JP6424825B2 - Fluorescent nanoparticle label, multiplex immunostain kit and multiplex immunostaining method - Google Patents

Description

  The present invention relates to a fluorescent nanoparticle label, a multiplex immunostaining kit comprising the fluorescent nanoparticle label, and a multiplex immunostaining method using the multiplex immunostain kit.

  A technique for binding an antibody bound with a dye or a chromogenic enzyme to an antigen in a tissue sample by an antigen-antibody reaction and causing color development to detect the antigen is widely known as a technique by immunohistochemistry (IMMUNOHISTOCHEMISTRY; IHC) There is. Such immunohistochemical techniques are sometimes referred to as "immunostaining" because they involve color processing that visualizes antigen-antibody reactions that are otherwise invisible. Immunostaining is widely used in the medical and life sciences fields to detect the whereabouts of biomolecules in tissue samples.

In DAB staining, which is an example of immunostaining, after peroxidase-linked antibody and antigen are bound by antigen-antibody reaction, hydrogen peroxide and DAB (Diaminobenzidine; diaminobenzidine) are added as a coloring treatment. As a result, hydrogen peroxide is decomposed by peroxidase, and a chemical reaction (chemical coloration) occurs in which DAB molecules are polymerized to develop color by O ₂ ²⁻ generated by the decomposition, and the antigen part is stained brown. By measuring the amount of color development, it is also possible to determine the approximate amount of the expressed antigen. In addition, it is also possible to identify the type and location of cells by immunostaining, and immunostaining that targets markers (such as CD34 antigen) specifically expressed in certain cells has been established as a general technique.

  Apart from the above-described immunostaining utilizing chemical coloring, nanoparticles (semiconductor fine particles etc.) that emit fluorescence by being irradiated with excitation light and an antibody are linked, and the antibody is allowed to bind to an antigen by an antigen-antibody reaction, Methods for fluorescently staining antigens (fluorescent immunostaining) are known (see, for example, Patent Documents 1 to 3).

  Patent Document 1 discloses semiconductor microparticles surface-modified with polyethylene glycol (PEG) having a molecular weight of 300 to 20,000. Also disclosed is the provision of a biomolecule at the tip of the PEG of the semiconductor fine particle.

  In Patent Documents 2 and 3, the surface of the semiconductor fine particle is modified with PEG (molecular weight 300 to 3000, etc.), and a functional group (amino group etc.) is provided at the tip of the PEG farthest from the surface. There is disclosed a biomolecule fluorescent labeling agent in which another biomolecule (such as DNA) capable of specifically binding to a biomolecule to be stained is bound to a group, and an immunofluorescent staining method using the same.

  Furthermore, in addition to the single staining described above, a multiplex immunostaining method is also known which simultaneously detects two or more different antigens (see, for example, Patent Document 4).

  In Patent Document 4, two or more types of antigens are detected on the same tissue section, and locations and amounts of expression of the respective antigens are evaluated. Furthermore, Patent Document 4 discloses that, as a colored substance or luminescent substance for the detection described above, a combination of a substance that emits a color or emits light upon reaction with a substrate of a labeling enzyme and a fluorescent dye is an optical microscope and It is described that it is preferable from the viewpoint that a fluorescence microscope or a laser confocal microscope can be used.

  Furthermore, the detection accuracy of biomolecules can be enhanced by performing multiple immunostaining using the same biomolecule with immunostaining utilizing chemical coloring and fluorescence immunostaining utilizing light emission by fluorescent dye in combination. It is disclosed.

JP, 2004-300253, A International Publication WO 2010/004777 International Publication WO 2007/086501 JP, 2005-17133, A

  The inventors of the present invention have found that, in order to stain a predetermined biomolecule in chemical coloration, the insoluble precipitate generated by the reaction of an enzyme and a predetermined substrate absorbs the fluorescence emitted from the excitation light and the phosphor that irradiates the phosphor. It was speculated that this was the cause of the problems inherent in multiple immunostaining as described above. Then, a fluorescent substance-accumulating nanoparticle is used in a fluorescent immunostaining state in which the fluorescent substance is accumulated, and a biomolecule recognition molecule is bound to the fluorescent substance-accumulating nanoparticle through a hydrophilic polymer chain of a specific length It has been found that fluorescence immunostaining of a given biomolecule can avoid the problems as described above, and that fluorescence observation of a given biomolecule can be performed at a level equivalent to that of single staining in which only fluorescence immunostaining is performed. The present invention has been completed.

  The present inventors compared the single-color immunostaining with the single-color immunostaining alone in the multiple immunostaining method in which the immunostaining utilizing chemical coloration and the fluorescence immunostaining utilizing light emission of the fluorescent dye are used in combination. We noticed a problem that the light emission intensity of or the number of bright spots observed decreased.

  That is, the present invention has been made in view of the above problems, and in the multiple immunostaining method using the immunostaining utilizing chemical coloring and the fluorescent immunostaining utilizing light emission of a fluorescent substance such as a fluorescent dye in combination, It is an object of the present invention to suppress the decrease in light emission intensity or the number of bright spots as described above.

  In order to realize the above-mentioned object, the fluorescent nanoparticle label reflecting one aspect of the present invention is bound to the surface of the fluorescent substance-concentrating nanoparticle having fluorescent substance in an accumulated state and the surface of the fluorescent substance-concentrating nanoparticle It is a fluorescent nanoparticle labeled body for multiplex immunostaining comprising a hydrophilic polymer chain (A) having a number average molecular weight of 3,000 or more and a biomolecule recognition molecule bonded to the end of the hydrophilic polymer chain.

  In order to achieve the above-mentioned object, a multiplex immunostain kit reflecting one aspect of the present invention comprises a fluorescent immunostain having the above fluorescent nanoparticle label and a chemical immunostain capable of bright field observation. And a multiple immunostaining kit.

  In order to realize the above-mentioned object, the multiplex immunostaining method reflecting one aspect of the present invention is a fluorescent immunostaining using a fluorescent immunostaining agent having the above-mentioned fluorescent nanoparticle labeled body, and chemical immunity which can be observed in bright field This is a multiple immunostaining method in which a chemical immunostaining using a staining agent is performed on the same section.

  According to the present invention, in the multiple immunostaining using fluorescence immunostaining and chemical immunostaining using chemical coloring in combination according to the present invention, the decrease of the light emission intensity or the number of bright points due to the insoluble precipitate generated by the chemical coloring is It can be suppressed. As a result, it is possible to secure the brightness score obtained when the fluorescent immunostaining is performed alone also in the multiplex immunostaining.

FIG. 1 (A) is a view showing a conceptual diagram of a conventional multiplex immunostaining method. FIG. 1 (B) shows an example of the multiple immunostaining method according to the present invention. FIG. 2 is a view showing an example of the labeled fluorescent nanoparticle according to the present invention. FIG. 3 is a view showing another example of the labeled fluorescent nanoparticle according to the present invention.

  Hereinafter, the fluorescent nanoparticle labeled body, the multiplex immunostaining agent kit and the multiplex immunostaining method according to the present invention will be described. The fluorescent nanoparticle labeled body according to the present invention is used for multiple immunostaining, and has a fluorescent substance-accumulating nanoparticle having fluorescent substances such as fluorescent dyes accumulated and a number average bound to the surface of the fluorescent substance-accumulating nanoparticle It has a hydrophilic polymer chain having a molecular weight of 3,000 or more, and a biomolecule recognition molecule etc. 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 agent together with a staining agent of a chemical color.

[Phosphor-integrated nanoparticles]
The phosphor-accumulated nanoparticles are obtained by accumulating phosphors and may be called fluorescent nanoparticle labels. By using such phosphor-accumulated 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, as compared to the phosphor itself.

<Phosphor>
In the present specification, “phosphor” refers to a substance in general that emits light in the process of being excited by receiving external X-ray, ultraviolet light or visible light, and reaching from the excited state to the ground state. Therefore, the "phosphor" referred to in the present invention is a substance that emits fluorescence in a narrow sense, which does not ask about the transition mode when returning from the excited state to the ground state, and emits light associated with deactivation from the singlet. It may be a substance that emits phosphorescence that is emission associated with deactivation from triplets. Further, the "phosphor" in the present invention is not limited by the light emission life after blocking the excitation light. Therefore, it may be a substance known as a luminous substance such as zinc sulfide or strontium aluminate. Such phosphors can be roughly classified into organic phosphors (fluorescent dyes) and inorganic phosphors.

[Organic fluorescent substance]
Examples of organic fluorescent substances that can be used as fluorescent substances include fluorescein-based dye molecules, rhodamine-based dye molecules, Alexa Fluor (registered trademark, manufactured by Invitrogen Corporation) -based pigment molecules, BODIPY (registered trademark, manufactured by Invitrogen Corporation) -based pigments 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 Substances known as organic fluorescent dyes such as dye molecules and oxazine dye molecules can be mentioned.

  Specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ', 4, 4', 5 ', 7, 7'-hexachlorofluorescein, 6 -Carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy Rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Lexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor, 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 Je And methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7 and the like. A single one or a mixture of multiple types may be used.

[Inorganic phosphor]
Examples of inorganic phosphors that can be used as phosphors include quantum dots containing II-VI compounds, III-V compounds, or IV group elements as components (respectively "II-VI quantum dots", " Examples of the group III-V quantum dot and the group IV quantum dot may also be mentioned. A single one or a mixture of multiple types may be used.

  Specific examples thereof include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge, but are not limited thereto.

It is also possible to use a quantum dot having the above-mentioned quantum dot as a core and a shell provided thereon. Hereinafter, as a notation of the quantum dot which has a shell, when a core is CdSe and a shell is ZnS, it describes as CdSe / ZnS. For example, CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO ₂ , Si / ZnS, Ge / GeO ₂ , Ge / ZnS, etc. can be used, but the invention is not limited thereto.

  The quantum dots may be those which have been surface-treated with an organic polymer or the like, as required. For example, CdSe / ZnS (manufactured by Invitrogen) having a surface carboxy group, CdSe / ZnS (manufactured by Invitrogen) having a surface amino group, and the like can be mentioned.

<Method of manufacturing phosphor-integrated nanoparticles>
The method for producing the phosphor-accumulated nanoparticles in which the phosphors are accumulated is not particularly limited, and can be produced by a known method. Generally, a production method can be used in which a phosphor is assembled using a resin or silica as a matrix (the phosphor is immobilized on the inside or the surface of the matrix).

<In the case of organic fluorescent substance>
As a method for producing phosphor-accumulated nanoparticles using an organic phosphor, mention is made of a method of forming resin particles having a diameter on the order of nanometers, in which a fluorescent dye that is a phosphor is fixed to the inside or surface of a matrix made of resin. Can. The method for preparing the phosphor-accumulated nanoparticles is not particularly limited, but, for example, (co) monomers for synthesizing a resin (thermoplastic resin or thermosetting resin) as a matrix of the phosphor-accumulated nanoparticles While (co) polymerizing, a fluorescent substance is added and the method of making the said fluorescent substance take in inside or the surface of the said (co) polymer can be used.

  As said thermoplastic resin, a polystyrene, a polyacrylonitrile, a polyfuran, or resin similar to this can be used suitably, for example. As the above-mentioned thermosetting resin, for example, polyxylene, polylactic acid, glycidyl methacrylate, polymelamine, polyurea, polybenzoguanamine, polyamide, phenol resin, polysaccharide or resin similar thereto can be suitably used. A thermosetting resin, in particular a melamine resin, is preferable in that the elution of the dye incorporated in the dye resin can be suppressed even by a treatment such as dehydration, clear, and encapsulation using an organic solvent such as xylene.

  For example, polystyrene nanoparticles incorporating an organic fluorescent dye (phosphor) can be obtained by copolymerization using an organic dye having a polymerizable functional group as described in US Pat. No. 4,326,008 (1982), US Pat. It can be prepared using the method of impregnating the fluorescent nanoparticle with polystyrene nanoparticles described in 1.).

  On the other hand, it is also possible to produce silica nanoparticles in which the organic fluorescent substance is immobilized on the inside or surface of a matrix made of silica. As such a production method, the method of synthesizing FITC-encapsulated silica particles described in Langmuir 8: 2921 (1992) can be referred to. Various fluorescent dye-containing silica nanoparticles can be synthesized by using a desired fluorescent dye instead of FITC.

<In the case of inorganic phosphor>
As a manufacturing method of the fluorescent substance accumulation | occurence | production nanoparticle using inorganic fluorescent substance, the method of forming the silica nanoparticle which fixed the quantum dot which is fluorescent substance on the inside or surface of the base | substrate consisting of silica is mentioned. This production method can be referred to the synthesis of CdTe-encapsulated silica nanoparticles described in New Journal of Chemistry 33, page 561 (2009).

  Moreover, as a manufacturing method of the fluorescent substance accumulation | occurence | production nanoparticle different from the above, a silica bead is processed with a silane coupling agent, the terminal is aminated, the semiconductor fine particle as a fluorescent substance which has a carboxyl group terminal is amido on the surface of a silica bead. There is also a method of accumulating by binding by binding to form a phosphor integrated nanoparticle.

  Further, as a method for producing a phosphor-accumulating nanoparticle, a reverse micelle method and a mixture of an organic alkoxysilane and an alkoxide having an organic functional group having good adsorptivity to a semiconductor nanoparticle at the end of the molecule as a precursor of glass By combining with the sol-gel method described above, an example in which glass-like particles in which semiconductor nanoparticles are dispersed and fixed inside is formed, and phosphor-integrated nanoparticles can be mentioned.

  As still another method of producing phosphor-accumulated nanoparticles, semiconductor nanoparticles of amino group terminal and carboxyl group terminal in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) There is an example in which semiconductor nanoparticles are mixed, and semiconductor nanoparticles are integrated via amide bond bonding between semiconductor nanoparticles to integrate the semiconductor nanoparticles, thereby producing phosphor integrated nanoparticles.

  Furthermore, it is also possible to produce an aggregate in which the inorganic fluorescent substance is immobilized on the inside or on the surface of a matrix made of a resin. For example, polymer nanoparticles containing quantum dots can be prepared using the method of impregnating polystyrene nanoparticles with quantum dots as described in Nature Biotechnology 19: 631 (2001).

[Hydrophilic polymer chain]
The hydrophilic polymer chain used for the surface modification of the phosphor-accumulated nanoparticles includes a hydrophilic polymer (having a predetermined number average molecular weight for linking the phosphor-accumulated nanoparticles and a biomolecule recognition molecule described later) A) and, if necessary, 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 (Figure See the examples of PEG chain 1 and PEG chain 2 of 3).

  Examples of 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, polyhydroxy propyl oxazoline, polyhydroxy propyl methacrylamide, poly methacrylamide, poly dimethyl acrylamide, poly hydroxy propyl methacrylate, poly hydroxyethyl acrylate, hydroxy methyl cellulose, hydroxyethyl cellulose, Polyaspartamide, synthetic polyamino acids and the like can be mentioned. As the hydrophilic polymer chains (A) and (B), one or two or more hydrophilic polymers selected from the group formed by the hydrophilic polymers exemplified above can be used. Among the above-described exemplified hydrophilic polymers, polyethylene glycol (PEG) is preferable from the viewpoint of easy setting of the chain length by the number of oxyethylene units and the like.

  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 carried out by performing immunostaining utilizing chemical coloration and fluorescent immunostaining, when performing multiplex immunostaining. It is possible to adjust so that the number of bright spots caused by the phosphor can be confirmed to the same extent. The confirmation of the number of bright spots can be performed with a fluorescence microscope or the like as described later. On the other hand, the number average molecular weight of the hydrophilic polymer chain (B) is usually less than 3,000, preferably 300 or more. The number average molecular weight of the hydrophilic polymer chain (B) can be adjusted while taking into consideration the effect of preventing aggregation of phosphor-accumulated nanoparticles.

As for the hydrophilic polymer chains (A) and (B), the polymer structural unit contained in the chemical structure of the hydrophilic polymer from the viewpoint of adjusting the length of the molecule to obtain the effect of the present invention Preferably it is adjusted by the number. For example, when the hydrophilic polymer is PEG, it is preferable to be adjusted by the number (n) of oxyethylene units (-CH ₂ -CH ₂ -O).

(Measurement of number average molecular weight)
The number average molecular weight of the phosphor-aggregated nanoparticles is measured by gel permeation chromatography (GPC) using tetrahydrofuran (THF) as a column solvent.
Specifically, 1 mL of THF is added to 1 mg of a measurement sample, and stirring is performed using a magnetic stirrer at room temperature to dissolve sufficiently. Then, it is injected into GPC after being treated with a membrane filter with a pore size of 0.45 to 0.50 μm. 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 μL of a sample at a concentration of 1 mg / mL. As the column, it is preferable to use a commercially available polystyrene gel column in combination. For example, combinations of Shodex GPC KF-801, 802, 803, 804, 805, 806, 807 manufactured by Showa Denko KK, TSKgel G1000H, G2000H, G3000H, G4000H, G5000H, G6000H, G7000H, TSK guard column manufactured by Tosoh Corporation. A combination etc. can be mentioned.
As a detector, a refractive index detector (RI detector) is preferably used. In the molecular weight measurement of a sample, the molecular weight distribution of the sample is calculated using a calibration curve or the like prepared using monodispersed polystyrene standard particles. It is preferable to use about 10 points as polystyrene or the like for preparing a calibration curve.

The bonding between the hydrophilic polymer and the phosphor-aggregated nanoparticle is not particularly limited, and bonding by a suitable bonding mode such as covalent bonding, ionic bonding, hydrogen bonding, coordination bonding, physical adsorption, or chemisorption, or bonding From the viewpoint of strength, covalent bonding such as amide bond, ester bond, imide bond, bond utilizing thiol addition to maleimide group, etc., or biotin-avidin bond or biotin-streptavidin bond is preferable. In particular, from the viewpoint of the strength of binding and the specificity of binding, biotin-avidin binding or biotin-streptavidin binding is more preferable.
Examples of coupling methods include biotin-avidin method, coupling reaction method of thiol group-maleimide group, method using a chemical linker, crosslinking agent (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), etc.) And a crosslinking reaction method, an ion binding method and the like.

As a specific example of the chemical linker, when an amino group (NH ₂ group) is present on the surface of a phosphor-accumulated nanoparticle, for example, it is bound to biotin (biomolecule recognition molecule) as shown in [Chemical formula 1] below A PEGylation reagent can be suitably used.

  In this case, the N-hydroxysuccinimidyl ester group of the PEGylation reagent (the functional group at the left end of [Chemical Formula 1]) and the amino group on the surface of the phosphor-accumulated nanoparticles react to produce phosphor-assembled nanoparticles. The PEG chain and biotin will be added (see FIG. 2).

  Also, unlike the above example, when a sulfhydryl group (SH group) is present on the surface of the phosphor-accumulated nanoparticle, it is bound to biotin (biomolecule recognition molecule) as shown in the following [Chemical formula 2], for example A PEGylation reagent can be suitably used. In this case, the maleimide group of the PEGylation reagent (the functional group at the left end of [Chem. 2]) and the sulfhydryl group (SH group) of the surface of the fluorescent substance-accumulating nanoparticles react to form a fluorescent substance-containing nanoparticle And biotin will be added (not shown).

In any of the PEGylation reagents of [Formula 1] and [Formula 2], the number average molecular weight of the PEG chain moiety “— (CH ₂ CH ₂ O) n-” derived from PEG (hydrophilic polymer) In order to make it 3000 or more, it is preferable to set to the number (n) of oxyethylene units.
In the case of using the above-mentioned hydrophilic polymer instead of polyethylene glycol (PEG), the chain length of the portion derived from the hydrophilic polymer is designed to have a number average molecular weight equal to or higher than that of the above PEG chain Can be manufactured and used as a material for fluorescent nanoparticle labels. Such a PEGylation reagent having a predetermined molecular weight and the like can be obtained, for example, by asking a synthetic maker (eg, CreativePEGWorks etc.) for synthesis.

  Before bonding the hydrophilic polymer chain to the phosphor-accumulating nanoparticle, a functional group necessary for bonding may be introduced in advance to the phosphor-accumulating nanoparticle or the hydrophilic polymer chain. For example, as an example of introducing a necessary functional group to a phosphor-accumulating nanoparticle in advance, when producing a phosphor-accumulating nanoparticle using a thermoplastic resin, glycidyl methacrylate is used as a monomer together with styrene and copolymerized The epoxy group is introduced onto the surface of the phosphor-accumulated nanoparticles by the above, and the epoxy group is converted to an amino group by amino group introduction treatment using an amino group introduction reagent (APS etc.) to react the above-mentioned PEGylation reagent etc. An example is given. The number of hydrophilic polymers to be bound per phosphor-accumulated nanoparticle can be adjusted by the number of functional groups as described above required for binding, or the reaction conditions with the hydrophilic polymer.

  On the other hand, in the case of producing a thermosetting dye resin particle, a dye resin particle of a melamine resin having an amino group on the surface by copolymerizing using a melamine resin raw material (for example, MX035 manufactured by Sanwa Chemical Co., Ltd.) as a monomer. Can be made to react with the above-mentioned PEGylation reagent and the like.

<Biomolecular recognition molecule>
The biomolecular recognition molecule used in the present invention means a molecule that specifically binds to a biomolecule such as an antigen. As a biomolecule recognition molecule, a primary antibody that recognizes a specific biomolecule to be stained, a secondary (-n order) antibody that recognizes this antibody, a specific one that is bound to the secondary (-n order) antibody It is a molecule that recognizes or is recognized by a biomolecule, and examples of the biomolecule recognition molecule include an antibody, biotin or avidin (including an analog such as streptavidin).

  In the present invention, the term "antibody" is used to mean any antibody fragment or derivative and includes Fab, Fab'2, CDR, humanized antibody, multifunctional antibody, single chain antibody (ScFv) and the like. Includes various antibodies.

  In the present invention, the term "antigen" refers to a biological substance, in particular, a molecule or molecular fragment consisting of a protein (polypeptide, oligopeptide, etc.), but it is a biological substance that can produce antibodies and can be immunostained. For example, molecules or molecular fragments consisting of other than proteins, such as nucleic acid (DNA, which may be single-stranded or double-stranded, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., or nucleoside, Nucleotides and their modifying molecules may also be able to be antigens.

  As a biomolecule recognition molecule, an antibody which is a component of an antibody drug can be used. Examples of antibody drugs include antibody drugs generally used for the treatment of autoimmune diseases such as rheumatoid arthritis, malignancies such as cancer, and viral infections.

  The bonding between the biomolecular recognition molecule and the end of the hydrophilic polymer chain is similar to the bonding between the phosphor-accumulated nanoparticle and the hydrophilic polymer chain, such as covalent bonding, ionic bonding, hydrogen bonding, coordination bonding, physical adsorption, From the viewpoint of bond strength by means of appropriate bonding mode such as chemisorption and bond strength, covalent bonds such as amide bond, ester bond, imide bond, and bonds utilizing thiol addition to a maleimide group are preferable.

  Specific binding methods of binding between the biomolecular 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 Cross-linking reaction method using an agent (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and the like), an ion binding method and the like can be mentioned.

  The biomolecules to be stained by each of the chemical immunostaining and the fluorescent immunostaining are not particularly limited, and may be the same or different. You want to detect multiple staining targets separately with different staining methods in cases where there are two or more staining targets (for example, the target of chemical immunostaining is CD34 antigen, the target of fluorescent immunostaining is VEGFR-2, etc.) In some cases, the binding mode of the biomolecule recognition molecule and the staining target in the fluorescence immunostaining is different from the binding mode of the biomolecule recognition molecule and the staining target in the chemical immunostaining to avoid unintended binding and competition of the molecules. Is appropriate. For example, in the case of chemical immunostaining, while the binding mode is such that a complex of antigen-primary antibody to be stained (mouse antibody) -enzyme-labeled secondary antibody (anti-mouse IgG antibody) is formed, The binding mode is such that a complex to be formed of a target antibody-primary antibody (rabbit antibody) -biotin labeled secondary antibody (anti-rabbit IgG antibody) -avidin as a biomolecular recognition molecule is formed. It can be done.

<Multiple immunostaining method>
The multiplex immunostaining method according to the present invention is a multiplex immunostaining method in which both staining utilizing chemical coloring and fluorescent immunostaining using a fluorescent nanoparticle label are performed. The target to be stained by the multiple staining method according to the present invention is a biomolecule targeted by general immunostaining, and a tumor marker, a signal transducer, a hormone, a cancer growth regulator, a metastasis regulator, a growth regulator acceptance The body, metastasis regulator receptor, etc. can be exemplified.

  Hereinafter, one embodiment of multiplex immunostaining of each of different biomolecules A and B by immunostaining using fluorescent coloring and fluorescent immunostaining will be described in the order of steps (1) to (5).

(1) Deparaffinization step The 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 more and 30 minutes or less. If necessary, xylene may be replaced during immersion.

  Then, 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 more and 30 minutes or less. In addition, if necessary, ethanol may be replaced during immersion.

  The pathological section is immersed in a water container to remove ethanol. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or more and 30 minutes or less. In addition, water may be replaced during immersion if necessary.

(2) Activation Process Step When immunohistochemical staining is performed as histochemical staining, it is preferable to perform an activation process of a target biomolecule, following known methods. Although the activation conditions are not particularly limited, as an activation liquid, 0.01 M citric acid buffer (pH 6.0), 1 mM ethylenediaminetetraacetic acid (EDTA) solution (pH 8.0), 5% urea, 0.1 M Tris Hydrochloric acid buffer etc. can be used. As a heating apparatus, 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 temperature of heat treatment of activation treatment can be 50 to 130 ° C., and the time of heat treatment can be 5 to 30 minutes.

  Then, the section after 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 more and 30 minutes or less. If necessary, the PBS may be replaced during immersion.

(3-1) Dyeing step A
Staining step A for chemical immunostaining comprises: binding step A in which an antibody or the like to which an enzyme for color development is linked is bound by an antigen-antibody reaction or the like to a specific biomolecule in a pathological section; And color-developing step A, in which a color-developing substrate is added to the reaction system to cause the color-developing enzyme to develop a color.

  In the binding step A, the above binding to a biomolecule can be carried out by a known method. It is preferable to add a known blocking agent such as BSA-containing PBS dropwise and incubate at room temperature for a predetermined time (for example, 1 hour) prior to this binding.

In the color forming step A, the substrate of the color forming enzyme and the color forming agent are added to the reaction system to cause a chemical color, but the color forming agent targeted by the present invention is to form an insoluble precipitate by color forming reaction Can be illustrated.
When horseradish peroxidase (HRP) is used as a coloring enzyme, TMB (3,3,5,5-tetramethylbenzidine), 3,3'-diaminobenzidine (DAB), 4-chloro-1-naphthol, etc. are targeted It becomes. Moreover, when using enzyme alkaline phosphatase as an enzyme for color development, neufuchsin etc. become object. In addition, as described above, a color-developing agent that produces an unwanted precipitate due to chemical coloring is targeted.

(3-2) Dyeing step B
The staining process step B for fluorescent immunostaining comprises a binding step B for binding the biomolecule recognition molecule of the fluorescent nanoparticle label to the biomolecule. Here, the biomolecule refers to an antigen to be stained by fluorescence immunostaining, a primary antibody specifically bound to the antigen, a secondary (n-order) antibody bound to the primary antibody, or the antigen or The term means any one of other biomolecules bound to the first to nth antibodies.
In addition, the binding between the above-described antigen, primary antibody, secondary (n-order) antibody, other biomolecules, and biomolecule recognition molecules may be performed before or after.
As an example of a specific procedure of the binding step B, a buffer (PBS etc.) dispersion of labeled fluorescent nanoparticle is prepared, placed on a pathological section, and a biomolecule recognition molecule of the labeled fluorescent nanoparticle and the biomolecule Combine the Next, the section after staining is immersed in a container containing buffer (PBS or the like) to remove unreacted fluorescent nanoparticle labeled body, antibody having a coloring enzyme, and the like. The immersion time is preferably 3 minutes to 30 minutes. If necessary, the buffer (such as PBS) may be exchanged during immersion.

(4) Fixation Treatment Step The fixation treatment step is a step of fixing the above-mentioned color former and fluorescent nanoparticle labeled body introduced in the tissue staining treatment step (1) to a tissue section.

  Examples of the fixing treatment solution include formalin, paraformaldehyde, glutaraldehyde, a crosslinking agent such as acetone, ethanol and methanol, and a cell membrane permeable substance.

  Fixation processing can be performed by a conventionally known method. The fixation treatment can be performed specifically by immersing the stained tissue section obtained by the histochemical staining process in the fixation treatment solution as described above. For example, it can be carried out by immersing the stained tissue section obtained by the histochemical staining process in a dilute paraformaldehyde aqueous solution for several minutes to several hours.

(5) Observation step (5-1) Bright field observation step In the bright field observation step, illumination light is applied to the tissue section stained in the above steps (1) to (4), and the pigment of the color former deposited on the tissue section Is a step of observing distribution information (coloring score etc.) of biomolecule A to be stained in cells or tissues.

  Bright field observation may be performed by a general method. For example, in bright field observation when histochemical staining is performed as a biomolecule to be stained with VEGFR-2 protein in esophageal cancer cells, a 4-times objective lens of an optical microscope is used under irradiation of appropriate illumination light. The VEGFR-2 protein positive staining image, the intensity of the positive staining, and the percentage of positive cells of cancer cells in the sample tissue are observed. Next, the objective lens is switched 10 times to check whether the positive findings are localized in the cell membrane or cytoplasm, and the objective lens is further searched with 20 times as necessary.

  In this step, the distribution information of biomolecule A and the distribution information of biomolecule B may be acquired from the lens barrel of a microscope so that rapid observation can be performed, or an image taken by a camera installed in the microscope may be separately obtained. It may be obtained by displaying it on a display means (such as a monitor) and observing it.

(5-2) Fluorescence observation process A fluorescence observation process is a biomolecule based on the fluorescence which the fluorescent substance emits by irradiating excitation light to the fluorescent substance of the fluorescent nanoparticle label in the tissue section stained at the above-mentioned process. It is a process of acquiring distribution information (brightness point etc.) of B.

  The said excitation light irradiates the excitation light suitable with respect to the said fluorescent substance, excites a fluorescent substance, and stains the biomolecule to be stained with fluorescence. Also, the means for irradiating the excitation light is not particularly limited. For example, excitation light of an appropriate wavelength and output may be irradiated to the stained tissue section 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 from the label is distinguishable in relation to the autofluorescence of the tissue section, but visual observation of the fluorescence is possible, and a substrate used for staining in bright field observation (for example, diamino Those having a wavelength of 450 nm to 700 nm are preferable from the viewpoint of avoiding absorption by benzidine) and preventing autofluorescence intensity from a tissue section becoming too high. In addition, as a fluorescent substance constituting the fluorescent substance used as the fluorescent label, a 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 To measure the fluorescence).

  Further, in the present step, each distribution information of biomolecules A and B may be acquired from the column of a (fluorescent) microscope so that rapid observation can be performed, or a camera installed in the (fluorescent) microscope captures an image The acquired image may be separately displayed on display means (such as a monitor) and acquired by observing it. According to the fluorescent substance which constitutes the fluorescent substance used as a label, even if biomolecular distribution information can not be sufficiently obtained by visual observation from the microscope column, biomolecular distribution information is obtained from the image taken by the camera There are cases where it is possible.

  Examples of acquiring the distribution information of the biomolecules A and B include measuring the number or density of biomolecules to be stained per cell based on the number of fluorescent spots or the emission brightness. For excitation of a fluorescent nanoparticle labeled body, an excitation light source and an optical filter for fluorescence detection corresponding to the absorption maximum wavelength and the fluorescence wavelength may be selected. It is preferable to use commercially available image analysis software (for example, all bright spot automatic measurement software “G-Count” (manufactured by G-Angstrom)) for measuring the number of bright spots or emission luminance, but the measuring means is particularly limited. It is not something to be done.

  In the embodiment of the multiple immunostaining described above, an example of the multiple immunostaining for staining two types of biomolecules is shown, but the number of biomolecules C ... Alternatively, multiple immunostaining may be used to detect

The actions and effects of the fluorescent nanoparticle labeled body, multiplex immunostaining agent and multiplex immunostaining method according to the present invention will be described below.
(1) The labeled fluorescent nanoparticle according to the present invention comprises a fluorescent substance-accumulating nanoparticle having fluorescent substance in an accumulated state, and a hydrophilic polymer having a number average molecular weight of 3000 or more bound to the surface of the fluorescent substance-accumulating nanoparticle It is a particle comprising a chain and a biomolecular recognition molecule attached to the end of the hydrophilic polymer chain.
When the fluorescent nanoparticle label according to the present invention is used for multiplex immunostaining to perform fluorescent immunostaining and immunostaining by chemical coloration for the purpose of detecting the same or different antigens, first, biomolecule recognition of the fluorescent nanoparticle label is carried out A molecule is bound to the antigen or a biomolecule (such as an antibody) bound thereto, and the antigen is fluorescently stained. On the other hand, an antibody in which an enzyme for chemical color development is added is bound to an antigen which is the same as or different from this antigen, and addition of a color former causes a reaction of chemical color development to stain the antigen. At this time, because the number average molecular weight of the hydrophilic polymer chain of the fluorescent nanoparticle labeled body is 3,000 or more, it physically separated from the insoluble precipitate (such as one obtained by polymerizing the color former) generated by the above-mentioned chemical coloration. The phosphors are arranged at the positions, and the insoluble precipitates are less likely to inhibit the light emission from the phosphors (see contrast between FIG. 1 (A) and (B)). As a result, it is possible to ensure the same degree of brightness as in the case of performing fluorescent immunostaining alone, and the detection accuracy of the antigen in multiplex immunostaining is improved.

  (2) By providing the hydrophilic polymer chain (B) having a number average molecular weight of less than 3000 bound to the surface of the above-mentioned phosphor-accumulated nanoparticles, non-specific binding of the labeled fluorescent nanoparticle to other molecules It can be suppressed.

  (3) When the hydrophilic polymer chain (A) and / or the hydrophilic polymer chain (B) is polyethylene glycol, activation of unintended biomolecules can be prevented.

  (4) If the biomolecular recognition molecule is avidin capable of binding to a primary antibody capable of specifically binding to an antigen to be stained or a biotin linked to a secondary antibody capable of binding to the primary antibody, Because a very strong and specific bond between avidin and avidin is used to bind a fluorescent nanoparticle label to a biomolecule, the time from the addition of the fluorescent nanoparticle label to the reaction system to the fluorescence observation is short. Thus, it is possible to suppress the influence of the decrease in the fluorescence intensity at the bright spot caused by the fading of the phosphor.

Example 1A
Synthesis of labeling agent A (streptavidin-bound Texas red dye-containing melamine resin nanoparticles having a number average molecular weight of 3400 polyethylene glycol) 2.5 mg of sulforhodamine 101 (“Sulforhodamine 101”, Sigma-Aldrich, Texas Red dye) in pure water 22. After dissolving in 5 mL, the solution was stirred for 20 minutes while maintaining the temperature of the solution at 70 ° C. by a hot stirrer. To the solution after stirring, 1.5 g of a melamine resin "Nicalac 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.

  After adding 100 μL of formic acid to the solution after stirring and stirring for 20 minutes while maintaining the temperature of the solution at 60 ° C., the solution was allowed to cool to room temperature. The solution after cooling is divided into a plurality of tubes for centrifugation, centrifuged at 12,000 rpm for 20 minutes, and the Texas red dye-containing melamine resin nanoparticles (hereinafter abbreviated as particle A) contained as a mixture in the solution ) Was precipitated and the supernatant removed. Thereafter, the precipitated particles A were washed with ethanol and water.

  0.1 mg of particles A after washing is dispersed in 1.5 mL of ethanol, 2 μL of aminopropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.) is added and reacted at room temperature for 8 hours with stirring to perform surface amination I did the processing.

  The concentration of particle A whose surface is aminated is adjusted to 3 nM with PBS (phosphate buffered saline) containing 2 mM EDTA (ethylenediaminetetraacetic acid) to give a final concentration of 10 mM for this solution Maleimido-PEG-carboxylate-NHS (manufactured by NOF Corporation, SUNBRIGHT MA-034TS, average molecular weight 3400) was mixed and reacted at room temperature for 1 hour.

  The reaction solution was centrifuged at 10,000 G for 20 minutes, and after removing the supernatant, PBS containing 2 mM EDTA was added, the precipitate was dispersed, and centrifugation was performed again under the same conditions. By washing three times according to the same procedure, a particle A surface-modified with a PEG chain having a maleimide group at the end was obtained.

On the other hand, preparation of streptavidin having a sulfhydryl group was performed as follows. First, for 40 μL 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 μL were reacted at room temperature for 1 hour. That is, the introduction of protected thiol group to the amino group of streptavidin _{(-NH-CO-CH 2 -S} -CO-CH 3).

  After that, a free thiol group (-SH) was generated from the protected thiol group by a known hydroxylamine treatment, and a treatment of adding a thiol group (-SH) to streptavidin was performed.

  The streptavidin solution was desalted with a gel filtration column (Zaba Spin Desalting Columns: Funakoshi) to obtain streptavidin capable of binding to the melamine-based particles.

  The PEG-modified particles A and streptavidin were mixed in PBS containing 2 mM EDTA, and allowed to react for 1 hour. The reaction was stopped by adding 10 mM mercaptoethanol. The resulting solution was concentrated using a centrifugal filter, and unreacted streptavidin and the like were removed using a gel filtration column for purification to obtain a labeling agent A.

Example 1 B (Multiple Immunostaining)
Using labeling agent A prepared in Example 1A and DAB staining kit for immunostaining general CD34, vascular growth factor vascular endothelial cell 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.

(1) Deparaffinization Step A tissue array slide (T022 manufactured by US Biomax) on which esophagus cancer tissue was fixed was immersed in xylene and deparaffinized.

(2) Activation Process After deparaffinization, this tissue array slide is immersed in a citrate buffer (pH 6.0) and autoclaved (for 15 minutes at 121 ° C.) to activate the antigen. The

(3-1) Dyeing step A
After washing the activated tissue array slide with PBS, 10% goat serum (manufactured by Nichirei) was added to prevent nonspecific binding, and left at room temperature for 1 hour. After washing this tissue array slide with PBS, an anti-CD34 antibody (Nichirei mouse antibody) was added and left at room temperature for 30 minutes. After washing the tissue array slide again with PBS, dextran polymer peroxidase labeled anti-mouse antibody (manufactured by Nichirei Corporation) was added, and left at room temperature for 30 minutes. Immunostaining using an enzyme having diaminobenzidine (DAB) as a chromogenic substrate was performed using a DAB substrate kit (manufactured by Nichirei).

(3-2) Dyeing step B
After washing the tissue array slide after the above (3-1) immunostaining with PBS, 10% goat serum (manufactured by Nichirei Co., Ltd.) was added, and left at room temperature for 1 hour. After washing this tissue array slide with PBS, an anti-VEGFR-2 antibody (Abcam rabbit antibody) was added and left at room temperature for 30 minutes. After washing with PBS, a biotin-labeled anti-rabbit antibody (manufactured by Nichirei Corporation) was added, and left at room temperature for 30 minutes. The labeling agent A was added and allowed to react at room temperature for 2 hours.

(4) Fixation treatment step The slide obtained from the immunohistochemical staining section obtained above is dipped in the order of ethanol and xylene, then a mounting agent (Merck Enteranu New) is added, a cover glass is placed, and fixation treatment is carried out. This was taken as an evaluation slide.

(5) Observation The double-stained section fixed in the above (4) was observed using a general-purpose fluorescence microscope BX53 manufactured by Olympus Corporation. The excitation wavelength of Texas red (phosphor) was 580 nm and the measurement wavelength was 630 nm when the number of bright spots was measured by fluorescence observation.

[Comparative Examples 1A, 1B]
Labeling agent B Synthesis of Streptavidin-Coupled Rexazolide dye-containing melamine resin nanoparticles having a molecular weight of 528 polyethylene glycol In Example 1A, instead of maleimide-PEG-carboxylate-NHS (manufactured by NOF Corporation SUNBRIGHT MA-034TS average molecular weight 3400) MAL-dPEG-NHS Ester (manufactured by Quanta, QB10284a, polyethylene glycol moiety, molecular weight 528) was used in the same manner as in the synthesis of labeling agent A to prepare labeling agent B, and multiple immunostaining as in Example 1A And the observation etc. which confirm the brightness score were performed. Table 1 shows the results of Example 1B and Comparative Example 1B.

[Examples 2A, 2B]
Labeling Agent C Synthesis of Streptavidin-Coupled Rexazolide Dye-Containing Melamine Resin Nanoparticles Containing Polyethylene Glycol with Molecular Weights of 5000 and 352 Maleimide-PEG-Carboxylate-NHS (Nippo Oil Corporation SUNBRIGHT MA-034 TS Average Molecular Weight 3400) Instead of Maleimide The final concentrations of -PEG-carboxylate-NHS (SUNBRIGHT MA-050TS by NOF Corporation, average molecular weight 5000) and MAL-dPEG-NHS Ester (QB10274a polyethylene glycol moiety molecular weight 352 by Quanta) become 1 mM and 10 mM, respectively. Thus, the labeling agent C was synthesized in the same manner as the labeling agent A except that a mixture of 1:10 (molar ratio) was used. Further, using this labeling agent C, multiple immunostaining, observation for confirming the brightness score and the like were performed as in Example 1A.

[Examples 3A, 3B]
Labeling Agent D Synthesis of Streptavidin-Coupled Rexazolide Dye-Containing Melamine Resin Nanoparticles Containing Polyethylene Glycol with Molecular Weight 20000 2000 and 2000 Maleimide-PEG-Carboxylate-NHS (Nippo Oil Co., Ltd. SUNBRIGHT MA-034 TS Average Molecular Weight 3400) instead of Maleimide -PEG-Carboxylate-NHS (Nippo Oil Corporation SUNBRIGHT MA-200 TS average molecular weight 20000) and Maleimido-PEG-Carboxylate-NHS (NOF Corporation SUNBRIGHT MA-020 TS average molecular weight 2000) to a final concentration of 1 mM respectively The labeling agent D was synthesized in the same manner as in the synthesis of the labeling agent A except that a mixture of 1:10 (molar ratio) was used so as to be 10 mM. Further, using this labeling agent D, multiple immunostaining, observation for confirming the brightness score, etc. were performed as in Example 1A.

[Examples 4A, 4B]
Labeling Agent E Synthesis of Streptavidin-Coupled Rexazolide Dye-Containing Melamine Resin Nanoparticles Containing Polyethylene Glycol with Molecular Weights of 40000 and 528 Maleimide-PEG-Carboxylate-NHS (Made by NOF SUNBRIGHT MA-034 TS Average Molecular Weight 3400) instead of Maleimide -The final concentrations of-PEG-Carboxylate-NHS (SUNBRIGHT MA-400TS, manufactured by NOF Corporation) and MAL-dPEG-NHS Ester (QB10284a, a polyethylene glycol moiety manufactured by Quanta, molecular weight 528) will be 1 mM and 10 mM respectively. Then, the labeling agent E was synthesized in the same manner as the labeling agent A except that a mixture of 1:10 (molar ratio) was used. Further, using this labeling agent E, multiple immunostaining, observation for confirming the brightness score, etc. were performed as in Example 1A.

  The fluorescent nanoparticle labeling substance according to the present invention, the multiplex immunostaining agent containing the fluorescent nanoparticle labeling body, and the multiplex immunostaining method using the multiplex immunostaining agent have been described based on the embodiments and examples. However, the present invention is not limited thereto, and design changes are permitted without departing from the scope of the present invention.

Claims (3)

Phosphor accumulated nanoparticles having phosphor accumulated therein;
A hydrophilic polymer chain (A) having a number average molecular weight of 3,000 or more bonded to the surface of the phosphor-aggregated nanoparticle;
A biomolecule recognition molecule bound to the end of the hydrophilic polymer chain (A) ;
A hydrophilic polymer chain (B) having a number average molecular weight of less than 3000 bound to the surface of the phosphor-aggregated nanoparticle;
A multi immunostaining method using immunofluorescence stain and bright field observation possible chemical immune stains, has a fluorescent nanoparticle labels provided with,
The biomolecular recognition molecule is avidin or streptavidin capable of binding to biotin linked to a secondary antibody capable of binding to a primary antibody capable of specifically binding to a biomolecule to be subjected to fluorescent immunostaining,
The chemical immunostaining agent is an antibody to which a coloring enzyme is linked,
The multiple immunostaining method is
Chemistry for chemical immunostaining, comprising a binding step of binding a biomolecule in a pathological section to the chemical immunostaining agent, and a color development step of adding a substrate for the color forming enzyme after the binding step to cause color development An immunostaining process,
A biomolecule in the pathological section, which is the same as or different from the biomolecule, is bound to the primary antibody, and the secondary antibody is bound to the primary antibody, and the secondary antibody Fluorescent immunostaining processing step of binding biotin linked to the biomolecular recognition molecule of the labeled fluorescent nanoparticle, and
Multiple immunostaining, wherein said chemical immunostaining and said fluorescent immunostaining are performed on the same pathological section. The multiplex immunostaining method according to claim 1 , wherein the hydrophilic polymer chain (A) and / or the hydrophilic polymer chain (B) is polyethylene glycol. A multiplex immunostaining kit for use in the multiplex immunostaining method according to claim 1 or 2 , comprising:
A multiple immunostaining kit comprising: a fluorescent immunostaining agent having the fluorescent nanoparticle label; and a chemical immunostaining agent capable of bright field observation.

Images