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WO2018151071A1 - Procédé de marquage fluorescent - Google Patents

Procédé de marquage fluorescent Download PDF

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Publication number
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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WO
WIPO (PCT)
Prior art keywords
aminocoumarin compound
particles
compound
aminocoumarin
encapsulated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2018/004802
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English (en)
Japanese (ja)
Inventor
賢司 西川
健作 高梨
武寿 磯田
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Konica Minolta Inc
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Konica Minolta Inc
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Filing date
Publication date
Application filed by Konica Minolta Inc filed Critical Konica Minolta Inc
Priority to JP2018568515A priority Critical patent/JP7095603B2/ja
Publication of WO2018151071A1 publication Critical patent/WO2018151071A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/02—Coumarine dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B67/00—Influencing 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
    • C09B67/0001—Post-treatment of organic pigments or dyes
    • C09B67/0004—Coated particulate pigments or dyes
    • C09B67/0008—Coated particulate pigments or dyes with organic coatings
    • C09B67/0013—Coated particulate pigments or dyes with organic coatings with polymeric coatings
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B67/00—Influencing 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
    • C09B67/0071—Process features in the making of dyestuff preparations; Dehydrating agents; Dispersing agents; Dustfree compositions
    • C09B67/0084—Dispersions of dyes
    • C09B67/0085—Non common dispersing agents
    • C09B67/009—Non common dispersing agents polymeric dispersing agent
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B67/00—Influencing 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
    • C09B67/0097—Dye preparations of special physical nature; Tablets, films, extrusion, microcapsules, sheets, pads, bags with dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the present invention relates to an immunostaining method and a fluorescent labeling method such as FISH, in which an aminocoumarin compound is used as a dye and labeling is performed with aminocoumarin compound-encapsulating resin particles encapsulating the aminocoumarin compound.
  • an antigen is fluorescently labeled by specifically binding a fluorescently labeled antibody to an in vivo molecule (antigen) whose expression level is increased or decreased depending on the presence or absence of the disease, and the amount of fluorescent signal is used to determine the disease. Quantifying the amount of antigen associated with.
  • an antibody is directly or indirectly bound to a nanoparticle encapsulating a fluorescent dye and bound to an antigen. Yes.
  • the fluorescent dye a dye that emits light in each of the green region, the red region, the orange region, and the far infrared region is used.
  • Examples of the dye that emits light in the green region include Pyrromethene 556 described in Patent Document 2.
  • fluorescent labeling is performed using nanoparticles containing Pyrromethene 556, the green bright spot is unclear, and in the case of multiple labels, leakage into other color regions is large, and effective observation cannot be performed. There was a problem.
  • the present invention seeks to solve the problems associated with the prior art as described above, and has a clear green luminescent spot, and in the case of multiple labels, a fluorescent labeling method with little leakage into other color regions.
  • the purpose is to provide.
  • the present inventors have used an aminocoumarin compound having a specific structure as a green pigment, and by labeling with an aminocoumarin compound-encapsulated particle encapsulating this aminocoumarin compound.
  • the present inventors have found that the above problems can be solved and have completed the present invention.
  • labeling is performed using aminocoumarin compound-encapsulated particles in which an aminocoumarin compound having a structure represented by the following formula (1) or (2) or a salt thereof is encapsulated in base particles.
  • This is a fluorescent labeling method.
  • each R independently represents 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.
  • A independently represents 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.
  • the average particle size of the aminocoumarin compound-containing particles is preferably 80 to 200 nm.
  • the fluorescent labeling method can perform multiple labeling including labeling using the aminocoumarin compound-encapsulated particles. Examples of the fluorescent labeling method include immunostaining and FISH.
  • Preferable embodiments of the immunostaining method include PDL1, CTLA4, CD8, CD30, CD48, CD59, IDO, TDO, CSF-1R, HDAC, CXCR4, FLT-3, TIGIT, INF- ⁇ , INF- ⁇ , INF- ⁇ , INF- ⁇ , INF- ⁇ , INF- ⁇ 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, Galle Cutin-9 (Galectin-9), LAG-3 (CD223), B7-H3
  • a green bright spot is clear, and in the case of multiple labels, leakage into other color areas, for example, a red area is small, and a green bright spot and a red bright spot are detected. A good balance with the points is obtained.
  • the fluorescent labeling method of the present invention is a fluorescent label for labeling using an aminocoumarin compound-encapsulated particle in which an aminocoumarin compound having a structure represented by the following formula (1) or (2) or a salt thereof is encapsulated in a base particle. Is the law.
  • R's each independently represent a hydrogen atom or a methyl group.
  • six A's 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.
  • the aminocoumarin compound of the present invention has a benzothiazole structure when Q in formula (1) or (2) is a sulfur atom, and has a benzoxazole structure when Q is an oxygen atom, and N—R 1 In some cases, it will have a benzimidazole structure.
  • the sulfonic acid group SO 3 H contained in the formulas (1) and (2) is any carbon atom among the four carbon atoms that can be bonded to the benzene ring contained in the benzothiazole structure, benzoxazole structure or benzimidazole structure. May be bonded to.
  • the aminocoumarin compound having the structure represented by the formula (1) and the aminocoumarin compound having the structure represented by the formula (2) have a sulfonated benzothiazole residue, benzoxazole residue or benzimidazole residue. Common in that it has an aminocoumarin structure.
  • the nitrogen atom bonded to the coumarin structure forms two 6-membered rings together with the four carbon atoms of the benzene ring contained in the coumarin structure, That is, the structure is different from a known sulfonated coumarin compound in that the amino group of aminocoumarin has a julolidine structure.
  • the aminocoumarin compound having the structure represented by the formula (1) has an excitation wavelength longer than that of a known sulfonated coumarin compound, and a wavelength giving a maximum excitation intensity is 475 nm or more, for example, at 475 to 510 nm. is there.
  • the aminocoumarin compound of the present invention also has a longer emission wavelength than known sulfonated coumarin compounds, and the wavelength that gives the maximum emission intensity is 510 nm or more, for example, 510 to 540 nm.
  • the aminocoumarin compound represented by the formula (1) has a feature that the emission intensity is higher than that of an aminocoumarin compound formed by substituting the sulfone group of the aminocoumarin compound with a hydrogen atom.
  • An aminocoumarin compound having a structure represented by the formula (1) can be produced, for example, by a method of sulfonating a coumarin compound having a structure represented by the following formula (3). Specifically, it can be produced by adding 1 ml of fuming sulfuric acid to 0.1 g of the coumarin compound represented by the formula (3) and reacting at 0 to 140 ° C. for 1 to 8 hours.
  • An aminocoumarin compound having a structure represented by the formula (2) can be produced, for example, by a method of sulfonating a coumarin compound having a structure represented by the following formula (4). Specifically, it can be produced by adding 1 ml of fuming sulfuric acid to 0.1 g of the coumarin compound represented by formula (4) and reacting at 0 to 140 ° C. for 1 to 8 hours.
  • the aminocoumarin compound-encapsulated particles have an aminocoumarin compound having a structure represented by formula (1) or formula (2) and base particles encapsulating the aminocoumarin compound.
  • the base particles encapsulating the aminocoumarin compound are organic particles or inorganic particles, and are not particularly limited as long as the aminocoumarin compound can be encapsulated.
  • the organic particles are preferably thermosetting resins. Since the thermosetting resin has a three-dimensional network structure, the aminocoumarin compound encapsulated in the thermosetting resin is not easily detached from the resin particles, and is suitable for fluorescent labeling such as immunostaining.
  • the thermosetting resin include melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin. Among these, amino resins such as melamine resin and urea resin are particularly preferable because they can more effectively suppress the release of the pigment from the resin particles.
  • the inorganic particles include silica particles and glass particles.
  • the amount of the aminocoumarin compound encapsulated in the base particle is not particularly limited, and may be an amount that can ensure a detectable luminance when the aminocoumarin compound-encapsulated particle is used for a fluorescent label such as immunostaining.
  • the average particle diameter of the aminocoumarin compound-encapsulated particles is not particularly limited, but when used for fluorescent labeling such as immunostaining, it is usually 20 to 500 nm, preferably 80 to 200 nm. If the average particle size of the aminocoumarin compound-containing particles exceeds 200 nm, there may be a problem with labeling properties, and if it is less than 80 nm, there may be problems with visibility.
  • the average particle size is calculated as an average value obtained by measuring the particle size of 1000 aminocoumarin compound-encapsulated particles by SEM observation.
  • the production method of the aminocoumarin compound-encapsulated particles is not particularly limited, and a known method can be adopted. In general, a method of forming a matrix such as a resin or silica in the presence of an aminocoumarin compound and encapsulating the aminocoumarin compound in the matrix particles can be used.
  • an aminocoumarin compound is added while (co) polymerizing the (co) monomer for synthesizing the base particle by emulsion polymerization, and the (co) polymer is added.
  • a method of incorporating an aminocoumarin compound into the inside or the surface of can be used.
  • the base particles are inorganic particles such as silica
  • the method for synthesizing FITC-encapsulated silica nanoparticles described in Langmuir Vol. 8, Vol. 9, page 2921 (1992) can be referred to.
  • Aminocoumarin compound-encapsulated silica nanoparticles can be synthesized by using an aminocoumarin compound instead of FITC.
  • the wavelength that gives the maximum excitation intensity is preferably 475 to 510 nm, and the wavelength that gives the maximum emission intensity is preferably 510 to 540 nm.
  • the aminocoumarin compound-encapsulating resin particle produced by encapsulating the aminocoumarin compound represented by the formula (1) in the resin is obtained by replacing the sulfone group of the aminocoumarin compound with a hydrogen atom.
  • the emission intensity tends to be strong. This is because the aminocoumarin compound represented by the formula (1) is more easily included in the resin than the aminocoumarin compound formed by replacing the sulfone group of the aminocoumarin compound with a hydrogen atom. It is presumed that it is taken in.
  • fluorescent labeling method of the present invention labeling is performed using the aminocoumarin compound-encapsulated particles.
  • the fluorescent labeling method include immunostaining and FISH.
  • the specific operation method of immunostaining and FISH is not particularly limited, and a known method can be used.
  • the aminocoumarin compound-encapsulated particles may be used as the dye particles.
  • the aminocoumarin compound-encapsulated particles can be used to stain not only HER2 and Ki67 but also proteins to be stained such as PDL1, CTLA4, CD8, CD30, CD48, and CD59.
  • the fluorescent labeling method of the present invention may be a multiple label including a label using the aminocoumarin compound-encapsulated particles. That is, multiple labeling can be performed on two or more labeling targets using different dyes, and at least one of the staining targets can be labeled using the aminocoumarin compound-encapsulated particles. For example, for a plurality of labeling targets, some of the labeling targets are labeled using the aminocoumarin compound-containing particles, and particles containing a dye that emits light other than green are used for other labeling targets. Thus, a plurality of objects to be labeled can be separately labeled with green and a color other than green.
  • multiple staining is performed on at least two proteins to be stained selected from PDL1, CTLA4, CD8, CD30, CD48 and CD59 using different dyes, and at least one of the proteins to be stained is selected.
  • PDL1 can be stained green with aminocoumarin compound-encapsulated particles
  • CTLA4 can be stained with red
  • PDL1 and CTLA4 can be labeled with different colors.
  • the aminocoumarin compound has a light emitting region close to a red region as compared with a coumarin compound other than the aminocoumarin compound.
  • a coumarin other than the aminocoumarin compound is used. The effect that the leakage into the red region is rather smaller than that of the pigment particles containing the compound is obtained.
  • the recovered precipitate was dispersed with ethanol, the dispersion was centrifuged, and the supernatant was removed to obtain an aminocoumarin compound I represented by the following formula (I) as a precipitate.
  • the yield of aminocoumarin compound I was 80%.
  • the obtained powder was added to pure water, then neutralized with an aqueous NaOH solution to dissolve the precipitate, and the pH of the solution was adjusted to 7-8.
  • This solution was dried with a freeze dryer to obtain Na salt of aminocoumarin compound I. It was confirmed that the aminocoumarin compound I dissolves quickly in water by using Na salt, whereas the sulfonic acid form has poor solubility in water.
  • the resulting organoalkoxysilane compound solution (0.3 mL) was mixed with 99% ethanol (24 mL), tetraethoxysilane (TEOS) (0.3 mL), ultrapure water (0.75 mL), and 28% by mass of ammonia water (0.75 mL) at 25 ° C. Mixed for hours.
  • TEOS tetraethoxysilane
  • the mixed solution prepared in the above step was centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. To this precipitate, ethanol was added to disperse the precipitate, and rinsing was performed again by centrifugation. Further, similar rinsing was repeated twice to obtain aminocoumarin compound-encapsulated particles I. When 1000 particles of the obtained particles were observed with an SEM and the average particle size was measured, the average particle size was 60 nm.
  • Aminocoumarin compound-encapsulated particles VI were obtained in the same manner as in Production Example 3 except that aminocoumarin compound II was used instead of aminocoumarin compound I. When 1000 particles of the obtained particles were observed with an SEM and the average particle size was measured, the average particle size was 150 nm.
  • the solution was heated to 70 ° C. while stirring on a hot stirrer, and then 0.65 g of melamine resin raw material Nicalak MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was added to the solution.
  • melamine resin raw material Nicalak MX-035 manufactured by Nippon Carbide Industries Co., Ltd.
  • To this solution was added 1000 ⁇ L of a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Ltd.) as a reaction initiator, and the mixture was heated and stirred at 70 ° C. for 50 minutes, then heated to 90 ° C. and stirred for 20 minutes.
  • aminocoumarin compound-encapsulated particles VII were obtained.
  • the obtained dispersion liquid of aminocoumarin compound-encapsulating resin particles VII was washed with pure water to remove impurities such as excess resin material and aminocoumarin compound. Specifically, the mixture was centrifuged at 20000 G for 15 minutes in a centrifuge (Kubota Micro Cooling Centrifuge 3740), and after removing the supernatant, ultrapure water was added and ultrasonically irradiated to redisperse. Centrifugation, supernatant removal, and washing by redispersion in ultrapure water were repeated 5 times. When 1000 particles of aminocoumarin compound-encapsulated particles VII were observed with SEM and the average particle size was measured, the average particle size was 150 nm.
  • Dye-containing particles i were obtained in the same manner as in Production Example 1 except that Pyrromethene 556, which is a green pigment, was used instead of aminocoumarin compound I. When 1000 particles of the obtained particles were observed with an SEM and the average particle size was measured, the average particle size was 150 nm.
  • Dye-encapsulated particles ii were obtained in the same manner as in Production Example 7 except that Pyrromethene 556, which is a green pigment, was used instead of aminocoumarin compound I. When 1000 particles of the obtained particles were observed with an SEM and the average particle size was measured, the average particle size was 150 nm.
  • Example 1 Immunostaining was performed by the following method. (Modification of dye-encapsulated particles with streptavidin) Aminocoumarin compound-encapsulated particles I were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and SM (PEG) was added to this solution to a final concentration of 10 mM. ) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleimidopropionamid) -dodecaneethyleneglycol] ester) was mixed and reacted at 5 ° C. for 1 hour.
  • the mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and the mixture was centrifuged again. By performing washing by the same procedure three times, aminocoumarin compound-encapsulated particles I having a maleimide group at the end were obtained.
  • streptavidin solution was desalted with a gel filtration column (Zaba Spin Desaling Columns: Funakoshi) to obtain streptavidin capable of binding to the silica particles.
  • This total amount of streptavidin (containing 0.04 mg) was mixed with 740 ⁇ L of the silica-based particles adjusted to 0.67 nM using PBS containing 2 mM of EDTA, and reacted at room temperature for 1 hour.
  • the reaction solution was purified by subjecting it to a desalting column “Zeba Desalt Spin Spin Columns” (Thermo Scientific Cat. # 89882). Absorption at a wavelength of 300 nm of the desalted reaction solution was measured with a spectrophotometer (Hitachi “F-7000”) to calculate the amount of protein contained in the reaction solution.
  • the reaction solution was adjusted to 250 ⁇ g / mL with a 50 mM Tris solution, and this solution was used as a biotinylated secondary antibody solution.
  • Specimen processing step (1-1) Deparaffinization processing step HER2 (3+) and HER2 (-) tissue array slides ("CB-A712 series” manufactured by Cosmo Bio) are used as tissue sections for staining. It was. The tissue array slide was deparaffinized.
  • Activation treatment step Washing was performed by replacing the deparaffinized tissue array slide with water.
  • the washed tissue array slide was autoclaved at 121 ° C. for 15 minutes in 10 mM citrate buffer (pH 6.0) to activate the antigen.
  • the tissue array slide after the activation treatment was washed with PBS, and the washed tissue array slide was subjected to blocking treatment with PBS containing 1% BSA for 1 hour.
  • tissue sections that had undergone the immunostaining step and the morphological observation staining step were immersed in pure ethanol for 5 minutes four times, and washed and dehydrated. Subsequently, the operation of immersing in xylene for 5 minutes was carried out 4 times to perform clearing. Finally, a tissue section slide of a sample for observation was prepared by enclosing a tissue section using an encapsulant (“Enteran New” manufactured by Merck).
  • the tissue section after the immobilization treatment step was irradiated with predetermined excitation light to emit fluorescence.
  • the tissue sections in this state were observed and imaged with a fluorescence microscope (OLYMPUS "BX-53") and a digital camera for microscope (OLYMPUS "DP73").
  • the excitation light was set to 575 to 600 nm by passing through an optical filter.
  • the range of the wavelength (nm) of fluorescence to be observed was also set to 612 to 692 nm by passing through an optical filter.
  • the conditions of the excitation wavelength during microscopic observation and image acquisition were such that the irradiation energy near the center of the field of view was 900 W / cm 2 for excitation at 580 nm.
  • the exposure time at the time of image acquisition was arbitrarily set so as not to saturate the brightness of the image (for example, set to 4000 ⁇ sec) and imaged.
  • the number of bright spots of the HER2 (3+) tissue was an average value of 1000 cells measured by the ImageJ FindMaxims method based on an image captured at 400 times.
  • the number S of bright spots on the cell membrane in the field of view and the number N of bright spots outside the field in the field of view were measured, and S / N was calculated. S / N is shown in Table 1.
  • Examples 2 to 5, 7, 8 (Modification of dye-encapsulated particles with streptavidin)
  • streptavidin-linked amino acid was prepared in the same manner as in Example 1 except that aminocoumarin compound-encapsulated particles II to VI and VIII were used instead of aminocoumarin compound-encapsulated particle I, respectively.
  • Coumarin compound-encapsulated particles II to VI and VIII were obtained, respectively.
  • Examples 2 to 5, 7, and 8 were the same as Example 1 except that streptavidin-conjugated aminocoumarin compound-encapsulated particles I were used instead of streptavidin-conjugated aminocoumarin compound-encapsulated particles I, respectively.
  • S / N was calculated by the method. S / N is shown in Table 1.
  • Example 6 Modification of dye-encapsulated particles with streptavidin
  • Streptavidin-linked aminocoumarin compound-encapsulated particles III were obtained in the same manner as in Example 1 except that aminocoumarin compound-encapsulated particles III were used instead of aminocoumarin compound-encapsulated particles I.
  • a biotinylated secondary antibody solution was obtained in the same manner as in Example 1.
  • tissue array slide of PDL1 was used as a tissue section for staining.
  • the tissue array slide was deparaffinized.
  • Activation treatment step Washing was performed by replacing the deparaffinized tissue array slide with water.
  • the washed tissue array slide was autoclaved in a 10 mM citrate buffer (pH 6.0) at 121 ° C. for 15 minutes to activate the antigen.
  • the tissue array slide after the activation treatment was washed with PBS, and the washed tissue array slide was subjected to blocking treatment with PBS containing 1% BSA for 1 hour.
  • tissue sections that had undergone the immunostaining step and the morphological observation staining step were immersed in pure ethanol for 5 minutes four times, and washed and dehydrated. Subsequently, the operation of immersing in xylene for 5 minutes was carried out 4 times to perform clearing. Finally, a tissue section slide of a sample for observation was prepared by enclosing a tissue section using an encapsulant (“Enteran New” manufactured by Merck).
  • Example 9 (Modification of dye-encapsulated particles with streptavidin) Streptavidin-linked aminocoumarin compound-encapsulated particles VIII were obtained in the same manner as in Example 1 except that aminocoumarin compound-encapsulated particles VIII were used instead of aminocoumarin compound-encapsulated particles I.
  • Step (1) The tissue specimen was immersed in a container containing xylene for 15 minutes. The xylene was changed twice during the process.
  • Step (2) The tissue specimen was immersed in a container containing ethanol for 10 minutes. The ethanol was changed twice during the process.
  • Step (3) The tissue specimen was immersed in a container containing water for 10 minutes.
  • Step (4) The tissue specimen was immersed in a 10 mM citrate buffer (pH 6.0).
  • Process (5) The autoclave process was performed for 5 minutes at 121 degreeC.
  • Step (6) The tissue specimen after the autoclave treatment was immersed in a container containing PBS for 15 minutes. The PBS was changed three times during the process.
  • Step (7) PBS containing 1% BSA was placed on the tissue specimen and allowed to stand for 1 hour.
  • Step (10) Anti-Ki67 antibody-binding dye-encapsulated particles adjusted to 0.1 nM with PBS containing 1% BSA were placed on a tissue specimen and allowed to stand overnight to label Ki67.
  • Step (11) The labeled tissue specimen was immersed in a container containing PBS for 30 minutes.
  • Step (12) The tissue specimen was fixed with a 4% neutral paraformaldehyde solution for 10 minutes, and then stained with HE.
  • Step (13) After dropping Aquack made by Merck, a cover glass was placed and a tissue specimen was enclosed.
  • the tissue specimen after immunostaining was placed on the stage, and each time the filter set was switched while switching between two types of filter sets for green and red, the number of fluorescent luminescent spots in the fluorescence image of the tissue specimen was measured. The results are shown in Table 3.
  • Examples 11 and 12 In Examples 11 and 12, multiple immunostaining was performed in the same manner as in Example 10 except that aminocoumarin compound-encapsulated particles IX and aminocoumarin compound-encapsulated particles VIII were used instead of aminocoumarin compound-encapsulated resin particles VII, respectively. went. The results are shown in Table 3.
  • Example 13 Multiple immunostaining of green and red was performed by the following method.
  • Aminocoumarin compound-encapsulated particle VIII is introduced with maleimide using NHS-PEG (polyethylene glycol) -maleimide reagent at the end, and thiolated anti-CTLA4 antibody is bound thereto to produce an anti-CTLA4 antibody-bound aminocoumarin compound-encapsulated particle did.
  • maleimide was introduced into the end of the dye-encapsulated particle iii, and a thiolated anti-PDL1 antibody was bound thereto to produce an anti-CTLA4 antibody-bound dye-encapsulated particle.
  • Step (1) The tissue specimen was immersed in a container containing xylene for 15 minutes. The xylene was changed twice during the process.
  • Step (2) The tissue specimen was immersed in a container containing ethanol for 10 minutes. The ethanol was changed twice during the process.
  • Step (3) The tissue specimen was immersed in a container containing water for 10 minutes.
  • Step (4) The tissue specimen was immersed in a 10 mM citrate buffer (pH 6.0).
  • Process (5) The autoclave process was performed for 5 minutes at 121 degreeC.
  • Step (6) The tissue specimen after the autoclave treatment was immersed in a container containing PBS for 15 minutes. The PBS was changed three times during the process.
  • Step (7) PBS containing 1% BSA was placed on the tissue specimen and allowed to stand for 1 hour.
  • Step (9) The labeled tissue specimen was immersed in a container containing PBS for 15 minutes.
  • Step (10) Anti-CTLA4 antibody-binding dye-encapsulated particles adjusted to 0.1 nM with PBS containing 1% BSA were placed on a tissue specimen and allowed to stand overnight to label PDL1.
  • Step (11) The labeled tissue specimen was immersed in a container containing PBS for 30 minutes.
  • Step (12) The tissue specimen was fixed with a 4% neutral paraformaldehyde solution for 10 minutes, and then stained with HE.
  • Step (13) After dropping Aquack made by Merck, a cover glass was placed and a tissue specimen was enclosed. (Microscopic observation) Microscopic observation was performed in the same manner as in Example 10. The results are shown in Table 4.
  • Example 14 Multiple immunostaining of green and red was performed by the following method.
  • Maleimide was introduced into the end of the aminocoumarin compound-encapsulated particles VIII using NHS-PEG (polyethylene glycol) -maleimide reagent, and thiolated anti-CD8 antibody ("DCD” anti-CD8 mouse monoclonal antibody (C8 / 144B )]) was bound to produce anti-CD8 antibody-bound aminocoumarin compound-encapsulated particles.
  • Step (1) The tissue specimen was immersed in a container containing xylene for 15 minutes. The xylene was changed twice during the process.
  • Step (2) The tissue specimen was immersed in a container containing ethanol for 10 minutes. The ethanol was changed twice during the process.
  • Step (3) The tissue specimen was immersed in a container containing water for 10 minutes.
  • Step (4) The tissue specimen was immersed in a 10 mM citrate buffer (pH 6.0).
  • Process (5) The autoclave process was performed for 5 minutes at 121 degreeC.
  • Step (6) The tissue specimen after the autoclave treatment was immersed in a container containing PBS for 15 minutes. The PBS was changed three times during the process.
  • Step (7) PBS containing 1% BSA was placed on the tissue specimen and allowed to stand for 1 hour.
  • Step (10) Anti-PDL1 antibody-binding dye-encapsulated particles adjusted to 0.1 nM with PBS containing 1% BSA were placed on a tissue specimen and allowed to stand overnight to label PDL1.
  • Step (11) The labeled tissue specimen was immersed in a container containing PBS for 30 minutes.
  • Step (12) The tissue specimen was fixed with a 4% neutral paraformaldehyde solution for 10 minutes, and then stained with HE.
  • Step (13) After dropping Aquack made by Merck, a cover glass was placed and a tissue specimen was enclosed. (Microscopic observation) Microscopic observation was performed in the same manner as in Example 10. The results are shown in Table 4.
  • Anti-CD30 antibody manufactured by Dako, “Anti-CD30 mouse monoclonal antibody (BerH2) obtained by introducing maleimide into the end of aminocoumarin compound-encapsulated particles VIII using NHS-PEG (polyethylene glycol) -maleimide reagent and thiolating it. Were bound to produce anti-CD30 antibody-bound aminocoumarin compound-encapsulated particles.
  • Step (1) The tissue specimen was immersed in a container containing xylene for 15 minutes. The xylene was changed twice during the process.
  • Step (2) The tissue specimen was immersed in a container containing ethanol for 10 minutes. The ethanol was changed twice during the process.
  • Step (3) The tissue specimen was immersed in a container containing water for 10 minutes.
  • Step (4) The tissue specimen was immersed in a 10 mM citrate buffer (pH 6.0).
  • Process (5) The autoclave process was performed for 5 minutes at 121 degreeC.
  • Step (6) The tissue specimen after the autoclave treatment was immersed in a container containing PBS for 15 minutes. The PBS was changed three times during the process.
  • Step (7) PBS containing 1% BSA was placed on the tissue specimen and allowed to stand for 1 hour.
  • Step (10) Anti-PDL1 antibody-binding dye-encapsulated particles adjusted to 0.1 nM with PBS containing 1% BSA were placed on a tissue specimen and allowed to stand overnight to label PDL1.
  • Step (11) The labeled tissue specimen was immersed in a container containing PBS for 30 minutes.
  • Step (12) The tissue specimen was fixed with a 4% neutral paraformaldehyde solution for 10 minutes, and then stained with HE.
  • Step (13) After dropping Aquack made by Merck, a cover glass was placed and a tissue specimen was enclosed. (Microscopic observation) Microscopic observation was performed in the same manner as in Example 10. The results are shown in Table 4.

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Abstract

La présente invention concerne un procédé de marquage fluorescent pour un marquage utilisant des particules contenant un composé aminocoumarine, qui sont obtenues par inclusion d'un composé aminocoumarine ou un sel de celui-ci ayant la structure représentée par la formule (1) ou (2) dans les particules de matrice. Dans la formule (1), R représentent chacun indépendamment un atome d'hydrogène ou un groupe méthyle, Q représente un atome de soufre, un atome d'oxygène, ou N-R1, et R1 représente un atome d'hydrogène ou un groupe méthyle. Dans la formule (2), A représentent chacun indépendamment un atome d'hydrogène ou un groupe méthyle, Q représente un atome de soufre, un atome d'oxygène, ou N-R1, et R1 représente un atome d'hydrogène ou un groupe méthyle. Selon ce procédé de marquage fluorescent, des points luminescents verts sont évidents, et dans des cas de coloration multicolore simultanée, il y a peu de fuite dans d'autres régions de couleur, par exemple, des régions rouges, et il est possible d'obtenir un excellent équilibre entre les points luminescents verts et les points luminescents rouges.
PCT/JP2018/004802 2017-02-14 2018-02-13 Procédé de marquage fluorescent Ceased WO2018151071A1 (fr)

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JPH069892A (ja) * 1992-06-22 1994-01-18 Nippon Kanko Shikiso Kenkyusho:Kk クマリン誘導体
JPH06271599A (ja) * 1993-01-29 1994-09-27 Bayer Ag スルホクマリン−含有ヌクレオチド及び核酸検出法におけるそれらの利用
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* Cited by examiner, † Cited by third party
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JP7261609B2 (ja) 2018-03-28 2023-04-20 日本化薬株式会社 クマリン化合物、及びこれを含んだ顔料組成物

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