein antibody solution was desalted with a gel filtration column (Zaba Spin Desaling Columns: Funakoshi) to obtain 0.04 mg of an anti-fluorescein antibody having an SH group.
  • Step (1) 1 mg of the above phosphor-integrated nanoparticles were dispersed in 5 mL of pure water to prepare a dispersion. Next, 20 ⁇ L of Tris (2-aminoethyl) amine was added to the dispersion, and the mixture was heated and stirred at 70 ° C. for 20 minutes.
  • Step (2) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
  • Step (3) Ethanol was added to disperse the sediment and centrifuged again. Washing with ethanol and pure water was performed once by the same procedure.
  • the obtained amino group-modified nanoparticles were subjected to FT-IR measurement and XPS analysis. As a result, absorption derived from the amino group was observed, and it was confirmed that the amino group was modified.
  • Example 1-1 ⁇ Immunostaining ⁇ Instead of the ⁇ immune reaction step ⁇ and ⁇ staining reaction step ⁇ in Example 1-1, the above-described fluorescein-modified anti-rabbit antibody and phosphor-integrated nanoparticles modified with the anti-fluorescein antibody were used, and the following steps (B ) Other than that, immunostaining and evaluation were performed in the same manner as in Example 1-1. The results are shown in Table 4.
  • the sulforhodamine 101 having an amino group introduced therein is dissolved in 1 mL of dichloromethane (CH 2 Cl 2 ), and an amount of “ALK-PEG corresponding to 1.1 mol per 1 mol of the sulforhodamine 101 having an amino group introduced thereinto.
  • -NHS (PG2-AKNS-2k, NANOCOS) was added and mixed and allowed to react overnight at room temperature.
  • the resulting reaction solution was purified by silica gel chromatography.
  • a solution of sulforhodamine 101 having an alkyne-derived carbon-carbon triple bond moiety via PEG was prepared. When FT-IR measurement was performed on this sulforhodamine 101, absorption derived from a carbon-carbon triple bond was observed, and it was confirmed that an alkyne compound was bound (not shown).
  • Comparative Example 1 using the biotin-streptavidin bond, the biotin-streptavidin bond is a strong bond as in the Husgen cycloaddition reaction and a strong fluorescence signal is obtained. Since it binds to endogenous biotin, nonspecific adsorption cannot be suppressed. Therefore, Comparative Example 1 cannot achieve both staining specificity and luminescence performance in immunostaining.
  • Comparative Example 2 using the binding between the hapten and the anti-hapten antibody, since the binding between the hapten and the anti-hapten antibody is specific, nonspecific binding is unlikely to occur.
  • the bond is weaker than the bond due to the Husgen cycloaddition reaction, and the binding between the fluorescent aggregate nanoparticle and the antibody is easily released accordingly. The decline cannot be suppressed. Therefore, Comparative Example 2 cannot achieve both staining specificity and luminescence performance in immunostaining.
  • the immunostaining method according to the present invention and the immunostaining reagent kit used therefor have been described in detail based on the embodiments and examples.
  • the present invention is not limited to these examples and the like, and is claimed. Changes in design are allowed without departing from the scope of the present invention described in the above.

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Abstract

Selon l'invention, un groupement azoture (-N3) est introduit dans l'un des membres du groupe constitué par un anticorps qui peut être immobilisé sur un antigène directement par une réaction antigène-anticorps et un autre anticorps qui peut être immobilisé sur l'antigène indirectement par l'intermédiaire de l'anticorps susmentionné et une nanoparticule à luminophore intégré et une partie à triple liaison carbone-carbone (C≡C) est introduite dans l'autre, l'anticorps est immobilisé sur l'antigène, une liaison à travers un cycle triazole est formée entre une molécule de l'anticorps et une molécule de la nanoparticule à luminophore intégré par une réaction de cycloaddition de Huisgen entre le groupement azoture et la partie à triple liaison carbone-carbone, et l'antigène est marqué de manière fluorescente par la nanoparticule à luminophore intégré via la formation susmentionnée. De cette manière, il devient possible d'empêcher l'apparition d'une adsorption non-spécifique d'une nanoparticule de colorant fluorescent qui peut provoquer la génération de bruit de signal, et il devient également possible d'empêcher la rupture de la liaison entre l'anticorps lié à l'antigène à détecter et la nanoparticule à luminophore intégré, et par conséquent, d'empêcher la réduction des signaux due à la rupture susmentionnée.
PCT/JP2015/080512 2014-11-06 2015-10-29 Procédé d'immunocoloration, et trousse de réactifs d'immunocoloration pour utilisation dans ledit procédé WO2016072341A1 (fr)

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WO2022030371A1 (fr) * 2020-08-05 2022-02-10 コニカミノルタ株式会社 Procédé de coloration immunologique et procédé de production d'un échantillon de tissu à coloration immunologique
EP3497445B1 (fr) * 2016-08-12 2022-02-16 IST Austria - Institute of Science and Technology Austria Procédés de microstructuration et dispositifs à microstructures
CN115754281A (zh) * 2022-11-28 2023-03-07 广东省大湾区华南理工大学聚集诱导发光高等研究院 一种荧光纳米粒子在免疫荧光组化染色中的应用
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JP2017227502A (ja) * 2016-06-21 2017-12-28 コニカミノルタ株式会社 組織切片から蛍光ナノ粒子の解離を防止する方法
EP3497445B1 (fr) * 2016-08-12 2022-02-16 IST Austria - Institute of Science and Technology Austria Procédés de microstructuration et dispositifs à microstructures
JPWO2018185943A1 (ja) * 2017-04-07 2020-02-13 コニカミノルタ株式会社 蛍光プレミックス粒子、それを含有する蛍光染色液、およびそれらを用いた蛍光染色法
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WO2022030371A1 (fr) * 2020-08-05 2022-02-10 コニカミノルタ株式会社 Procédé de coloration immunologique et procédé de production d'un échantillon de tissu à coloration immunologique
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CN115754281A (zh) * 2022-11-28 2023-03-07 广东省大湾区华南理工大学聚集诱导发光高等研究院 一种荧光纳米粒子在免疫荧光组化染色中的应用
CN115754281B (zh) * 2022-11-28 2023-08-15 广东省大湾区华南理工大学聚集诱导发光高等研究院 一种荧光纳米粒子在免疫荧光组化染色中的应用

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