WO2018151071A1 - Fluorescent labeling method - Google Patents

Fluorescent labeling method Download PDF

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WO2018151071A1
WO2018151071A1 PCT/JP2018/004802 JP2018004802W WO2018151071A1 WO 2018151071 A1 WO2018151071 A1 WO 2018151071A1 JP 2018004802 W JP2018004802 W JP 2018004802W WO 2018151071 A1 WO2018151071 A1 WO 2018151071A1
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amino coumarin
particles
formula
inf
represents
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PCT/JP2018/004802
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French (fr)
Japanese (ja)
Inventor
賢司 西川
健作 高梨
武寿 磯田
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コニカミノルタ株式会社
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Priority to JP2017024875 priority
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Publication of WO2018151071A1 publication Critical patent/WO2018151071A1/en

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    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09BORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00Other synthetic dyes of known constitution
    • C09B57/02Coumarine dyes
    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09BORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B67/00Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
    • C09B67/0001Post-treatment of organic pigments or dyes
    • C09B67/0004Coated particulate pigments or dyes
    • C09B67/0008Coated particulate pigments or dyes with organic coatings
    • C09B67/0013Coated particulate pigments or dyes with organic coatings with polymeric coatings
    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09BORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B67/00Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
    • C09B67/0071Process features in the making of dyestuff preparations; Dehydrating agents; Dispersing agents; Dustfree compositions
    • C09B67/0084Dispersions of dyes
    • C09B67/0085Non common dispersing agents
    • C09B67/009Non common dispersing agents polymeric dispersing agent
    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09BORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B67/00Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
    • C09B67/0097Dye preparations of special physical nature; Tablets, films, extrusion, microcapsules, sheets, pads, bags with dyes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • G01N33/532Production of labelled immunochemicals
    • G01N33/533Production of labelled immunochemicals with fluorescent label

Abstract

The present invention is a fluorescent labeling method for a labeling using particles containing an aminocoumarin compound, which are obtained by including an aminocoumarin compound or salt thereof having the structure represented by formula (1) or (2) in matrix particles. In formula (1), R each independently represent a hydrogen atom or a methyl group, Q represents a sulfur atom, an oxygen atom, or N-R1, and R1 represents a hydrogen atom or a methyl group. In formula (2), A each independently represent a hydrogen atom or a methyl group, Q represents a sulfur atom, an oxygen atom, or N-R1, and R1 represents a hydrogen atom or a methyl group. According to this fluorescent labeling method, green luminescent spots are evident, and in cases of simultaneous multi-color dyeing, there is little leakage into other color regions, e.g., red regions, and it is possible to attain an excellent balance between green luminescent spots and red luminescent spots.

Description

Fluorescence labeling method

The present invention uses an amino coumarin compound as a dye, performs labeling by amino coumarin compounds containing resin particles The amino coumarin compound is encapsulated, for fluorescent labeling method such as immunostaining and FISH.

Currently, fluorescence labeling method has been widely used, such as immunostaining and FISH.
For example, in the medical, in order to provide data for determining whether a subject is suffering from a target disease, immunostaining is widely used for tissue sections and the like of the subject. This immunostaining, for example, a molecule (antigen) in vivo the amount of expression is increased or decreased by the presence or absence of the diseased, the antigen fluorescently labeled by specifically binding the fluorescence-labeled antibody, the disease from the amount of fluorescent signal it is performed to quantify the amount of the relevant antigen. Fluorescence labeled antibodies as a technology for binding to an antigen, the fluorescent dye directly or indirectly bound to antibody nanoparticles were encapsulated in particles, which is a technique to bind to an antigen, for example, disclosed in Patent Document 1 there.

Examples of the fluorescent dye, a green region, a dye that emits light in the region of the red region, an orange region and the far infrared region is used, respectively. Using two or more dyes emitting at different areas, multiple labeling carry both labels in two or more regions is a very effective indicator means, it is expected the development of the technology.

Examples of the dye that emits in the green region, for example, include Pyrromethene556 described in Patent Document 2.
However, when the fluorescent labeled using nanoparticles containing an Pyrromethene556, not clear green bright spot, and if the multiply labeled, large leakage to other color regions can not effectively observed there is a problem in that.

WO2015 / 159776 WO2012 / 133920

The present invention is intended to solve the problems with the prior art as described above, a clear green bright spot, if the multiple labeling, fluorescent labeling leakage is small to other color regions an object of the present invention is to provide a.

The present inventors have made intensive studies to solve the above problems, using amino coumarin compound having a specific structure as a green pigment, by performing labeling by the amino coumarin compound aminocoumarin compound containing particles contained the It can solve the above problems, and have completed the present invention.

That is, the fluorescence labeling method of the present invention performs labeling using the following equation (1) or amino coumarin compound having a structure represented by (2) or amino coumarin compounds containing particles formed by encapsulating the salt base particles it is a fluorescent labeling method.

Figure JPOXMLDOC01-appb-C000003
(In the formula (1), R each independently represent a hydrogen atom or a methyl group, Q represents a sulfur atom, an oxygen atom or N-R 1, R 1 represents a hydrogen atom or a methyl group.)

Figure JPOXMLDOC01-appb-C000004
(In the formula (2), A represents each independently a hydrogen atom or a methyl group, Q represents a sulfur atom, an oxygen atom or N-R 1, R 1 represents a hydrogen atom or a methyl group.)

In the fluorescent labeling method, it is preferable that the average particle size of the amino coumarin compounds containing particles is 80 ~ 200 nm.
The fluorescent labeling method, it is possible to perform multiple labels, including labeling with the amino coumarin compound containing particles.
The fluorescent labeling method, for example, immunostaining and FISH.

As a preferred embodiment of the immunostaining, PDL1, CTLA4, CD8, CD30, CD48, CD59, IDO, TDO, CSF-1R, HDAC, CXCR4, FLT-3, TIGIT, INF-α, INF-β, INF- ω, INF-ε, INF-κ, INF-γ, INF-λ CSF, EPO, EGF, FGF, PDGF, HGF, TGF, CD3, CD4, CD25, CD28, CD80, CD86, CD160, CD57, OX40 (CD134 ), OX40L (CD252), ICOS (CD278), ICOSL (CD275), CD155, CD226, CD112, CD27, CD70,4-1BB (CD137), 4-1BBL (CD137L), GITR (CD357), GITRL, BTLA ( CD272), HVEM (CD270), TIM-3, Gareku Down -9 (Galectin-9), LAG-3 (CD223), B7-H3 (CD276), B7-H4, B7-H5, CD40, CD40L, PD-1, PD-L2,2B4 (CD244), KLRG- 1, E-Cadherin, performs multiple staining with N-Cadherin, R-Cadherin, CD68, different dye for at least two dyeing the protein of interest is selected from CD163 and CSF1-R, the stained protein of interest at least one, it can be cited fluorescent labeling staining with an amino coumarin compound containing particles.

Doing fluorescence labeled with a fluorescent labeling method of the present invention, a green bright points clearly, in the case of multiple labeling, other color areas, for example small leakage of the red region, the green bright spots and red bright good balance of points is obtained.

Fluorescence labeling method of the present invention, a fluorescent label which performs labeling using the following equation (1) or amino coumarin compound having a structure represented by (2) or amino coumarin compounds containing particles formed by encapsulating the salt base particles it is the law.

Figure JPOXMLDOC01-appb-C000005

Figure JPOXMLDOC01-appb-C000006
In the formula (1), thirteen R represents each independently a hydrogen atom or a methyl group.
In the formula (2), 6 A each independently represent a hydrogen atom or a methyl group.

In the formula (1) and (2), Q represents a sulfur atom, an oxygen atom or N-R 1. Wherein R 1 represents a hydrogen atom or a methyl group. Amino coumarin compounds of the present invention, when Q of formula (1) or (2) is a sulfur atom, having a benzothiazole structure, if an oxygen atom, having a benzoxazole structure, with N-R 1 some cases, will have a benzimidazole structure.

Sulfonic acid group SO 3 H contained in formula (1), and (2), the benzothiazole structure, which carbon atom of the four carbon atoms capable of binding to the benzene ring in the benzoxazole structure or a benzimidazole structure have it may be bonded to.

The amino coumarin compounds having the structure represented by Formula amino coumarin compound of formula having the structure represented by (1) (2), having sulfonated benzothiazole residue, benzoxazole residues or benzimidazole residue common in that a aminocoumarin structure.

Amino coumarin compound having a structure represented by the formula (1) are that the nitrogen atom bonded to the coumarin structure, with four carbon atoms of the benzene ring included in the coumarin structure, and form two 6-membered ring, that amino group of the amino coumarins known sulfonated coumarin compounds and structure that has a julolidine structure is different.

Amino coumarin compound having a structure represented by the formula (1), rather than the known sulfonated coumarin compound, an excitation wavelength long wavelength, the wavelength giving the maximum excitation intensity of not less than 475 nm, for example 475 ~ 510 nm is there. Further, amino coumarin compounds of the present invention, the emission wavelength is also longer wavelength than known sulfonated coumarin compound, a wavelength giving the maximum emission intensity is not less than 510 nm, for example, 510 ~ 540 nm.

Amino coumarin compound represented by formula (1), compared to the amino coumarin compound formed by substituting a sulfone group of the amino coumarin compound with hydrogen atoms, it has a feature that emission intensity is high.

Amino coumarin compound having a structure represented by the formula (1) may, for example, can be produced by a method of sulfonating the coumarin compound having a structure represented by the following formula (3). Specifically, in addition 1ml fuming sulfuric acid relative to the coumarin compound 0.1g of the formula (3), at 0 ~ 140 ° C., it can be prepared by reacting 1-8 hours.

Figure JPOXMLDOC01-appb-C000007
(R and Q in formula (3) has the same meaning as R and Q, respectively formula (1).)

Amino coumarin compound having a structure represented by the formula (2), for example, can be produced by a method of sulfonating the coumarin compound having a structure represented by the following formula (4). Specifically, in addition 1ml fuming sulfuric acid relative to the coumarin compound 0.1g of the formula (4), at 0 ~ 140 ° C., it can be prepared by reacting 1-8 hours.

Figure JPOXMLDOC01-appb-C000008
(A and Q in the formula (4) has the same meaning as R and Q in each formula (2).)

The amino coumarin compound encapsulated particles includes a base particles containing the formula (1) or amino coumarin compound and the amino coumarin compound having a structure represented by the formula (2).
Mother particles containing an amino coumarin compounds are organic or inorganic particles is not particularly limited as long as possible containing an amino coumarin compound.
Examples of the organic particles is preferably thermosetting resin. Since the thermosetting resin has a three-dimensional network structure, amino coumarin compounds encased This difficulty in removing from the resin particles, it is preferred in fluorescent labels such as immunostaining. The thermosetting resin, melamine resin, urea resin, aniline resin, guanamine resins, phenol resins, xylene resins and furan resins. Among these, melamine resins, amino resins such as urea resin, in that it can suppress the separation of the resin particles of dye more effectively, particularly preferred.

Examples of the inorganic particles, silica particles, glass particles and the like.
The amount of amino coumarin compounds contained in the base particles is not particularly limited, an amino coumarin compound encapsulated particles when used in fluorescent labeling of immunostaining or the like may be an amount capable of ensuring a detectable intensity.

The average particle size of the amino coumarin compounds containing particles is not particularly limited, in the case of using fluorescent labels such as immunostaining, usually 20 ~ 500 nm, preferably 80 ~ 200 nm. The average particle size of the amino coumarin compounds containing particles may encounter problems labeled property exceeds 200 nm, is less than 80 nm, there are cases where problems visibility occurs.

The average particle diameter is determined by measuring the particle size for 1000 amino coumarin compounds containing particles in SEM observation, it is calculated as the average value.
Method for producing an amino coumarin compounds containing particles is not particularly limited, it may be a known method. In general, maternal and the formation of such a resin or silica in the presence of an amino coumarin compounds, amino coumarin compounds can be used a method for encapsulating in the base particles.

If the base particles are organic particles, for example, by emulsion polymerization, while (co) monomer (co) polymerizing for synthesizing base particles, were added amino coumarin compound, the (co) polymer the method of incorporating the amino coumarin compound in or on can be used.

If the base particles are inorganic particles such as silica, for example, it can be a method for the synthesis of FITC encapsulated silica particles described in Langmuir Volume 8 2921 pages (1992) reference. It can be synthesized amino coumarin compound containing silica particles by using an amino coumarin compound instead of FITC.

Amino coumarin compounds containing resin particles, a wavelength is 475 ~ 510 nm which gives the maximum excitation intensity, it is preferable wavelength giving the maximum emission intensity is 510 ~ 540 nm.
Amino coumarin compounds containing resin particles of amino coumarin compound was prepared by being incorporated into a resin represented by formula (1) is an amino coumarin compound to a resin formed by substituting the sulfonic group of the amino coumarin compounds with hydrogen atoms compared to the amino coumarin compounds containing resin particles prepared by being incorporated, emission intensity is strong tendency. This amino coumarin compound represented by the formula (1), rather than an amino coumarin compound formed by substituting a sulfone group of the amino coumarin compound with hydrogen atoms, there is a property of easily encapsulated in the resin, more to the resin it is presumed to be because captured.

Fluorescence labeling method of the present invention performs labeled with an amino coumarin compound containing particles. The fluorescent labeling method include immunostaining and FISH like. Specific instructions for immunostaining and FISH is not particularly limited, and may be a known method. In conventional immunostaining or FISH performing labeled with dye particles, it may be used an amino coumarin compound encapsulated particles as the pigment particles.

For immunostaining, using an amino coumarin compounds containing particles, other HER2 and Ki67, it can also be stained for PDLl, CTLA4, CD8, CD30, CD48 and CD59 staining protein of interest such as.

Fluorescence labeling method of the present invention may be a multi-labels, including labeling with the amino coumarin compound containing particles. That performs a multiply labeled with a different dye for the two or more labeled target, at least one of its dyeing target, can be labeled with an amino coumarin compound containing particles. For example, for a plurality of labeled target performed labeled with an amino coumarin compounds containing particles for some of the labeled target of which, using particles containing a dye showing an emission other than green for the other labeled target Te performing labeling, it may be separately labeled multiple labeled target in the green and green other colors.

For example, in the immunostaining, PDLl, CTLA4, CD8, CD30, CD48 and perform multiple staining with different dyes respectively for at least two dyeing the protein of interest is selected from CD59, at least one of the stained protein of interest One and can be dyed using the amino coumarin compound containing particles. That way, for example, stained green by an amino coumarin compounds containing particles PDL1, to stain the CTLA4 in red, it is possible that labeled with different colors and PDL1 and CTLA4.
In this multiple staining, PDL1, CTLA4, CD8, CD30, CD48, CD59, IDO, TDO, CSF-1R, HDAC, CXCR4, FLT-3, TIGIT, INF-α, INF-β, INF-ω, INF- ε, INF-κ, INF-γ, INF-λ CSF, EPO, EGF, FGF, PDGF, HGF, TGF, CD3, CD4, CD25, CD28, CD80, CD86, CD160, CD57, OX40 (aka CD134), OX40L (also known as CD252), ICOS (also known as CD278), ICOSL (also known as CD275), CD155, CD226, CD112, CD27, CD70,4-1BB (also known as CD137), 4-1BBL (aka CD137L), GITR (also known as CD357), GITRL , BTLA (also known as CD272), HVEM (also known as CD2 0), TIM-3, Galectin-9, LAG-3 (also known as CD223), B7-H3 (also known as CD276), B7-H4, B7-H5, CD40, CD40L, PD-1, PD-L2,2B4 (aka CD244), performs multiple staining with KLRG-1, E-Cadherin, N-Cadherin, R-Cadherin, CD68, different dye for at least two dyeing the protein of interest is selected from CD163 and CSF1-R, at least one of the stained protein of interest, can be dyed using the amino coumarin compound containing particles.

Doing labeled with an amino coumarin compounds containing particles, you can see a clear green bright spot is small leakage to other color regions, such as the red region. Therefore, it makes green labeled with an amino coumarin compound encapsulated particles to a specific labeled target, when the red labeled with color particles containing a red pigment to other staining subject, obtained the green bright spots small leakage of the red region, good balance of green bright spots and red bright points are obtained.

The amino coumarin compound, compared to coumarin compounds other than the amino coumarin compound has a light-emitting region close to the red region, when the labeled with an amino coumarin compounds containing particles, coumarin other than the amino coumarin compound than the color particles containing a compound, leakage of the red region is effect that rather small.

[Synthesis Example 1]
Put compound 600mg represented by the following formula 20mL vial tube bottle (5), by adding fuming sulfuric acid 6 mL, and stirred for 4 hours at 25 ° C., the reaction was carried out. The progress of the reaction was confirmed by TLC. Specifically, a portion of the reaction solution was neutralized with NaOH aqueous solution, ethanol was added to the reaction solution was subjected to TLC using a solution obtained by mixing with CHCl 3 2, MeOH at a ratio of 3. To Rf values ​​0.88 of the raw material, a Rf value 0.73 of the object, from the data of this TLC, confirmed the formation of the convergence and the desired product of the reaction.

8 minutes eyes ice in 50mL vial tube bottle put up (30 mL), was added and the reaction mixture little by little into this. Suspension formed dye was suspended was obtained. The suspension was centrifuged and collected as a precipitate a dye by removing the supernatant. The precipitate was dispersed in pure water 10 mL, the dispersion was centrifuged to remove the supernatant, the precipitate was collected again and dispersed in pure water 10 mL, the supernatant of this dispersion was centrifuged It was removed, and the precipitate was collected. The recovered precipitate was dispersed in ethanol, the dispersion was centrifuged to remove the supernatant, to obtain the amino coumarin compound I represented by the following formula (I) as precipitates. The yield of the amino coumarin compound I was 80%.

After drying the resulting precipitate, and the obtained powder was added to pure water, neutralized with aqueous NaOH to dissolve the precipitate, the pH of the solution was 7-8. By drying the solution in a freeze drier to obtain a Na salt of the amino coumarin compound I. Amino coumarin compounds I, in the sulfonic acid form whereas poor solubility in water, by the Na salt, it was confirmed that rapidly dissolve in water.

Figure JPOXMLDOC01-appb-C000009

Figure JPOXMLDOC01-appb-C000010

[Synthesis Example 2]
By the same method as in Production Example 1 except for using an amino coumarin compound i represented by the following formula (6) instead of the compound of formula (5), amino coumarin compound II represented by the following formula (II) It was obtained.

Figure JPOXMLDOC01-appb-C000011

Figure JPOXMLDOC01-appb-C000012

[Synthesis Example 3]
By the same method as in Production Example 1 except for using an amino coumarin compound i represented by the following formula (7) instead of the compound of formula (5), amino coumarin compound III represented by the following formula (III) It was obtained.

Figure JPOXMLDOC01-appb-C000013

Figure JPOXMLDOC01-appb-C000014

[Production Example 1]
Amino coumarin compound I 3.4 mg to thionyl chloride 0.1mL was added, 65 ° C. 4 hours, after heating and mixing, to remove the excess thionyl chloride by performing vacuum drying. And resulting amino coumarin compound and reaction products of thionyl chloride 3-aminopropyltrimethoxysilane (3-aminopropyltrimetoxysilane, Shin-Etsu Silicone Co., Ltd., KBM903) 3μL a 1.2mL of N, in N- dimethylformamide (DMF) mixture to obtain a organoalkoxysilane compound.

The resulting organoalkoxysilane compound solution 0.3 mL, 99% ethanol 24 mL, tetraethoxysilane (TEOS) 0.3 mL, ultrapure water 0.75mL, and 28 mass% aqueous ammonia 0.75mL and at 25 ° C. 3 and mixed time.

The mixture prepared in the above step were centrifuged 20 minutes at 10000 G, the supernatant was removed. For this precipitate, the addition of ethanol, the precipitate was dispersed, was subjected to rinsing to centrifugation again. Further repeated twice same rinsing, to give the amino coumarin compounds containing particles I. About 1,000 of the obtained particles were observed by SEM, was measured an average particle size, the average particle size was 60 nm.

[Production Example 2]
Ultrapure water 0.85 mL, in the same manner as in Production Example 1 except that the ammonia water 0.85 mL, to give the amino coumarin compound encapsulated particles II. About 1,000 of the obtained particles were observed by SEM, was measured an average particle size, the average particle size was 80 nm.

[Production Example 3]
Ultrapure water 1.10 mL, in the same manner as in Production Example 1 except that the ammonia water 1.10 mL, to give the amino coumarin compound encapsulated particles III. About 1,000 of the obtained particles were observed by SEM, was measured an average particle size, the average particle size was 150 nm.

[Production Example 4]
Ultrapure water 1.15 mL, in the same manner as in Production Example 1 except that the ammonia water 1.15 mL, to give the amino coumarin compound encapsulated particles IV. About 1,000 of the obtained particles were observed by SEM, was measured an average particle size, the average particle size was 195 nm.

[Production Example 5]
Ultrapure water 1.20 mL, except that the ammonia water 1.20 mL in the same manner as in Production Example 1, to give the amino coumarin compound encapsulated particles V. About 1,000 of the obtained particles were observed by SEM, was measured an average particle size, the average particle size was 220 nm.

[Production Example 6]
Except for using an amino coumarin compound II in place of the amino coumarin compounds I in the same manner as in Production Example 3, to give the amino coumarin compound encapsulated particles VI. About 1,000 of the obtained particles were observed by SEM, was measured an average particle size, the average particle size was 150 nm.

[Production Example 7]
The amino coumarin compounds I 14.4 mg was added thereto and dissolved in water 22 mL. The solution emulsifier for emulsion polymerization of emulsion (R) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation) 5% aqueous solution of was added 2 mL.

This solution was allowed to warm to 70 ° C. with stirring on a hot stirrer, was added 0.65g of melamine resin material NIKALAC MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) to the solution. To this solution a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Inc.) was added 1000μL as a reaction initiator, and heated and stirred at 70 ° C. 50 min, then the mixture was heated with stirring the temperature was raised to 90 ° C. 20 min. By the above operation, to give the amino coumarin compound encapsulated particles VII.

From the dispersion of the resulting amino coumarin compounds containing resin particles VII, it was washed with pure water to remove impurities such as excess resin material or an amino coumarin compound. Specifically, a centrifuge (Kubota Co. micro refrigerated centrifuge 3740) for 15 minutes at 20000 G, then centrifuged, after removal of the supernatant, redispersed by sonication added ultrapure water. Centrifugation was repeated five times washing with redispersion in supernatant was removed and ultrapure water.
Perform SEM observation for 1000 amino coumarin compounds containing particles VII, result of measurement of the average grain diameter and the average particle size was 150 nm.

[Production Example 8]
Except for using an amino coumarin compound II in place of the amino coumarin compounds I in the same manner as in Production Example 7, to give the amino coumarin compound encapsulated particles VIII.
Perform SEM observation for 1000 amino coumarin compounds containing particles VIII, was measured an average particle size, the average particle size was 150 nm.

[Production Example 9]
Except for using an amino coumarin compound III in place of the amino coumarin compounds I in the same manner as in Production Example 7, to give the amino coumarin compound encapsulated particles IX.
Perform SEM observation for 1000 amino coumarin compounds containing particles IX, result of measurement of the average grain diameter and the average particle size was 150 nm.

[Production Example 10]
Except for using Pyrromethene556 is green dye in place of the amino coumarin compounds I in the same manner as in Production Example 1 to obtain a dye encapsulated particles i. About 1,000 of the obtained particles were observed by SEM, was measured an average particle size, the average particle size was 150 nm.

[Production Example 11]
Except for using Pyrromethene556 is green dye in place of the amino coumarin compounds I in the same manner as in Production Example 7, to obtain a dye encapsulated particles ii. About 1,000 of the obtained particles were observed by SEM, was measured an average particle size, the average particle size was 150 nm.

[Production Example 12]
Except that in place of the amino coumarin compounds I using sulforhodamine 101 is a red dye in the same manner as in Production Example 7, to obtain a dye encapsulated particles iii. About 1,000 of the obtained particles were observed by SEM, was measured an average particle size, the average particle size was 150 nm.

[Example 1]
Immunostaining was performed by the following method.
(Streptavidin-modified dye encapsulated particles)
The amino coumarin compounds containing particles I, EDTA (ethylenediaminetetraacetic acid) and using PBS (phosphate buffer saline) containing 2mM adjusted to 3 nM, SM (PEG to a final concentration of 10mM to this solution ) 12 (thermo Scientific Co., succinimidyl - mixing [(N-maleimidopropionamid) -dodecanethyleneglycol] ester), and reacted for 1 hour at 5 ° C..

The mixed solution for 20 minutes centrifugation at 10000 G, after removing the supernatant, adding PBS was 2mM containing EDTA, a sediment is dispersed and centrifuged again. To give the amino coumarin compounds containing particles I of the terminal maleimide groups attached by three times washing with the same procedure.

After adding streptavidin was adjusted to 1 mg / mL (manufactured by Wako Pure Chemical Industries Ltd.) 40 [mu] L in borate buffer 210 uL, 64 mg / mL to adjust the 2-iminothiolane hydrochloride (manufactured by Sigma-Aldrich Co.) 70 [mu] L was added, at room temperature It was allowed to react for 1 hour. Thus, a thiol group to the amino group of streptavidin (-NH-C (= NH 2 + Cl -) -CH 2 -CH 2 -CH 2 -SH) was introduced.

The streptavidin solution gel filtration column (Zaba Spin Desalting Columns: Funakoshi) to desalting to obtain linkable streptavidin to the silica-based particles. The streptavidin total volume of (0.04 mg containing) the EDTA with PBS containing 2mM mixing the silica-based particles 740μL adjusted to above 0.67 nM, were incubated at room temperature for one hour.

Was added 10mM mercaptoethanol, the reaction was stopped. After concentration solution obtained in centrifugal filter, unreacted streptavidin, etc. were removed using a purification gel filtration column, to obtain a streptavidin binding amino coumarin compounds containing particles I.

(Preparation of secondary antibody biotin-modified)
It was dissolved anti-rabbit IgG antibody 50μg to 50 mM Tris-HCl solution (pH 7.5). To the solution, to a final concentration of 3mM was mixed DTT (dithiothretol) solution. Thereafter, the solution was reacted for 30 minutes at 37 ° C.. It was then purified secondary antibody deoxidization with DTT using a desalting column. Was obtained antibody solution 200μL of purified antibody entire amount was dissolved in 50 mM Tris-HCl solution (pH 7.5). And - "Biotin-PEG 6 -NH-Mal ( +) " (PurePEG Co., product number 2461006-250) was converted, using DMSO 0.4 mM while the linker reagent the length of the spacer is 30 Å It was adjusted so as to. The solution 8.5μL was added to the antibody solution and reacted for 30 minutes at mixing to 37 ° C..

The reaction solution was desalted column "Zeba Desalt Spin Columns" (Thermo Scientific, Inc., Cat. # 89882) were purified and subjected to. And calculate the amount of protein contained in to the reaction solution measured by an altimeter spectral absorption wavelength 300nm of desalted reaction solution (manufactured by Hitachi, "F-7000"). 50mMTris solution by adjusting the reaction solution to 250 [mu] g / mL, and the solution with a solution of biotinylated secondary antibody.

(staining)
(1) as tissue sections specimen processing step (1-1) deparaffinized step for dyeing, HER2 (3+) and HER2 - use a tissue array slides (Cosmo Bio Co., Ltd., "Series CB-A712") () It had. The tissue array slides were deparaffinized.

(1-2) the activation process deparaffinized tissue array slides were washed to replace the water. 121 ° C. The washed tissue array slides in 10mM citrate buffer (pH 6.0), by autoclaving for 15 minutes, was subjected to activation treatment of the antigen. The tissue array slides after activation treatment were washed with PBS, and subjected to 1 hour blocking treatment with PBS containing 1% BSA respect cleaned tissue array slides.

(2) with PBS containing 1% of immunostaining process (2-1) Primary reaction BSA, adjusted Ventana Co. The "anti-HER2-rabbit monoclonal antibody (4B5)" to 0.05 nM, the 1 was overnight reaction at 4 ° C. to a solution of the above blocking treated tissue array slides following antibody.

(2-2) a tissue array slides were secondary reaction the primary reaction were washed with PBS, and reacted 1% BSA was diluted to 6 [mu] g / mL in PBS containing the above biotinylated secondary antibody at room temperature for 30 minutes .

(2-3) the fluorescent label processing secondary reaction was carried out tissue array slides containing 1% BSA streptavidin binding amino coumarin compounds containing particles I diluted 0.02nM with PBS, and neutral pH environment (pH 6.9 ~ 7.4) was allowed to react for 3 hours under the conditions of room temperature. The tissue array slides after the reaction and washed with PBS.

(3) after morphological observation staining step immunostaining was performed with hematoxylin-eosin staining (HE staining). The immune stained sections were stained with Mayer hematoxylin solution for 5 minutes was performed hematoxylin staining. Thereafter, the tissue sections were washed for 3 minutes with running 45 ° C.. Next, it eosin stained 5 minutes in 1% eosin solution.

(4) with respect to tissue sections after fixing processing step immunostaining process and morphological observation dyeing step, performed 4 times an operation of immersing 5 minutes in pure ethanol was subjected to washing and dehydration. Then carried out 4 times the operation of immersing 5 minutes in xylene, it was subjected to clearing. Finally, using a mounting medium (Merck "Entellan New"), and with the tissue array slide samples for observation by sealing the tissue sections.

(5) irradiating a predetermined excitation light to tissue sections after the observation and measurement step fixing process, light is emitted fluorescence. Fluorescence microscopy of tissue sections of state (Olympus "BX-53"), were observed and photographed by a microscope digital camera (Olympus Corp. "DP73"). It said excitation light was set to 575 ~ 600 nm by passing the optical filter. As for the range of wavelengths of fluorescence observed (nm), were set to 612 ~ 692 nm by passing the optical filter. Microscopy, condition of the excitation wavelength at the time of image acquisition, the irradiation energy around the central portion of the visual field was set to be 900 W / cm 2 in the excitation of 580 nm. Exposure time for image acquisition is, arbitrarily set so that the brightness of the image is not saturated (e.g. set to 4000μ seconds) and were imaged. Bright points of tissue HER2 (3+) were the average of 1000 cells was measured by based on ImageJ FindMaxims method an image taken by 400 times.
The extracellular bright points N in the bright points S and the field on the cell membrane in the field of view was measured to calculate the S / N. The S / N shown in Table 1.

[Examples 2 to 5, 7, 8]
(Streptavidin-modified dye encapsulated particles)
In Examples 2 to 5, 7, 8, amino coumarin compound encapsulated particles II-VI, streptavidin binding amino in the same manner as in Example 1 except for using each of the VIII instead of amino coumarin compounds containing particles I coumarin compounds containing particles II ~ VI, VIII were respectively obtained.

(Preparation of secondary antibody biotin-modified)
In Examples 2 to 5, 7, 8, to obtain a solution of biotinylated secondary antibody in the same manner as in Example 1.

(staining)
In Examples 2 to 5, 7, 8, streptavidin binding amino coumarin compounds containing particles I instead streptavidin binding amino coumarin compounds containing particles II-VI, except for using each of VIII in the same manner as in Example 1 in a way, to calculate the S / N. The S / N shown in Table 1.

[Example 6]
(Streptavidin-modified dye encapsulated particles)
Except for using an amino coumarin compound encapsulated particles III instead of the amino coumarin compounds containing particles I got streptavidin binding amino coumarin compound encapsulated particles III in the same manner as in Example 1.
(Preparation of secondary antibody biotin-modified)
To obtain a solution of biotinylated secondary antibody in the same manner as in Example 1.

(staining)
(1) as tissue sections specimen processing step (1-1) deparaffinized process for dyeing using tissue array slides PDLl. The tissue array slides were deparaffinized.

(1-2) the activation process deparaffinized tissue array slides were washed to replace the water. 121 ° C. The washed tissue array slides in 10mM citrate buffer (pH 6.0), by autoclaving for 15 minutes, was subjected to activation treatment of the antigen. The tissue array slides after activation treatment were washed with PBS, and subjected to 1 hour blocking treatment with PBS containing 1% BSA respect cleaned tissue array slides.

(2) Immunostaining processing step (2-1) primary reaction BSA with PBS containing 1%, Cell Signaling Technology, Inc. "anti PD-L1 rabbit monoclonal antibody (E1L3N)" adjustments to 0.05nM and it was allowed to react overnight at 4 ° C. the solution of the primary antibody against the above-described blocked tissue array slides.

(2-2) a tissue array slides were secondary reaction the primary reaction were washed with PBS, and reacted 1% BSA was diluted to 6 [mu] g / mL in PBS containing the above biotinylated secondary antibody at room temperature for 30 minutes .

(2-3) the fluorescent label processing secondary reaction was carried out tissue array slides and 1% BSA streptavidin binding amino coumarin compounds containing particles III diluted 0.02nM of PBS containing, neutral pH environment (pH 6.9 ~ 7.4) was allowed to react for 3 hours under the conditions of room temperature. The tissue array slides after the reaction and washed with PBS.

(3) after morphological observation staining step immunostaining was performed with hematoxylin-eosin staining (HE staining). The immune stained sections were stained with Mayer hematoxylin solution for 5 minutes was performed hematoxylin staining. Thereafter, the tissue sections were washed for 3 minutes with running 45 ° C.. Next, it eosin stained 5 minutes in 1% eosin solution.

(4) with respect to tissue sections after fixing processing step immunostaining process and morphological observation dyeing step, performed 4 times an operation of immersing 5 minutes in pure ethanol was subjected to washing and dehydration. Then carried out 4 times the operation of immersing for 5 minutes in xylene, it was subjected to clearing. Finally, using a mounting medium (Merck "Entellan New"), and with the tissue array slide samples for observation by sealing the tissue sections.

(5) Observation and measurement step the same method as in Example 1 was calculated S / N. The S / N shown in Table 1.

[Example 9]
(Streptavidin-modified dye encapsulated particles)
Except for using an amino coumarin compound encapsulated particles VIII in place of the amino coumarin compounds containing particles I got streptavidin binding amino coumarin compound encapsulated particles VIII in the same manner as in Example 1.

(Preparation of secondary antibody biotin-modified)
To obtain a solution of biotinylated secondary antibody in the same manner as in Example 1.
(staining)
Except for using streptavidin-conjugated amino coumarin compound encapsulated particles VIII instead of streptavidin binding amino coumarin compound containing silica particles III is in the same manner as in Example 6, was calculated S / N. The S / N shown in Table 1.

[Comparative Example 1]
(Streptavidin-modified dye encapsulated particles)
Except for using the dye encapsulated particles i in place of the amino coumarin compounds containing particles I give the streptavidin binding dye encapsulated particles i in the same manner as in Example 1.

(Preparation of secondary antibody biotin-modified)
To obtain a solution of biotinylated secondary antibody in the same manner as in Example 1.
(staining)
Except for using streptavidin binding dye encapsulated particles i instead of streptavidin binding amino coumarin compounds containing particles I in the same manner as in Example 1, was calculated S / N. The S / N shown in Table 1.

[Comparative Example 2]
(Streptavidin-modified dye encapsulated particles)
Except for using the dye encapsulated particles i in place of the amino coumarin compounds containing particles I give the streptavidin binding dye encapsulated particles i in the same manner as in Example 1.

(Preparation of secondary antibody biotin-modified)
To obtain a solution of biotinylated secondary antibody in the same manner as in Example 1.
(staining)
Except for using streptavidin binding dye encapsulated particles i instead of streptavidin binding amino coumarin compounds containing particles III is in the same manner as in Example 6, was calculated S / N. The S / N shown in Table 1.

Figure JPOXMLDOC01-appb-T000015

From Table 1, when the immunostained with amino coumarin compounds containing particles containing an amino coumarin compound I ~ III having the structure represented by the formula (1) or (2), in the formula (1) or (2) compared with the case of using a dye encapsulated particles containing therein the Pyrromethene556 a dye other than amino coumarin compound having a structure represented, it was confirmed that the improved S / N.

[Example 10]
It was green and red multi immunostaining by the following methods.
(Modification of the dye-containing particles)
Terminus NHS-PEG amino coumarin compounds containing resin particles VII (polyethylene glycol) - maleimide reagent to introduce a maleimide with which the bound anti-HER2 antibody thiolated anti-HER2 antibody binding amino coumarin compounds containing resin particles It was produced.
Similar to the above, introducing a maleimide-terminated dye encapsulated particles iii, this was bound to anti-Ki67 antibodies thiolated to prepare an anti-Ki67 antibody binding dye encapsulated particles.

(Immune staining of tissue specimens)
Immunostaining of human breast tissue specimens (IHC) method was carried out by following steps (1) to (13).
Step (1): and the tissue specimen in a container filled with xylene was immersed 15 minutes. It was replaced in the middle twice xylene.
Step (2): ethanol in a container filled with the tissue specimen is immersed for 10 minutes. Was the middle exchange twice ethanol.
Step (3): a tissue specimen container containing water was immersed for 10 minutes.
Step (4): it was immersed tissue specimens in 10mM citrate buffer (pH 6.0).
Step (5): 121 for 5 minutes autoclaving was performed at ° C..
Step (6): a vessel containing PBS, and the tissue specimen after autoclaving was immersed for 15 minutes. It was replaced in the middle three times PBS.
Step (7): a 1% BSA-containing PBS and placed on tissue specimen was left for 1 hour.
Step (8): the PBS containing 1% BSA anti-HER2 antibody binding amino coumarin compounds containing resin particles was adjusted to 0.1nM in left overnight put the tissue specimen were labeled HER2.
Step (9): and the tissue specimen after labeling the container with PBS it was immersed 15 minutes.
Step (10): 0.1 nM Anti-Ki67 antibody binding dye encapsulated particles adjusted to with 1% BSA-containing PBS, and allowed to stand overnight placed on tissue specimen were labeled Ki67.
Step (11): and the tissue specimen after labeling the container with PBS was immersed 30 minutes.
Step (12): after fixing for 10 minutes in 4% neutral paraformaldehyde solution tissue specimens were performed HE staining.
Step (13): it was added dropwise to Merck Co. Aquatex, placing a cover glass was filled with tissue specimen.

(Microscopy)
The Carl Zeiss Co., Ltd. fluorescence microscope as a fluorescent microscope was used Semrock steel filter set as a filter set. Filter sets were used following two types corresponding to the immune stain (green and for red).

Figure JPOXMLDOC01-appb-T000016

Tissue specimens after immunostaining was placed on the stage, while switching the green and two filter sets for red, each time switching the filter set was measured fluorescence bright points of the fluorescent image of the tissue specimen. The results are shown in Table 3.

[Examples 11 and 12]
In Examples 11 and 12, a multi-immunostaining by the same method as in Example 10 except that the used respective amino coumarin compounds containing particles IX and amino coumarin compound encapsulated particles VIII in place of the amino coumarin compounds containing resin particles VII went. The results are shown in Table 3.

[Comparative Example 3]
Except for using the dye encapsulated particles ii instead of amino coumarin compounds containing resin particles VII was subjected to multiple immunostaining in the same manner as in Example 10. The results are shown in Table 3.

Figure JPOXMLDOC01-appb-T000017

From Table 3, double staining results of HER2 and Ki67, Doing immunostained with amino coumarin compounds containing particles containing an amino coumarin compound I ~ III having the structure represented by the formula (1) or (2) , as compared with the case of using a dye encapsulated particles containing therein the Pyrromethene556 a dye other than amino coumarin compound having a structure represented by the formula (1) or (2), leakage of the red bright spot green bright spot it has been confirmed is less.

Furthermore, when performing immunostaining with an amino coumarin compounds containing particles containing an amino coumarin compound I ~ III having the structure represented by the formula (1) or (2), the influence of the double staining of red bright points it was confirmed from Table 3, there is little.

Example 13
It was green and red multi immunostaining by the following methods.
(Modification of the dye-containing particles)
Terminus NHS-PEG amino coumarin compounds containing particles VIII (polyethylene glycol) - using a maleimide reagent introducing maleimide, this was bound to anti-CTLA4 antibodies thiolated, producing an anti-CTLA4 antibody binds amino coumarin compound encapsulated particles did.
Similar to the above, introducing a maleimide-terminated dye encapsulated particles iii, this was bound to anti-PDL1 antibodies thiolated to prepare an anti-CTLA4 antibody binding dye encapsulated particles.

(Immune staining of tissue specimens)
Immunostaining of human breast tissue specimens (IHC) method was carried out by following steps (1) to (13).
Step (1): and the tissue specimen in a container filled with xylene was immersed 15 minutes. It was replaced in the middle twice xylene.
Step (2): ethanol in a container filled with the tissue specimen is immersed for 10 minutes. Was the middle exchange twice ethanol.
Step (3): a tissue specimen container containing water was immersed for 10 minutes.
Step (4): it was immersed tissue specimens in 10mM citrate buffer (pH 6.0).
Step (5): 121 for 5 minutes autoclaving was performed at ° C..
Step (6): a vessel containing PBS, and the tissue specimen after autoclaving was immersed for 15 minutes. It was replaced in the middle three times PBS.
Step (7): a 1% BSA-containing PBS and placed on tissue specimen was left for 1 hour.
Step (8): the PBS containing 1% BSA anti-CTLA4 antibodies bind an amino coumarin compounds containing particles was adjusted to 0.1nM in left overnight put the tissue specimen were labeled CTLA4.
Step (9): and the tissue specimen after labeling the container with PBS it was immersed 15 minutes.
Step (10): 0.1 nM Anti-CTLA4 antibody binding dye encapsulated particles adjusted to with 1% BSA-containing PBS, and allowed to stand overnight placed on tissue specimen were labeled PDLl.
Step (11): and the tissue specimen after labeling the container with PBS was immersed 30 minutes.
Step (12): after fixing for 10 minutes in 4% neutral paraformaldehyde solution tissue specimens were performed HE staining.
Step (13): it was added dropwise to Merck Co. Aquatex, placing a cover glass was filled with tissue specimen.
(Microscopy)
It was microscopic observation in the same manner as in Example 10. The results are shown in Table 4.

[Example 14]
It was green and red multi immunostaining by the following methods.
(Modification of the dye-containing particles)
Terminus NHS-PEG amino coumarin compounds containing particles VIII (polyethylene glycol) - introducing a maleimide using a maleimide reagent, to which the thiolated anti-CD8 antibody (Dako Co., Ltd., "anti-CD8 mouse monoclonal antibody (C8 / 144B ) ") was bound were prepared anti-CD8 antibody-bound amino coumarin compound containing particles.
Similar to the above, introducing a maleimide-terminated dye encapsulated particles iii, this was bound to anti-PDL1 antibodies thiolated to prepare an anti-PDL1 antibody binding dye encapsulated particles.

(Immune staining of tissue specimens)
Immunostaining of human breast tissue specimens (IHC) method was carried out by following steps (1) to (13).
Step (1): and the tissue specimen in a container filled with xylene was immersed 15 minutes. It was replaced in the middle twice xylene.
Step (2): ethanol in a container filled with the tissue specimen is immersed for 10 minutes. Was the middle exchange twice ethanol.
Step (3): a tissue specimen container containing water was immersed for 10 minutes.
Step (4): it was immersed tissue specimens in 10mM citrate buffer (pH 6.0).
Step (5): 121 for 5 minutes autoclaving was performed at ° C..
Step (6): a vessel containing PBS, and the tissue specimen after autoclaving was immersed for 15 minutes. It was replaced in the middle three times PBS.
Step (7): a 1% BSA-containing PBS and placed on tissue specimen was left for 1 hour.
Step (8): the PBS containing 1% BSA anti-CD8 antibody-bound amino coumarin compounds containing particles was adjusted to 0.1nM in left overnight put the tissue specimen were labeled CD8.
Step (9): and the tissue specimen after labeling the container with PBS it was immersed 15 minutes.
Step (10): containing 1% BSA anti PDL1 antibody binding dye encapsulated particles was adjusted to 0.1nM in PBS and left overnight put the tissue specimen were labeled PDL1.
Step (11): and the tissue specimen after labeling the container with PBS was immersed 30 minutes.
Step (12): after fixing for 10 minutes in 4% neutral paraformaldehyde solution tissue specimens were performed HE staining.
Step (13): it was added dropwise to Merck Co. Aquatex, placing a cover glass was filled with tissue specimen.
(Microscopy)
It was microscopic observation in the same manner as in Example 10. The results are shown in Table 4.

[Example 15]
It was green and red multi immunostaining by the following methods.
(Modification of the dye-containing particles)
Terminus NHS-PEG amino coumarin compounds containing particles VIII (polyethylene glycol) - using a maleimide reagent introducing maleimide, this thiolated anti-CD30 antibody (Dako Co., Ltd., "anti-CD30 mouse monoclonal antibody (BerH2)) It is bound, to produce an anti-CD30 antibody binds amino coumarin compound containing particles.
Similar to the above, introducing a maleimide-terminated dye encapsulated particles iii, this was bound to anti-PDL1 antibodies thiolated to prepare an anti-PDL1 antibody binding dye encapsulated particles.

(Immune staining of tissue specimens)
Immunostaining of human breast tissue specimens (IHC) method was carried out by following steps (1) to (13).
Step (1): and the tissue specimen in a container filled with xylene was immersed 15 minutes. It was replaced in the middle twice xylene.
Step (2): ethanol in a container filled with the tissue specimen is immersed for 10 minutes. Was the middle exchange twice ethanol.
Step (3): a tissue specimen container containing water was immersed for 10 minutes.
Step (4): it was immersed tissue specimens in 10mM citrate buffer (pH 6.0).
Step (5): 121 for 5 minutes autoclaving was performed at ° C..
Step (6): a vessel containing PBS, and the tissue specimen after autoclaving was immersed for 15 minutes. It was replaced in the middle three times PBS.
Step (7): a 1% BSA-containing PBS and placed on tissue specimen was left for 1 hour.
Step (8): the PBS containing 1% BSA anti-CD30 antibody binds amino coumarin compounds containing particles was adjusted to 0.1nM in left overnight put the tissue specimen were labeled CD30.
Step (9): and the tissue specimen after labeling the container with PBS it was immersed 15 minutes.
Step (10): containing 1% BSA anti PDL1 antibody binding dye encapsulated particles was adjusted to 0.1nM in PBS and left overnight put the tissue specimen were labeled PDL1.
Step (11): and the tissue specimen after labeling the container with PBS was immersed 30 minutes.
Step (12): after fixing for 10 minutes in 4% neutral paraformaldehyde solution tissue specimens were performed HE staining.
Step (13): it was added dropwise to Merck Co. Aquatex, placing a cover glass was filled with tissue specimen.
(Microscopy)
It was microscopic observation in the same manner as in Example 10. The results are shown in Table 4.

[Comparative Example 4]
Except for using the dye encapsulated particles ii instead of amino coumarin compounds containing particles VIII was green and red multi immunostaining in the same manner as in Example 13. The results are shown in Table 4.

[Comparative Example 5]
Except for using the dye encapsulated particles ii instead of amino coumarin compounds containing particles VIII was green and red multi immunostaining in the same manner as in Example 14. The results are shown in Table 4.

[Comparative Example 6]
Except for using the dye encapsulated particles ii instead of amino coumarin compounds containing particles VIII was green and red multi immunostaining in the same manner as in Example 15. The results are shown in Table 4.

Figure JPOXMLDOC01-appb-T000018

From Table 4, the result of double staining with PDL1 and CTLA4, CD8 or CD30, when performing immunostaining with an amino coumarin compounds containing particles containing an amino coumarin compound II having the structure represented by the formula (2), compared with the case of using a dye encapsulated particles containing therein the Pyrromethene556 a dye other than amino coumarin compound having a structure represented by the formula (1) or (2), the leakage of the red bright spot green bright spot less it has been confirmed.

Furthermore, when performing immunostaining with an amino coumarin compounds containing particles containing an amino coumarin compound II having the structure represented by the formula (2), as in the case of double staining of HER2 and Ki67, double stained impact on the Akateru number of rarely been confirmed from Table 4.

Claims (5)

  1. Fluorescent labeling method which performs labeled with amino coumarin compound or an amino coumarin compound containing particles with a salt formed by enclosing the base particles having a structure represented by the following formula (1) or (2).
    Figure JPOXMLDOC01-appb-C000001
    (In the formula (1), R each independently represent a hydrogen atom or a methyl group, Q represents a sulfur atom, an oxygen atom or N-R 1, R 1 represents a hydrogen atom or a methyl group.)
    Figure JPOXMLDOC01-appb-C000002
    (In the formula (2), A represents each independently a hydrogen atom or a methyl group, Q represents a sulfur atom, an oxygen atom or N-R 1, R 1 represents a hydrogen atom or a methyl group.)
  2. Fluorescence labeling method according to claim 1, wherein the average particle size of the amino coumarin compounds containing particles is 80 ~ 200 nm.
  3. Fluorescence labeling method according to claim 1 or 2 for multiplexing labels, including labeling with the amino coumarin compound containing particles.
  4. Fluorescence labeling method according to any one of claims 1 to 3, which is immunostaining or FISH.
  5. The immunostaining is, PDL1, CTLA4, CD8, CD30, CD48, CD59, IDO, TDO, CSF-1R, HDAC, CXCR4, FLT-3, TIGIT, INF-α, INF-β, INF-ω, INF- ε, INF-κ, INF-γ, INF-λ CSF, EPO, EGF, FGF, PDGF, HGF, TGF, CD3, CD4, CD25, CD28, CD80, CD86, CD160, CD57, OX40, OX40L, ICOS, ICOSL , CD155, CD226, CD112, CD27, CD70,4-1BB, 4-1BBL, GITR, GITRL, BTLA, HVEM, TIM-3, Galectin-9, LAG-3, B7-H3, B7-H4, B7-H5 , CD40, CD40L, PD-1, PD-L2,2B4, KLRG-1, E-Cadhe in, N-Cadherin, it performs multiple staining with different dyes respectively R-Cadherin, CD68, CD163 and CSF1-R at least two dyeing target protein selected from at least one of the stained protein of interest , fluorescent labeling method according to claim 4, stained with the amino coumarin compound containing particles.
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH069892A (en) * 1992-06-22 1994-01-18 Nippon Kanko Shikiso Kenkyusho:Kk Coumarin derivative
JPH06271599A (en) * 1993-01-29 1994-09-27 Bayer Ag Sulfocoumarin-containing nucleotide and its use in detection of nucleic acid
JPH07508309A (en) * 1992-05-13 1995-09-14
JP2004309458A (en) * 2003-03-27 2004-11-04 Institute Of Physical & Chemical Research Time-resolved fluorescence microscope
JP2006045314A (en) * 2004-08-03 2006-02-16 Asahi Denka Kogyo Kk Salt composed of fluorescent anion and fluorescent cation and color conversion membrane using the same
WO2015045961A1 (en) * 2013-09-26 2015-04-02 コニカミノルタ株式会社 Fluorescent nanoparticle label, multiple immunostain kit, and multiple immunostaining method
JP2015093878A (en) * 2013-11-08 2015-05-18 コニカミノルタ株式会社 Resin particle containing fluorescent dye, tissue multiple dyeing resin particle set containing fluorescent dye including the same, and tissue multiple dyeing method using the same
WO2016129444A1 (en) * 2015-02-12 2016-08-18 コニカミノルタ株式会社 Antibody-conjugated integrated phosphor nanoparticles, method for manufacturing antibody-conjugated integrated phosphor nanoparticles, and immunostaining kit

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07508309A (en) * 1992-05-13 1995-09-14
JPH069892A (en) * 1992-06-22 1994-01-18 Nippon Kanko Shikiso Kenkyusho:Kk Coumarin derivative
JPH06271599A (en) * 1993-01-29 1994-09-27 Bayer Ag Sulfocoumarin-containing nucleotide and its use in detection of nucleic acid
JP2004309458A (en) * 2003-03-27 2004-11-04 Institute Of Physical & Chemical Research Time-resolved fluorescence microscope
JP2006045314A (en) * 2004-08-03 2006-02-16 Asahi Denka Kogyo Kk Salt composed of fluorescent anion and fluorescent cation and color conversion membrane using the same
WO2015045961A1 (en) * 2013-09-26 2015-04-02 コニカミノルタ株式会社 Fluorescent nanoparticle label, multiple immunostain kit, and multiple immunostaining method
JP2015093878A (en) * 2013-11-08 2015-05-18 コニカミノルタ株式会社 Resin particle containing fluorescent dye, tissue multiple dyeing resin particle set containing fluorescent dye including the same, and tissue multiple dyeing method using the same
WO2016129444A1 (en) * 2015-02-12 2016-08-18 コニカミノルタ株式会社 Antibody-conjugated integrated phosphor nanoparticles, method for manufacturing antibody-conjugated integrated phosphor nanoparticles, and immunostaining kit

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