WO2015045961A1 - Marqueur à nanoparticules fluorescentes, kit d'immunocoloration multiple, et procédé d'immunocoloration multiple - Google Patents

Marqueur à nanoparticules fluorescentes, kit d'immunocoloration multiple, et procédé d'immunocoloration multiple Download PDF

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WO2015045961A1
WO2015045961A1 PCT/JP2014/074418 JP2014074418W WO2015045961A1 WO 2015045961 A1 WO2015045961 A1 WO 2015045961A1 JP 2014074418 W JP2014074418 W JP 2014074418W WO 2015045961 A1 WO2015045961 A1 WO 2015045961A1
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immunostaining
fluorescent
phosphor
hydrophilic polymer
polymer chain
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PCT/JP2014/074418
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English (en)
Japanese (ja)
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拓司 相宮
健作 高梨
古澤　直子
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コニカミノルタ株式会社
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals

Definitions

  • the present invention relates to a fluorescent nanoparticle label, a multiple immunostaining kit containing the fluorescent nanoparticle label, and a multiple immunostaining method using the multiple immunostaining kit.
  • a technique for binding an antibody to which a dye or a coloring enzyme is bound to an antigen in a tissue sample by an antigen-antibody reaction and causing the color to develop and detecting the antigen is widely known as a technique by immunohistochemistry (IMUNOHITOSHEMISTRY; IHC). Yes.
  • immunohistochemical method is sometimes referred to as “immunostaining” because it involves a color development process that visualizes an antigen-antibody reaction that is inherently invisible. Immunostaining is widely used in the medical and life science fields for the purpose of detecting the location of biomolecules in tissue samples.
  • DAB staining which is an example of immunostaining
  • DAB Diaminobenzidine; diaminobenzidine
  • DAB Diaminobenzidine
  • a chemical reaction chemical color development
  • DAB molecules are polymerized by O 2 2 ⁇ generated by the decomposition, and the antigen portion is stained brown.
  • immunostaining targeting markers such as CD34 antigen
  • Patent Document 1 discloses semiconductor fine particles whose surface is modified with polyethylene glycol (PEG) having a molecular weight of 300 to 20000. It is also disclosed that a biomolecule is provided at the tip of PEG of the semiconductor fine particles.
  • PEG polyethylene glycol
  • the surface of the semiconductor fine particles is modified with PEG (molecular weight of 300 to 3000, etc.), and a functional group (amino group, etc.) is provided at the tip of the PEG farthest from the surface.
  • PEG molecular weight of 300 to 3000, etc.
  • a functional group amino group, etc.
  • a biomolecule fluorescent labeling agent in which another biomolecule (DNA or the like) that can specifically bind to a biomolecule to be stained is bound to a group, and an immunofluorescent staining method using the same are disclosed.
  • Patent Document 4 two or more types of antigens are detected on the same tissue section, and the location and amount of each antigen are evaluated. Furthermore, Patent Document 4 discloses that, as a colored substance or a luminescent substance for the detection, a substance that colors or emits light by reaction with a substrate of a labeling enzyme and a fluorescent dye are used in combination with an optical microscope. It is described that it is preferable from the viewpoint that a fluorescence microscope or a laser confocal microscope can be used.
  • the detection accuracy of biomolecules can be improved by performing multiple immunostaining on the same biomolecule using immunostaining that uses chemical coloration and fluorescence immunostaining that uses luminescence by a fluorescent dye. Disclosure.
  • the present inventors absorb insoluble precipitates generated by the reaction between an enzyme and a predetermined substrate, the excitation light that irradiates the phosphor and the fluorescence emitted by the phosphor. It was speculated that this was the cause of the problems specific to multiple immunostaining as described above.
  • fluorescent immunostaining phosphor-integrated nanoparticles having phosphors accumulated are used, and biomolecule recognition molecules are bound to the phosphor-aggregated nanoparticles via a hydrophilic polymer chain having a specific length.
  • fluorescent immunostaining of a predetermined biomolecule can avoid the above-mentioned problems, and fluorescence observation of the predetermined biomolecule can be performed at the same level as single staining that performs only fluorescent immunostaining.
  • the present invention has been completed.
  • the present inventors have compared the fluorescent dye as compared with the single staining in which only fluorescent immunostaining is performed. I noticed a problem that the luminescence intensity or the number of observed bright spots decreased.
  • the present invention has been made in view of the above problems, and in the multiple immunostaining method using both immunostaining using chemical color development and fluorescent immunostaining using fluorescence of a fluorescent substance such as a fluorescent dye,
  • An object of the present invention is to suppress the decrease in emission intensity or the number of bright spots.
  • a fluorescent nanoparticle label reflecting one aspect of the present invention is bound to a phosphor-integrated nanoparticle having phosphors in an accumulated state and a surface of the phosphor-integrated nanoparticle.
  • a fluorescent nanoparticle label for multiple immunostaining comprising a hydrophilic polymer chain (A) having a number average molecular weight of 3000 or more and a biomolecule recognition molecule bonded to the end of the hydrophilic polymer chain.
  • a multiple immunostaining agent kit comprises a fluorescent immunostaining agent having the fluorescent nanoparticle label and a chemical immunostaining agent capable of bright field observation.
  • the multiple immunostaining method reflecting one aspect of the present invention includes fluorescent immunostaining using the fluorescent immunostaining agent having the fluorescent nanoparticle label and chemical immunity capable of bright field observation.
  • This is a multiple immunostaining method in which chemical immunostaining using a staining agent is performed on the same section.
  • the above structure reduces the emission intensity or the number of bright spots due to the insoluble precipitate generated by the chemical color development. Can be suppressed. As a result, the number of bright spots obtained when fluorescent immunostaining is performed alone can be ensured even in multiple immunostaining.
  • FIG. 1A is a diagram showing a conceptual diagram of a conventional multiple immunostaining method.
  • FIG. 1B is a diagram showing an example of the multiple immunostaining method according to the present invention.
  • FIG. 2 is a diagram showing an example of a fluorescent nanoparticle label according to the present invention.
  • FIG. 3 is a diagram showing another example of the fluorescent nanoparticle label according to the present invention.
  • the fluorescent nanoparticle label according to the present invention is used for multiple immunostaining, and has phosphor integrated nanoparticles having fluorescent substances such as fluorescent dyes integrated therein, and the number average bonded to the surface of the fluorescent integrated nanoparticles. It has a hydrophilic polymer chain having a molecular weight of 3000 or more and a biomolecule recognition molecule bonded to the end of the hydrophilic polymer chain.
  • the fluorescent immunostaining agent having this fluorescent nanoparticle label can be used as a multiple immunostaining together with a chemical coloring stain.
  • the phosphor-integrated nanoparticles are those in which phosphors are accumulated, and are sometimes referred to as fluorescent nanoparticle labels. 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. Further, the “phosphor” referred to in the present invention is not limited by the emission lifetime after the excitation light is blocked.
  • phosphorescent substance such as zinc sulfide or strontium aluminate.
  • 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.
  • CdSe CdS, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge, but are not limited thereto.
  • 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 in which the phosphor is integrated 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 the method of impregnating polystyrene nanoparticles with a fluorescent organic dye is described.
  • 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.
  • Various fluorescent dye-containing silica nanoparticles can be synthesized by using a desired fluorescent dye instead of FITC.
  • 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 the semiconductor fine particles as phosphors having carboxyl 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
  • an aggregate in which an inorganic phosphor is immobilized inside or on the surface of a matrix made of resin can be made using the method of impregnating quantum nanoparticles into polystyrene nanoparticles described in Nature Biotechnology Vol. 19, p. 631 (2001).
  • the hydrophilic polymer chain used for the surface modification of the phosphor-integrated nanoparticles is a hydrophilic polymer having a predetermined number average molecular weight for linking the phosphor-integrated nanoparticles and a biomolecule recognition molecule described later ( A) and optionally a hydrophilic polymer (B) having a number average molecular weight other than the hydrophilic polymer (A) (usually smaller than the hydrophilic polymer (A)) are used (see FIG. 3 see PEG chain 1 and PEG chain 2 example).
  • hydrophilic polymers (A) and (B) include, but are not limited to, polyethylene glycol, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, polyvinyl Pyrrolidone, polyvinyl methyl ether, polyvinyl methyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, Examples include polyaspartamide and synthetic polyamino acids.
  • hydrophilic polymer chains (A) and (B) one type or two or more types of hydrophilic polymers selected from the group formed by the exemplified hydrophilic polymers can be used.
  • hydrophilic polymers exemplified above polyethylene glycol (PEG) is preferable from the viewpoint of easy setting of the chain length depending on the number of oxyethylene units.
  • the number average molecular weight of the hydrophilic polymer chain (A) is 3000 or more, preferably 5000 or more, and preferably 20000 or less.
  • the number average molecular weight of this hydrophilic polymer chain (A) is compared with the case where fluorescent immunostaining is performed alone when multiple immunostaining is performed using immunostaining using chemical coloring and fluorescent immunostaining.
  • the number of bright spots resulting from the phosphor can be adjusted to be confirmed to the same extent.
  • the number of bright spots can be confirmed with a fluorescence microscope or the like as will be described later.
  • the number average molecular weight of the hydrophilic polymer chain (B) is usually less than 3000, preferably 300 or more.
  • the number average molecular weight of the hydrophilic polymer chain (B) can be adjusted in consideration of the effect of preventing aggregation of the phosphor-integrated nanoparticles.
  • the polymer structural unit contained in the chemical structure of the hydrophilic polymer is used. It is preferable to adjust by the number.
  • the hydrophilic polymer is PEG, it is preferably adjusted by the number (n) of oxyethylene units (—CH 2 —CH 2 —O).
  • the number average molecular weight of the phosphor-integrated nanoparticles is measured by a gel permeation chromatography (GPC) method using tetrahydrofuran (THF) as a column solvent. Specifically, 1 mL of THF is added to 1 mg of a measurement sample, and the mixture is sufficiently dissolved by stirring using a magnetic stirrer at room temperature. Next, after processing with a membrane filter having a pore size of 0.45 to 0.50 ⁇ m, it is injected into the GPC.
  • GPC gel permeation chromatography
  • the measurement conditions of GPC are measured by stabilizing the column at 40 ° C., flowing THF at a flow rate of 1 mL / min, and injecting about 100 ⁇ L of a sample having a concentration of 1 mg / mL.
  • the column it is preferable to use a combination of commercially available polystyrene gel columns.
  • Shodex GPC KF-801, 802, 803, 804, 805, 806, 807 made by Showa Denko KK, TSKgel G1000H, G2000H, G3000H, G4000H, G5000H, G6000H, G7000H, TSK guard column made by Tosoh Corporation Combinations can be mentioned.
  • a refractive index detector (RI detector) is preferably used as the detector.
  • the molecular weight distribution of the sample is calculated using a calibration curve or the like created using monodisperse polystyrene standard particles. It is preferable to use about 10 points as polystyrene for preparing a calibration curve.
  • the bond between the hydrophilic polymer and the phosphor-integrated nanoparticles is not particularly limited, and may be bonded or bonded by an appropriate bonding mode such as a covalent bond, an ionic bond, a hydrogen bond, a coordination bond, a physical adsorption, or a chemical adsorption.
  • an amide bond, an ester bond, an imide bond, a covalent bond such as a bond using thiol addition to a maleimide group, or the like, or a biotin-avidin bond or a biotin-streptavidin bond is preferable.
  • biotin-avidin bond or biotin-streptavidin bond is more preferable from the viewpoint of strength of binding force and specificity of binding.
  • binding methods include biotin-avidin method, thiol group-maleimide group coupling reaction method, method using chemical linker, cross-linking agent (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), etc.) Examples of the crosslinking reaction method and ion binding method using
  • an amino group (NH 2 group) is present on the surface of the phosphor-integrated nanoparticle, for example, it is bound to biotin (biomolecule recognition molecule) as shown in [Chemical Formula 1] below.
  • a PEGylation reagent can be preferably used.
  • the N-hydroxysuccinimidyl ester group of the PEGylating reagent (the functional group at the left end of [Chemical Formula 1]) reacts with the amino group on the surface of the phosphor-integrated nanoparticle to form the phosphor-integrated nanoparticle.
  • a PEG chain and biotin are added (see FIG. 2).
  • a sulfhydryl group (SH group) is present on the surface of the phosphor-integrated nanoparticle, for example, it binds to biotin (biomolecule recognition molecule) as shown in [Chemical Formula 2] below.
  • a PEGylation reagent can be preferably used.
  • the maleimide group of the PEGylation reagent (functional group at the left end of [Chemical Formula 2]) reacts with the sulfhydryl group (SH group) on the surface of the phosphor-integrated nanoparticle, and the phosphor-aggregated nanoparticle has a PEG chain.
  • biotin will be added (not shown).
  • the number average molecular weight of the PEG chain moiety “— (CH 2 CH 2 O) n—” derived from PEG (hydrophilic polymer) is calculated. In order to make it 3000 or more, it is preferable to set to the number (n) of oxyethylene units.
  • the chain length of the portion derived from the hydrophilic polymer is designed to be equal to or more than the number average molecular weight of the PEG chain. And can be used as a material for fluorescent nanoparticle labels.
  • a PEGylation reagent having a predetermined molecular weight can be obtained, for example, by requesting synthesis from a synthesis manufacturer (for example, CreativePEGWorks).
  • a functional group necessary for binding may be introduced into the phosphor-integrated nanoparticles or the hydrophilic polymer chain in advance.
  • a functional group necessary for phosphor-collected nanoparticles in advance when producing phosphor-collected nanoparticles using a thermoplastic resin, copolymerization is performed using glycidyl methacrylate as a monomer together with styrene.
  • the number of hydrophilic polymers to be bonded per phosphor-integrated nanoparticle can be adjusted by the number of functional groups as described above required for bonding, reaction conditions with the hydrophilic polymer, or the like.
  • thermosetting dye resin particles when producing thermosetting dye resin particles, melamine resin dye resin particles having an amino group on the surface are copolymerized using a melamine resin raw material (for example, MX035 manufactured by Sanwa Chemical Co., Ltd.) as a monomer. Can also be reacted with the PEGylation reagent and the like.
  • a melamine resin raw material for example, MX035 manufactured by Sanwa Chemical Co., Ltd.
  • Biomolecule recognition molecule used in the present invention means a molecule that specifically binds to a biomolecule such as an antigen.
  • Biomolecule recognition molecules include a primary antibody that recognizes a specific biomolecule to be stained, a secondary (to nth) antibody that recognizes this antibody, and a specific antibody that binds to the secondary (to nth) antibody.
  • Examples of biomolecule recognition molecules include antibodies, biotin, and avidin (including analogs such as streptavidin).
  • antibody is used to include any antibody fragment or derivative, and includes Fab, Fab′2, CDR, humanized antibody, multifunctional antibody, single chain antibody (ScFv) and the like. Contains various antibodies.
  • the term “antigen” refers to a biological material, particularly a molecule or molecular fragment (polypeptide, oligopeptide, etc.) consisting of a protein, but any biological material capable of producing antibodies and immunostaining can be used.
  • a molecule or molecular fragment other than a protein such as a nucleic acid (DNA that may be single-stranded or double-stranded, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., or nucleoside, It is also possible that nucleotides and their modified molecules) can be antigens.
  • Antibodies that are constituents of antibody drugs can be used as biomolecule recognition molecules.
  • Examples of antibody drugs include antibody drugs generally used for the treatment of autoimmune diseases such as rheumatoid arthritis, malignant tumors such as cancer, and viral infections.
  • Biomolecular recognition molecules and hydrophilic polymer chain ends and bonds are covalent bonds, ionic bonds, hydrogen bonds, coordinate bonds, physical adsorption, as well as the bonds between phosphor-integrated nanoparticles and hydrophilic polymer chains. From the viewpoint of bonding by an appropriate bonding mode such as chemisorption and the strength of bonding force, a covalent bond such as an amide bond, an ester bond, an imide bond, and a bond using thiol addition to a maleimide group is preferable.
  • Specific binding methods for binding between the biomolecule recognition molecule and the end of the hydrophilic polymer chain include biotin-avidin method, thiol group-maleimide group coupling reaction method, method using existing chemical linker, crosslinking Examples thereof include a crosslinking reaction method using an agent (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and the like), an ionic bond method, and the like.
  • the biomolecules to be stained for chemical immunostaining and fluorescent immunostaining are not particularly limited, and may be the same or different.
  • staining targets for example, when the target of chemical immunostaining is CD34 antigen and the target of fluorescent immunostaining is VEGFR-2
  • multiple staining targets are to be detected separately by different staining methods
  • the binding mode of the biomolecule recognition molecule and the target to be stained in fluorescent immunostaining is different from the binding mode of the biomolecule recognition molecule and the target to be stained in chemical immunostaining to avoid unintentional binding and competition between molecules. Is appropriate.
  • the binding mode is such that a complex of antigen to be stained—primary antibody (mouse antibody) —enzyme-labeled secondary antibody (anti-mouse IgG antibody) is formed
  • primary antibody molecular antibody
  • anti-mouse IgG antibody enzyme-labeled secondary antibody
  • fluorescent immunostaining Is a binding mode that forms a complex of antigen-primary antibody to be stained (rabbit antibody) -biotinylated secondary antibody (anti-rabbit IgG antibody) -phosphor-integrated nanoparticles having avidin as a biomolecule recognition molecule It can be.
  • the multiple immunostaining method according to the present invention is a multiple immunostaining method that performs both staining using chemical color development and fluorescent immunostaining using fluorescent nanoparticle labels.
  • the target of staining in the multiple staining method according to the present invention is a biomolecule targeted by a general immunostaining method, and is a tumor marker, a signal transmitter, a hormone, a cancer growth regulator, a metastasis regulator, a growth regulator acceptance. Body, metastasis regulator receptor and the like.
  • Deparaffinization treatment step A pathological 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 pathological 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. Further, 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. 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
  • 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.
  • 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.
  • Staining treatment step A for chemical immunostaining includes a binding step A in which an antibody linked to a coloring enzyme is bound to a specific biomolecule in a pathological section by an antigen-antibody reaction, and the like. And a color development step A in which a color development substrate is added to the reaction system and color is developed by the color development enzyme.
  • the above binding to the biomolecule can be performed by a known method.
  • a known blocking agent such as PBS containing BSA is preferably added dropwise and incubated at room temperature for a predetermined time (for example, 1 hour).
  • the coloring enzyme substrate and the coloring agent are added to the reaction system for chemical coloration.
  • the coloring agent that is the object of the present invention is to generate an insoluble precipitate in the coloring reaction, and Can be illustrated.
  • HRP horseradish peroxidase
  • TMB 3,3,5,5-tetramethylbenzidine
  • DAB 3,3′-diaminobenzidine
  • 4-chloro-1-naphthol, etc. are targeted It becomes.
  • an enzyme alkaline phosphatase is used as a color-developing enzyme, new fuchsin and the like are targeted.
  • color formers that generate unnecessary precipitates by chemical color development are targeted.
  • the staining process B for fluorescent immunostaining includes a binding process B for binding the biomolecule recognition molecule of the fluorescent nanoparticle label and the biomolecule.
  • the biomolecule here means an antigen to be stained by fluorescent immunostaining, a primary antibody specifically bound to the antigen, a secondary (n-order) antibody bound to the primary antibody, or the antigen or It means any of the other biomolecules bound to the 1st to nth antibodies. Further, there is no limitation on the order of binding between the aforementioned antigen, primary antibody, secondary (n-order) antibody, other biomolecules, and biomolecule recognition molecules.
  • a fluorescent nanoparticle labeled buffer (PBS or the like) dispersion is prepared, placed on a pathological section, and the fluorescent nanoparticle labeled biomolecule recognition molecule and the biomolecule Are combined.
  • the stained section is immersed in a container containing a buffer (PBS or the like) to remove unreacted fluorescent nanoparticle labels, antibodies having a coloring enzyme, and the like.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, the buffer (such as PBS) may be exchanged during the immersion.
  • the fixing treatment step is a step of immobilizing the color former and fluorescent nanoparticle label introduced in the tissue staining treatment step (1) on a tissue section.
  • fixing treatment solution examples include cross-linking agents such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol, and cell membrane permeants.
  • cross-linking agents such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol, and cell membrane permeants.
  • the fixing process can be performed by a conventionally known method.
  • the fixation treatment can be performed by immersing the stained tissue section obtained by the histochemical staining step in the fixation treatment solution as described above.
  • it can be performed by immersing a stained tissue section obtained by the histochemical staining step in a dilute paraformaldehyde aqueous solution for about several minutes to several hours.
  • Observation step (5-1) Bright field observation step In the bright field observation step, the coloring agent dye deposited on the tissue section by applying illumination light to the tissue section stained in steps (1) to (4). And obtaining distribution information (number of coloring points, etc.) of the biomolecule A to be stained in the cell or tissue.
  • Bright field observation may be performed by a general method. For example, in bright-field observation when histological staining is performed as a biomolecule for staining VEGFR-2 protein in esophageal cancer cells, a 4 ⁇ objective lens of an optical microscope is used under irradiation with appropriate illumination light. Use to observe the VEGFR-2 protein positive staining image, the intensity of positive staining, and the positive cell rate of cancer cells in the specimen tissue. Next, 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.
  • the distribution information of the biomolecule A and the distribution information of the biomolecule B may be obtained from a microscope barrel so that rapid observation can be performed, or an image taken by a camera installed in the microscope is separately obtained. You may make it acquire by displaying on a display means (monitor etc.) and observing it.
  • the fluorescence observation step is a biomolecule based on the fluorescence emitted by the fluorescent nanoparticle labeled body in the tissue section stained in the above-described step by irradiating the fluorescent body with excitation light. This is a process of acquiring B distribution information (such as the number of bright spots).
  • the excitation light irradiates a suitable excitation light to the phosphor to excite the phosphor, and stains a biomolecule to be stained with fluorescence.
  • the excitation light irradiation means is not particularly limited.
  • a stained tissue section may be irradiated with excitation light having an appropriate wavelength and output from a laser light source included in a fluorescence microscope using a filter that selectively transmits a predetermined wavelength as necessary.
  • the excitation light is not particularly limited as long as the fluorescence emitted by the label can be identified in relation to the autofluorescence of the tissue section.
  • the excitation light is a substrate (for example, diamino) that allows visual observation of fluorescence and allows bright field observation. From the viewpoint of avoiding absorption by benzidine) and preventing the autofluorescence intensity from the tissue section from becoming too high, those having a wavelength of 450 nm to 700 nm are preferable.
  • the fluorescent substance constituting the fluorescent substance used as the fluorescent label a fluorescent substance that emits fluorescence having a peak in the range of 480 nm or more, preferably in the range of 580 to 690 nm by the excitation light is used (therefore, the emission wavelength in this region). ) To measure fluorescence.
  • the distribution information of the biomolecules A and B may be acquired from a (fluorescence) microscope barrel so that rapid observation can be performed, or a camera installed in the (fluorescence) microscope takes a picture.
  • the obtained image may be separately displayed on a display means (a monitor or the like) and acquired by observing it.
  • the biomolecule distribution information can be obtained from the image taken by the camera even if the biomolecule distribution information cannot be obtained sufficiently by visual observation from the microscope barrel. Sometimes it is possible to do.
  • Obtaining the distribution information of the biomolecules A and B includes, for example, measuring the number or density of biomolecules to be stained per cell based on the number of fluorescent luminescent spots or emission luminance.
  • an excitation light source and an optical filter for fluorescence detection corresponding to the absorption maximum wavelength and the fluorescence wavelength may be selected.
  • the measurement of the number of bright spots or emission luminance it is preferable to use commercially available image analysis software (for example, all bright spot automatic measurement software “G-Count” (manufactured by Zeonstrom)), but the measuring means is particularly limited. Is not to be done.
  • multiple immunostaining an example of multiple immunostaining for staining two types of biomolecules has been shown, but more biomolecules C can be obtained by changing the emission wavelength range of the color former or the phosphor. Multiple immunostaining may be used to detect.
  • the fluorescent nanoparticle label according to the present invention comprises a phosphor-integrated nanoparticle having a phosphor integrated state, and a hydrophilic polymer having a number average molecular weight of 3000 or more bonded to the surface of the phosphor-integrated nanoparticle.
  • a particle comprising a chain and a biomolecule recognition molecule bonded to the end of the hydrophilic polymer chain.
  • the fluorescent nanoparticle label according to the present invention is used for multiple immunostaining in which fluorescent immunostaining and chemical coloring are used for the purpose of detecting the same or different antigens
  • biomolecule recognition of the fluorescent nanoparticle label is first performed. Molecules bind to the antigen and biomolecules (antibodies, etc.) bound thereto, and the antigen is stained with fluorescence.
  • an antibody to which an enzyme for chemical color development is added to the same or different antigen as this antigen is bound, and a chemical color reaction occurs by adding a color former to stain the antigen.
  • the number average molecular weight of the hydrophilic polymer chain of the fluorescent nanoparticle labeling body is 3000 or more, it is physically separated from the insoluble precipitates (such as those obtained by polymerizing the color former) generated by the chemical color development.
  • the phosphor is arranged at the position, and the light emission from the phosphor is not easily inhibited by the insoluble precipitate (refer to FIGS. 1A and 1B for comparison). As a result, the same number of bright spots as when fluorescent immunostaining is performed alone can be secured, and the antigen detection accuracy in multiple immunostaining is improved.
  • hydrophilic polymer chain (A) and / or the hydrophilic polymer chain (B) is polyethylene glycol, unintended activation of biomolecules can be prevented.
  • biomolecule recognition molecule is avidin that can bind to biotin linked to a primary antibody that can specifically bind to the antigen to be stained or a secondary antibody that can bind to the primary antibody
  • biotin And avidin are used for the binding of fluorescent nanoparticle label and biomolecules, so the time from adding the fluorescent nanoparticle label to the reaction system to observing fluorescence is short. Thus, it is possible to suppress the influence of the decrease in the fluorescence intensity at the bright spot due to the fading of the phosphor.
  • Example 1A Synthesis of Labeling Agent A (Streptavidin-Linked Texas Red Dye Encapsulated Melamine Resin Nanoparticles with Polyethylene Glycol with Number Average Molecular Weight 3400) 2.5 mg of sulforhodamine 101 (“Sulforhodamine 101”, Sigma Aldrich, Texas Red dye) After dissolving in 5 mL, the solution was stirred for 20 minutes while maintaining the temperature of the solution at 70 ° C. with a hot stirrer. To the stirred solution, 1.5 g of melamine resin “Nicalak MX-035” (manufactured by Nippon Carbide Industries Co., Ltd.) was added, and the mixture was further heated and stirred under the same conditions for 5 minutes.
  • melamine resin “Nicalak MX-035” manufactured by Nippon Carbide Industries Co., Ltd.
  • the solution After adding 100 ⁇ L of formic acid to the stirred solution and stirring the solution for 20 minutes while maintaining the temperature of the solution at 60 ° C., the solution was left to cool to room temperature.
  • the cooled solution is dispensed into a plurality of centrifuge tubes, centrifuged at 12,000 rpm for 20 minutes, and Texas Red dye-encapsulated melamine resin nanoparticles (hereinafter abbreviated as Particle A) contained in the solution as a mixture. .) was precipitated and the supernatant was removed. Thereafter, the precipitated particles A were washed with ethanol and water.
  • the concentration of the particle A whose surface was aminated was adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid), so that the final concentration of this solution was 10 mM.
  • PBS phosphate buffered saline
  • EDTA ethylenediaminetetraacetic acid
  • Maleimide-PEG-carboxylate-NHS (SUNBRIGHT MA-034TS average molecular weight 3400 manufactured by NOF Corporation) was mixed and reacted for 1 hour at room temperature with stirring.
  • the reaction solution was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again under the same conditions. By performing washing by the same procedure three times, particles A whose surface was modified with a PEG chain having a maleimide group at the terminal were obtained.
  • streptavidin having a sulfhydryl group was produced as follows. First, 40 ⁇ L of streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL, N-succinimidyl-S-acetylthioacetate (N-succinimidyl S-acetylthioacetate, SATA, Pirce) adjusted to 64 mg / mL 70 ⁇ L) was reacted at room temperature for 1 hour. That is, a thiol group (—NH—CO—CH 2 —S—CO—CH 3 ) protected against the amino group of streptavidin was introduced.
  • a free thiol group (—SH) was generated from the protected thiol group by a known hydroxylamine treatment, and a treatment for adding a thiol group (—SH) to streptavidin was performed.
  • the streptavidin solution was desalted with a gel filtration column (Zaba Spin Desaling Columns: Funakoshi) to obtain streptavidin capable of binding to the melamine particles.
  • PEG-modified particles A and streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react for 1 hour. 10 mM mercaptoethanol was added to stop the reaction. After concentrating the obtained solution with a centrifugal filter, unreacted streptavidin and the like were removed using a gel filtration column for purification to obtain labeling agent A.
  • Example 1B Multiple immunostaining
  • vascular growth factor vascular endothelial growth factor receptor-2 VEGFR-2
  • esophagus vascular growth factor vascular endothelial growth factor receptor-2 (VEGFR-2) and esophagus are as follows: Multiple immunostaining was performed to detect the surface antigen CD34 of cancer cells in cancer tissues.
  • Example 1A Synthesis of streptavidin-conjugated lexathread dye-encapsulated melamine resin nanoparticles having molecular weight of 528 polyethylene glycol
  • Example 1B instead of maleimide-PEG-carboxylate-NHS (SUNBRIGHT MA-034TS average molecular weight 3400 manufactured by NOF Corporation) , except that MAL-dPEG-NHS Ester (QB10284a polyethylene glycol part molecular weight 528, manufactured by Quanta) was used, the labeling agent B was prepared, and multiple immunostaining was performed as in Example 1A. Observations for confirming the number of bright spots were performed. Table 1 shows the results of Example 1B and Comparative Example 1B.
  • Labeling agent C Synthesis of streptavidin-linked lexathread dye-encapsulated melamine resin nanoparticles with polyethylene glycol having molecular weights of 5000 and 352
  • Maleimide-PEG-carboxylate-NHS manufactured by NOF Corporation, SUNBRIGHT MA-034TS average molecular weight 3400
  • -PEG-carboxylate-NHS SUNBRIGHT MA-050TS average molecular weight 5000 manufactured by NOF Corporation
  • MAL-dPEG-NHS Ester Quanta QB10274a polyethylene glycol part molecular weight 352 have final concentrations of 1 mM and 10 mM, respectively.
  • the labeling agent C was synthesized in the same manner as the synthesis of the labeling agent A, except that a mixture of 1:10 (molar ratio) was used. Further, using this labeling agent C, multiple immunostaining and observation for confirming the number of bright spots were performed in the same manner as in Example 1A.
  • Labeling agent D Synthesis of streptavidin-linked lexathread dye-encapsulated melamine resin nanoparticles with polyethylene glycol having molecular weights of 20000 and 2000 Maleimide-PEG-carboxylate-NHS (NOF Corporation, SUNBRIGHT MA-034TS average molecular weight 3400) instead of maleimide -PEG-carboxylate-NHS (NOF SUNBRIGHT MA-200TS average molecular weight 20000) and maleimide-PEG-carboxylate-NHS (NOF SUNBRIGHT MA-020TS average molecular weight 2000) each with a final concentration of 1 mM.
  • Maleimide-PEG-carboxylate-NHS NOF Corporation, SUNBRIGHT MA-034TS average molecular weight 3400
  • maleimide-PEG-carboxylate-NHS NOF SUNBRIGHT MA-200TS average molecular weight 20000
  • maleimide-PEG-carboxylate-NHS NOF SU
  • the labeling agent D was synthesized in the same manner as the synthesis of the labeling agent A, except that the mixture was mixed at 1:10 (molar ratio) so as to be 10 mM. In addition, this labeling agent D was used for multiple immunostaining and observation for confirming the number of bright spots as in Example 1A.
  • Labeling agent E Synthesis of streptavidin-conjugated lexathread dye-encapsulated melamine resin nanoparticles with polyethylene glycol having molecular weights of 40000 and 528 Maleimide-PEG-carboxylate-NHS (manufactured by NOF Corporation, SUNBRIGHT MA-034TS average molecular weight 3400) -PEG-carboxylate-NHS (SUNBRIGHT MA-400TS average molecular weight 40000 manufactured by NOF Corporation) and MAL-dPEG-NHS Ester (Quanta QB10284a polyethylene glycol part molecular weight 528) so that the final concentrations are 1 mM and 10 mM, respectively.
  • the labeling agent E was synthesized in the same manner as the synthesis of the labeling agent A except that a mixture of 1:10 (molar ratio) was used.
  • a mixture of 1:10 (molar ratio) was used.
  • multiple immunostaining and observation for confirming the number of bright spots were performed as in Example 1A.
  • the fluorescent nanoparticle label according to the present invention As described above, the fluorescent nanoparticle label according to the present invention, the multiple immunostaining containing the fluorescent nanoparticle label, and the multiple immunostaining method using the multiple immunostaining have been described based on the embodiments and examples. However, the present invention is not limited to these, and design changes are allowed without departing from the gist of the present invention.

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Abstract

Le but de la présente invention est de supprimer une dégradation du niveau d'émission lumineuse dans un procédé d'immunocoloration multiple qui utilise à la fois l'immunocoloration utilisant la chimioluminescence ainsi que l'immunocoloration fluorescente utilisant l'émission lumineuse d'un colorant fluorescent ou du phosphore. A cet effet, l'invention concerne un marqueur à nanoparticules fluorescentes pour l'immunocoloration multiple comprenant une nanoparticule de phosphore accumulé contenant du phosphore dans un état accumulé, une chaîne polymère hydrophile liée à la surface de la nanoparticule de phosphore accumulé et ayant un poids moléculaire moyen en nombre de 3000 ou plus, et une molécule de reconnaissance biomoléculaire liée à l'extrémité de la chaîne polymère hydrophile.
PCT/JP2014/074418 2013-09-26 2014-09-16 Marqueur à nanoparticules fluorescentes, kit d'immunocoloration multiple, et procédé d'immunocoloration multiple WO2015045961A1 (fr)

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WO2022059303A1 (fr) * 2020-09-17 2022-03-24 コニカミノルタ株式会社 Nanoparticules fluorescentes et procédé de préparation utilisant lesdites nanoparticules fluorescentes

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