JP6493085B2 - Method for selecting fluorescent dye-containing resin particle solution and method for adjusting concentration - Google Patents

Description

  The present invention relates to a selection method and a concentration adjustment method of a fluorescent dye-containing resin particle solution used for immunostaining and the like.

  With the recent spread of molecular targeted drug therapies centering on antibody drugs, there is an increasing need for accurate diagnostic methods for using molecular targeted drugs more efficiently. Conventionally, hematoxylin-eosin [HE] staining using a dye and DAB staining using an enzyme have been widely used as tissue staining methods, but the staining concentration depends greatly on environmental conditions such as temperature and time, and is accurate. Quantitative measurement is difficult.

  Therefore, fluorescent dyes have been used instead of dyes as labeling reagents for labeling biological material probes that can bind to biomolecules to be detected. However, when a normal fluorescent dye is used, there may be a problem of deterioration in quantitativeness due to fluorescent fading. For this reason, sensitization of fluorescence and luminescence by the enzyme is performed, but nonspecific fluorescence due to nonspecific adsorption of the enzyme to the tissue section is a problem. Under such circumstances, nanoparticles with integrated phosphors (fluorescent dye-containing resin particles) have become one of the options for fluorescence sensitization and can be used with conventional fluorescent dyes by staining them. It is possible to evaluate with high accuracy and quantitativeness that cannot be obtained by conventional staining.

  For example, Patent Document 1 (International Publication WO2012 / 029342 pamphlet) discloses a tissue staining method using a fluorescent substance-encapsulating resin particle bound to a biological material probe as a staining reagent. An embodiment in which an object to be detected is immunostained using an antibody in which a fluorescent dye-containing resin particle is bound by a covalent bond via a linker (polyethylene glycol chain) is disclosed. In addition, the biological material probe and the fluorescent dye-containing resin particle are not directly bonded by a covalent bond, but are indirectly bonded via an affinity molecule that specifically binds, for example, streptavidin (SA) and biotin. Techniques are also known. For example, in Patent Document 2 (International Publication WO2014 / 203614 pamphlet), detection is performed using a primary antibody, a secondary antibody bound to biotin, and fluorescent dye-containing resin particles bound to streptavidin in Examples. Embodiments for immunostaining a subject are disclosed.

  Here, in the conventional technique as described in Patent Document 1, when a fluorescent dye-containing resin particle solution whose surface is modified is used as a staining liquid for immunostaining, the staining liquid is prepared with a constant concentration or luminance. It was. However, in this method in which the concentration of particles, the brightness, and the amount of binding with the one that modifies the surface of the particles are different for each lot, the surface-modified fluorescent dye-containing resin particles of different lots have the same brightness. When dyeing is performed, there is a problem that the number of bright spots varies about ± 20%, and the variation is about ± 15% even if the density is constant. Further, even if the surface state of the particles is observed, the staining result cannot be predicted, and in order to evaluate the performance of the particles after the surface modification, it is necessary to perform pathological staining for each lot.

  In addition, as described in Patent Document 3 (Japanese Patent Laid-Open No. 2011-141232), the measurement of the affinity amount of avidin for biotin of a fluorescent dye using a biotinylated plate is performed for one molecule of avidin. There is no example used for evaluation of fluorescent dye-containing resin particles in which fluorescent dyes are integrated and a plurality of affinity molecules are bonded to the surface as in the present invention.

International Publication WO2012 / 029342 Pamphlet International Publication WO2014 / 203614 Pamphlet JP 2011-141232 A

  In order to solve the above-mentioned problems, the present invention is compared with a conventional product when a staining solution is prepared using fluorescent dye-containing resin particles whose surface has been modified with an affinity molecule such as streptavidin (SA). Another object of the present invention is to provide a method for preparing a fluorescent dye-containing resin particle solution with little variation in the number of bright spots after dyeing.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor made a plurality of different results when the fluorescence emitted by reacting with the biotinylated plate and the SA surface-modified fluorescent dye-containing resin particles was used as a signal value. When the SA surface-modified fluorescent dye-containing resin particle solution of a lot is selected from those having a signal value within a predetermined range, and such SA-modified fluorescent dye-containing resin particle solution is used as a staining solution The reason is that the variation in staining can be suppressed because the SA surface-modified fluorescent dye-containing resin particles that emit a predetermined signal value as described above have a streptavidin binding amount per surface area of each fluorescent dye-containing resin particle. Since this is considered to be the same, this selection method is very effective as a method for preparing the surface-modified fluorescent dye-containing resin particle solution. It was found that.

  The present invention can solve the above-mentioned problems, for example, having the following configuration, a method for selecting a fluorescent dye-containing resin particle solution, and a method for adjusting the concentration of the fluorescent dye-containing resin particle solution using the selection method, And a fluorescent dye-containing resin particle solution obtained by these methods.

[Claim 1]
A fixed amount of a solution containing a fluorescent dye-containing resin particle having a first affinity molecule bound to the surface at a fixed concentration is fixed at a fixed density with a second affinity molecule that can specifically bind to the first affinity molecule. A method for producing a fluorescent dye-encapsulating resin particle solution, comprising a step of selecting a signal value (S) to be measured that satisfies the following formula when reacted with a plate that has been converted into a fluorescent plate.
0.5 × A × B × (C / D) ≦ S ≦ 1.2 × A × B × (C / D)
A: The same number of fluorescent dyes as those contained in all fluorescent dye-containing resin particles contained in a fixed amount of the fixed concentration solution bound with the first affinity molecule were reacted with the plate. Signal value measured at the time B: Divide the total number of first affinity molecules bound to the entire surface of all fluorescent dye-encapsulating resin particles contained in a fixed amount of the fixed solution by the number of fluorescent dye-encapsulating resin particles Further, the number of first affinity molecules bonded per fluorescent dye-containing resin particle C: emission intensity per one fluorescent dye-containing resin particle D: included in one fluorescent dye-containing resin particle Luminescence intensity of several fluorescent dyes

[Section 2]
A fixed amount of a solution containing a fluorescent dye-containing resin particle having a first affinity molecule bound to the surface at a fixed concentration is fixed at a fixed density with a second affinity molecule that can specifically bind to the first affinity molecule. The total surface area of the fluorescent dye-containing resin particles contained in the fluorescent dye-containing resin particle solution with respect to the fluorescent dye-containing resin particle solution in which the measured signal value (S) satisfies the following formula when reacted with the plate The value (S / T) of the ratio of the signal value (S) measured when the fluorescent dye-containing resin particle solution is dispersed on a plate on which the second affinity molecule is fixed at a constant density with respect to (T). A method for producing a fluorescent dye-containing resin particle solution, the method comprising the step of diluting to a predetermined value and adjusting the concentration of the fluorescent dye-containing resin particle solution.
0.5 × A × B × (C / D) ≦ S ≦ 1.2 × A × B × (C / D)
A: The same number of fluorescent dyes as those contained in all fluorescent dye-containing resin particles contained in a fixed amount of the fixed concentration solution bound with the first affinity molecule were reacted with the plate. Signal value measured at the time B: Divide the total number of first affinity molecules bound to the entire surface of all fluorescent dye-encapsulating resin particles contained in a fixed amount of the fixed solution by the number of fluorescent dye-encapsulating resin particles Further, the number of first affinity molecules bonded per fluorescent dye-containing resin particle C: emission intensity per one fluorescent dye-containing resin particle D: included in one fluorescent dye-containing resin particle Luminescence intensity of several fluorescent dyes

[Section 3]
Item 3. The production method according to Item 1 or 2, wherein the resin that is a matrix constituting the fluorescent dye-containing resin particles is a resin selected from the group consisting of a melamine resin and a styrene resin.

[Claim 4]
Item 4. The production method according to any one of Items 1 to 3, wherein the fluorescent dye constituting the fluorescent dye-containing resin particles is an organic dye.

[Section 5]
Item 5. The production method according to any one of Items 1 to 4, wherein the fluorescent dye constituting the fluorescent dye-containing resin particles is selected from the group consisting of perylene diimide and fluorescein isothiocyanate.

[Claim 6]
Item 1-5, wherein the first affinity molecule bound to the fluorescent dye-containing resin particles is selected from the group consisting of biotin, avidin, streptavidin and neutravidin, an anti-dinitrophenol antibody and an anti-digoxigenin antibody. The method according to one item.

[Claim 7]
Item 7. The production method according to any one of Items 1 to 6, wherein the first affinity molecule is bound to the fluorescent dye-containing resin particles via a polymer-derived spacer.

[Section 8]
Item 8. The production method according to Item 7, wherein the spacer is polyethylene glycol (PEG).

  By selecting a fluorescent dye-containing resin particle solution having sufficient staining performance by the production method of the present invention, and further adjusting the concentration of the fluorescent dye-containing resin particles based on the signal value obtained by a predetermined method, A fluorescent encapsulating resin particle solution having a certain dyeing performance can be obtained. For example, when using biotinylated plates and fluorescent dye-encapsulating resin particles modified with SA, the signal values obtained from the plates and the intensity of luminance actually immunostained using the particles are used. Therefore, the dyeing performance of the particles can be judged without actually dyeing. Furthermore, the ratio value (S / T) of the signal value (S) measured when the fluorescent dye-containing resin particle solution is dispersed on the biotinylated plate with respect to the total surface area (T) of the fluorescent dye-containing resin particles is predetermined. It is possible to adjust the concentration of the fluorescent dye-containing resin particle solution so as to have the same dyeing performance by diluting the solution so as to have the above value. Therefore, even when pathological staining is performed using fluorescent dye-containing resin particle solutions of different lots, it is possible to obtain a staining result without variation.

The left figure shows the case where the SA-binding fluorescent dye-containing resin particle solution selected in Example 1 is used as a staining liquid, and the right figure shows the case where the SA-binding fluorescent dye solution selected in Comparative Example 1 is used as a staining liquid. Is an immunostaining image.

-Method for producing fluorescent dye-containing resin particle solution-
The production method of the fluorescent dye-containing resin particle solution of the present invention includes a predetermined “selection step” and / or “concentration adjustment step” described below. The predetermined fluorescent dye-containing resin particle solution used in the “concentration adjusting step” is typically selected in the “selecting step”, and therefore the production method of the fluorescent dye-containing resin particle solution of the present invention is “selected”. The embodiment may include a “density adjustment step” subsequent to the “step”. On the other hand, the predetermined fluorescent dye-containing resin particle solution used in the “concentration adjusting step” is selected by means different from the “selecting step” in the present invention, or the fluorescent dye-containing encapsulating method including the “concentration adjusting step” It may be selected by a “selection step” by an implementer different from the implementer of the production method of the resin particle solution (that is, discontinuous with the “concentration adjustment step”).

  Further, the fluorescent dye-containing resin particles used in the method for producing the fluorescent dye-containing resin particle solution of the present invention may be those obtained by the production process of the fluorescent dye-containing resin particles included as part of the production method. Alternatively, it may be obtained by a production process by an implementer different from the implementer of the production method (that is, discontinuous with the “density adjustment process”). The production process of the surface-modified fluorescent dye-containing resin particles is typically constituted by a synthesis process of the fluorescent dye-containing resin particles and a surface modification process thereof.

  Hereinafter, a synthesis step, a surface modification step, and a surface modification relating to a fluorescent dye-containing resin particle (which may be referred to as “surface-modified fluorescent dye-containing resin particle” in this specification) having a first affinity molecule bonded to the surface thereof. The steps related to the method for producing the fluorescent dye-containing resin particle solution of the present invention will be described in the order of the concentration adjusting step and the selecting step regarding the solution of the fluorescent dye-containing resin particles.

[Synthesis process of fluorescent dye-containing resin particles]
(Fluorescent dye-containing resin particles)
Specifically, the fluorescent dye-containing resin particles are nano-sized particles having a structure in which a relatively large amount of fluorescent dye is included (integrated) in a resin particle (matrix). The fluorescent dye is preferably confined in the particles by the molecular structure of the resin (for example, a three-dimensional network structure of a thermosetting resin such as a melamine resin). Alternatively, an electrostatic interaction (ionic bond) works between a functional group or part of the fluorescent dye and a functional group or part of the resin, or a covalent bond is formed between them. preferable. The fluorescent dye-encapsulating resin particles in which the fluorescent dye is firmly fixed to the resin particles in this way are noise because they are less likely to cause concentration quenching and are not easily discolored even if they are exposed to fluorescence for a certain period of time during fluorescence observation. It has extremely high brightness against eosin fluorescence and cell autofluorescence. In addition, the organic dye (xylene) used in the tissue staining penetration step is also prevented from eluting out of the fluorescent dye, making it an excellent fluorescent label.

  Examples of the resin forming the fluorescent dye-containing resin particles include styrene resin, acrylonitrile resin, furan resin, or similar resins, xylene resin, polylactic acid, polyglycidyl methacrylate, melamine resin, urea resin, benzoguanamine resin, and polyamide. , Phenol resins, polysaccharides, and the like that can stably encapsulate fluorescent dyes. By encapsulating the fluorescent dye in such particles, it is possible to produce fluorescent dye-containing resin particles that are less likely to be deteriorated by irradiation with excitation light (strong in light resistance) than the fluorescent dye alone. In particular, a styrene resin and a melamine resin are preferable as a base material for fluorescent dye-containing resin particles from the viewpoint of light resistance.

  The step of synthesizing the fluorescent dye-containing resin particles used for the production of the surface-modified fluorescent dye-containing resin particles can be performed using a known polymerization method similar to general fluorescent dye-containing resin particles.

  For example, when a thermosetting resin is used as a matrix, fluorescent dye-containing resin particles can be synthesized by using a fluorescent dye basically according to an emulsion polymerization method, and a polymerization method using a surfactant and a polymerization reaction accelerator. It is preferable to synthesize. When a thermoplastic resin is used as the matrix, the fluorescent dye-containing resin particles can be synthesized by using a fluorescent dye basically according to radical polymerization, ionic polymerization (anionic polymerization, etc.), for example, soap-free emulsification weight It is preferable to synthesize by a polymerization process according to a legal method.

  In the encapsulated fluorescent labeling resin particles obtained by such a synthesis method, most of the fluorescent dyes are desirably immobilized in a state where substantially all of the fluorescent dyes are encapsulated in the resin particles. It is not excluded that the fluorescent dye of the part is immobilized in a state of being bonded or adhered to the surface of the resin particle. In addition, there is no limitation on what kind of chemical or physical action the fluorescent dye is immobilized on the resin particles in the state where the fluorescent dye is encapsulated. According to the polymerization method exemplified above, prior to the synthesis step, a derivatization step for covalently bonding the resin raw material and the fluorescent dye in advance or actively introducing a charged substituent into the resin raw material is performed. Even if it is not provided, fluorescent dye-containing resin particles having excellent light emission intensity and light resistance can be obtained, but it is not excluded to use such a process in combination as desired.

(Fluorescent dye)
In this specification, the “fluorescent dye” generally refers to a substance that emits light in the process from an excited state to a ground state when excited by irradiation with external X-rays, ultraviolet rays, or visible light. Therefore, the “fluorescent dye” referred to in the present invention is not limited to the transition mode when returning from the excited state to the ground state, and is a substance that emits narrowly defined fluorescence that is light emission accompanying deactivation from the excited singlet. It may be a substance that emits phosphorescence, which is light emission accompanying deactivation from a triplet.

  In addition, the “fluorescent dye” referred to in the present invention is not limited by the emission lifetime after blocking the excitation light. Therefore, it may be a substance known as a phosphorescent substance such as zinc sulfide or strontium aluminate. As the fluorescent dye included in the fluorescent dye-containing resin particles used in the present invention, either an organic fluorescent dye or an inorganic fluorescent dye may be selected, but the particle diameter is small, and it can be efficiently included in the base resin. Since it is possible, it is more preferable to use an organic fluorescent dye.

(Organic fluorescent dye)
Examples of organic fluorescent dyes that can be used as fluorescent dyes include perylene dye molecules, fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (registered trademark, manufactured by Invitrogen), and BODIPY (registered trademark, Invitrogen). Manufactured) dye molecules, Cy (registered trademark, manufactured by GE Healthcare) dye molecules, coumarin dye molecules, NBD (registered trademark) dye molecules, eosin dye molecules, and pyrene dye molecules. .

  Specific examples of the perylene dye molecule include perylene diimide, and specific examples of the fluorescein dye molecule include fluorescein isothiocyanate, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy- Fluorescein, 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein, 6-carboxy-2 ′, 4,7,7′-tetrachlorofluorescein, 6-carboxy-4 ′, 5'-dichloro-2 ', 7'-dimethoxyfluorescein, naphthofluorescein, etc. are mentioned.

  Specific examples of the rhodamine dye molecule include 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine and the like.

  Specific examples of Alexa Fluor dye molecules include Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 532, Alexa Fluor 532, Alexa Fluor 532, Alexa Fluor 532, Alexa Fluor 514, Alexa Fluor 514 Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 7 and the like.

  Specific examples of the BODIPY dye molecules include BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, 3061 , BODIPY 650/665 and the like.

Specific examples of the Cy-based dye molecule include Cy5, Cy5.5, Cy7, and the like.
Specific examples of coumarin dye molecules include methoxycoumarin, eosin dye molecules as eosin, NBD (registered trademark) dye molecules as NBD, and pyrene dye molecules as pyrene. Is mentioned.

  These organic fluorescent dyes may be used alone or in combination. Further, as long as it can emit fluorescence, it may be used after being derivatized in consideration of reactivity with a compound serving as a base of fluorescent dye-containing resin particles. In particular, perylene diimide and fluorescein isothiocyanate are preferably used.

(Inorganic fluorescent dye)
Examples of inorganic fluorescent dyes that can be used as fluorescent dyes include quantum dots. Quantum dots include quantum dots containing II-VI group compounds, III-V group compounds, or IV group elements as components ("II-VI group quantum dots", "III-V group quantum dots", " Any of the “Group IV quantum dots” can be used. Specifically, particle dots such as CdSe exemplified in International Publication WO2012 / 133447 can be mentioned. These inorganic fluorescent dyes may be used alone or in combination.

  The average particle size of the fluorescent dye-containing resin particles is not particularly limited, but is usually 10 to 500 nm, preferably 30 to 200 nm. Further, the coefficient of variation indicating the variation in particle diameter is not particularly limited, but is usually 20% or less, preferably 5 to 15%.

  The average particle diameter of the produced fluorescent dye-containing resin particles can be measured by a method known in the art. For example, an electron micrograph is taken using a scanning electron microscope (SEM), and the fluorescent dye-containing resin particles are taken. The cross-sectional area of the resin particles can be measured, and the measured value can be measured as the diameter (area equivalent circle diameter) when the area is the corresponding circle. The average (average particle size) and variation coefficient of the particle size of the population of fluorescent dye-containing resin particles are measured as described above for a sufficient number (for example, 1000) of fluorescent dye-containing resin particles. After that, the average particle diameter is calculated as its arithmetic average, and the coefficient of variation is calculated by the formula: 100 × standard deviation of particle diameter / average particle diameter.

[Modification process of resin particles encapsulating fluorescent dye]
The modification step for producing the surface-modified fluorescent dye-containing resin particles from the fluorescent dye-containing resin particles can be performed using a known modification method similar to general surface-modified fluorescent dye-containing resin particles.

(First affinity molecule)
The first affinity molecule in the present invention is a molecule that modifies the surface of the fluorescent dye-containing resin particle, and is particularly limited as long as it is a molecule that can modify the fluorescent dye-containing resin particle and binds to the second affinity molecule described later. Is not to be done.

  The first group of first affinity molecules includes hapten affinity molecules such as biotin, avidin and variants thereof (streptavidin, neutravidin, etc.), and anti-hapten antibodies (anti-dinitrophenol antibody, anti-digoxigenin antibody, etc.) Is mentioned. Such a first group of first affinity molecules is an immunocomplex that includes, in immunostaining, a primary antibody or a secondary antibody to which a hapten is bound, and fluorescent dye-containing resin particles to which the hapten affinity molecule is bound. Used in embodiments that form the body. Alternatively, in FISH, it is used in an embodiment in which a complex including a nucleic acid molecule (probe) to which a hapten is bound and a fluorescent dye-containing resin particle to which a hapten affinity molecule is bound is formed. In view of such versatility and high staining efficiency, the first affinity molecule of the present invention is preferably one selected from the first group.

  The second group of first affinity molecules is an antibody that specifically recognizes a biomolecule intended for detection of immunostaining such as pathological diagnosis among antigen affinity molecules excluding anti-hapten antibodies, so-called primary. An antibody is mentioned. Such a second group of first affinity molecules is used in an embodiment of immunostaining to form an immune complex including an antigen and a fluorescent dye-containing resin particle to which a primary antibody is bound. In this case, a primary antibody corresponding to a biomolecule to be detected can be used. For example, when immunostaining for detecting HER2 is performed, an anti-HER2 antibody, preferably an anti-HER2 antibody is preferably used as the primary antibody. A HER2 monoclonal antibody may be used.

  As the third group of the first affinity molecules, among the antigen affinity molecules excluding the anti-hapten antibody, antibodies that specifically recognize the primary antibody described above as an antigen, so-called secondary antibodies can be mentioned. In such an embodiment, the third group of first affinity molecules forms an immunocomplex in immunostaining that includes an antigen, a primary antibody, and fluorescent dye-containing resin particles bound to the secondary antibody. Used. In this case, the secondary antibody may be any antibody that recognizes the primary antibody (IgG) as an antigen, and corresponds to the animal species that produced the primary antibody, such as anti-mouse, rabbit, cow, goat, sheep, and dog. Chicken IgG antibodies can be used. For example, when an anti-HER2 rabbit monoclonal antibody is used as the primary antibody, an anti-rabbit IgG antibody may be used as the secondary antibody.

  The bond between the fluorescent dye-containing resin particles and the first affinity molecule may be a direct bond or an indirect bond through which another molecule is interposed. The first affinity molecule to which the fluorescent dye-containing resin particles are bound can be produced using a general production method.

  The mode of directly binding the first affinity molecule to the fluorescent dye-containing resin particle is not particularly limited, and examples thereof include covalent bond, ionic bond, hydrogen bond, coordinate bond, physical adsorption and chemical adsorption. Can be carried out by a known method. From the viewpoint of bond stability, the functional group possessed by the resin (which may be inherently contained or introduced after synthesis of the resin by a predetermined method) and the functionality possessed by the first affinity molecule A bond having a strong binding force is preferred, such as a covalent bond formed by reacting with a group.

  As a mode in which the first affinity molecule is indirectly bound to the fluorescent dye-containing resin particles, a polymer having functional groups at both ends is used, and the functional groups of the resin and the functional groups of the resin are used. An embodiment in which the fluorescent dye-containing resin particles and the first affinity molecule are bonded via a spacer (linker) derived from the polymer by reacting with each of the functional groups possessed by the one affinity molecule. In particular, in such an embodiment, since the amount of the first affinity molecule bound per unit area on the surface of the fluorescent dye-containing resin particle is likely to vary, the effectiveness of applying the present invention is high.

  For example, using amidation by reaction of amine and carboxylic acid, sulfidation by reaction of maleimide and thiol, imination by reaction of aldehyde and amine, amination by reaction of epoxy and amine, etc. The fluorescent dye-containing resin particles can be covalently bonded (if necessary, with a spacer). By introducing a functional group involved in the above-described reaction into each of the first affinity molecule, both ends of the spacer used as necessary, and the surface of the fluorescent dye-containing resin particle, and reacting under a predetermined condition Can be linked by covalent bonds.

  The (high) molecule used as a spacer for indirectly binding the first affinity molecule to the fluorescent dye-encapsulating resin particles is not particularly limited, and an appropriate length generally used as a spacer. It is possible to select from (high) molecules having a thickness. Examples of the hydrophobic polymer spacer include polyamide, saturated hydrocarbon, hydrophobic polyamino acid, polystyrene, polymethacrylic acid ester, etc., and examples of the hydrophilic polymer spacer include polyethylene glycol, polypropylene glycol, Ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, polyvinyl pyrrolidone, polyvinyl methyl ether, polyvinyl methyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl meta Acrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethyl ester Loin, hydroxyethylcellulose, polyaspartamide, and synthetic polyamino acid. In addition, the spacer may be formed from one kind of substance, or may be formed from two or more kinds of substances.

  Among these spacers, a hydrophilic polymer is preferable from the viewpoint of suppressing nonspecific adsorption, and polyethylene glycol (PEG) is particularly preferable from the viewpoint of easily setting the chain length depending on the number of oxyethylene units. When using PEG, for example, you can purchase a commercially available product such as Thermo Scientific Co., Ltd., or set the number of repeating units of the chemical structure and request production from a reagent manufacturer. You can get the right one.

  The length of the spacer may be adjusted within an appropriate range according to the particle size of the fluorescent dye-containing resin particles to be bonded. A spacer having a single length may be used, or spacers having two or more different lengths may be used in combination. When a single spacer is used, the length of the spacer is preferably 1 to 100 nm. When using the spacer which has two or more types of length, it is preferable that the length of at least 1 type of those spacers is 1-100 nm, and the length of another spacer is 0.1-10 nm.

(Second affinity molecule)
In the present invention, the second affinity molecule is a molecule used for measuring fluorescence emitted as a signal value by reacting with the fluorescent dye-containing resin particles immobilized on the plate and bound to the first affinity molecule. Any molecule can be used as long as it corresponds to the first affinity molecule selected and specifically binds to it. Such a second affinity molecule is bound to the first affinity molecule by directly or indirectly binding to the biomolecule to be detected at the time of immunostaining. Used to mediate bonds between biomolecules and biomolecules.

  The first group of second affinity molecules, corresponding to the first group of first affinity molecules (hapten affinity molecules), is a molecule that specifically binds to the hapten affinity molecule, ie a hapten, for example biotin , Dinitrophenol, digoxigenin and the like.

  The second group of second affinity molecules, corresponding to the second group of first affinity molecules (primary antibodies), is a molecule that specifically binds to the primary antibody and is intended for detection of immunostaining. It may be a biomolecule or a secondary antibody having a primary antibody as an antigen. However, since the fluorochrome-encapsulating resin particles to which the primary antibody is bound are directly bound to the biomolecule to be detected at the time of immunostaining, the biomolecule to be detected is evaluated in order to evaluate the binding ability as accurately as possible. Is more suitably the second group of second affinity molecules.

  The third group of second affinity molecules corresponding to the third group of first affinity molecules (secondary antibody) is a molecule that specifically binds to the secondary antibody, ie, the antigen of the secondary antibody. Primary antibody.

  A commercially available plate may be used as the plate on which the second affinity molecule is immobilized, or the plate may be prepared using the plate and the second affinity molecule. A plate on which biotin is immobilized as the second affinity molecule is commercially available as a biotinylated plate (for example, a product of G-BIOSCIENCE). In addition, the plate on which the primary antibody or the secondary antibody is immobilized as the second affinity molecule is a commercially available microplate (for example, a product of Maxisorp, FluoroNunk Module & Plate), according to a predetermined protocol. It can be prepared by adding a solution containing a desired antibody to a well, allowing it to react and immobilizing.

[Selection process of fluorescent dye-containing resin particle solution]
In the selection step according to the present invention, the second affinity capable of specifically binding a certain amount of a solution containing the fluorescent dye-containing resin particles having the first affinity molecule bound to the surface at a constant concentration to the first affinity molecule. This is a step of selecting a signal value (S) that satisfies the following formula when a sex molecule is reacted with a plate immobilized at a constant density.

0.5 × A × B × (C / D) ≦ S ≦ 1.2 × A × B × (C / D)
In the formula, A reacted with the plate the same number of fluorescent dyes as the number contained in all fluorescent dye-containing resin particles contained in the fixed amount of solution to which the first affinity molecule was bound. Shows the signal value measured when
B is a fluorescent dye-encapsulating resin particle obtained by dividing the total number of first affinity molecules bound to the entire surface of all fluorescent dye-encapsulating resin particles contained in a fixed amount of the fixed solution by the number of fluorescent dye-encapsulating resin particles. Shows the number of first affinity molecules bound per
C represents the emission intensity per particle of the fluorescent dye-containing resin particles,
D represents the emission intensity of the number of fluorescent dyes contained in one fluorescent dye-containing resin particle.

Prior to the selection step, the fluorochrome-encapsulating resin particle solution prepared by preparing an arbitrary number of lots of fluorochrome-encapsulating resin particles bound to the first affinity molecule and suspending each in an appropriate solvent and adjusting to a predetermined concentration Prepare. Examples of the solvent used in this case include PBS, BSA-containing PBS, and H ₂ O. Particularly, from the viewpoint that nonspecific adsorption of proteins to the plate can be suppressed, about 1% BSA-containing PBS is preferable. It is.

Included in the provision of A above, “the number of fluorescent dyes encapsulated in all fluorescent dye-encapsulating resin particles contained in a fixed amount of the solution having the first affinity molecule bound thereto” ( a) can be calculated as follows. That is, a certain amount of fluorescent dye-containing resin particle solution (fluorescent dye-containing resin particle standard solution) having a constant concentration (for example, 0.01 nM, that is, 6.02 × 10 ⁹ particles / mL), and the same constant concentration (for example, 0.8. A fluorescent dye solution (fluorescent dye standard solution) of 01 nM, that is, 6.02 × 10 ⁹ molecules / mL) is prepared, and the fluorescence intensity at the emission peak wavelength of each solution is measured. Here, since the fluorescent dyes are accumulated at a high concentration in the fluorescent dye-containing resin particles, there is a possibility that the fluorescence emitted from the fluorescent dye-containing resin particles is weakened by a phenomenon called concentration quenching. Therefore, by multiplying "fluorescence intensity of fluorescent dye-containing resin particle standard solution" by "quantum yield of fluorescent dye alone / quantum yield of fluorescent dye-containing resin particles", the fluorescent dye corrected for the effect of concentration quenching The fluorescence intensity of the encapsulated resin particle standard solution can be calculated. Dividing this “fluorescence intensity of the fluorescent dye-embedded resin particle standard solution” by “fluorescence intensity of the fluorescent dye standard solution”, in other words, “fluorescence intensity of the fluorescent dye-embedded resin particle standard solution / fluorescence of the fluorescent dye standard solution” “Intensity” × “Quantum yield of fluorescent dye alone / Quantum yield of fluorescent dye-containing resin particles” is calculated to calculate “in a fixed amount of solution having the first affinity molecule bound thereto. The number of all fluorescent dye-containing resin particles contained is determined. The quantum yield can be measured by a known means, for example, using “Quantaurus-QY” (absolute PL quantum yield measuring device, Hamamatsu Photonics Co., Ltd.).

  If the value of a can be calculated, a solution containing that number of fluorescent dyes is prepared and reacted by, for example, applying it to a plate on which the second affinity molecule is immobilized. Signal value) (A) can be measured.

  The “total number of first affinity molecules bound to the entire surface of all fluorescent dye-containing resin particles contained in a fixed amount of a fixed solution” (b1) included in the provision of B above is the total surface It can be calculated by dividing the total weight of the first affinity molecule bound to, by the molecular weight of the first affinity molecule. The first affinity molecule typified by streptavidin is a protein, whereas the matrix of the fluorescent dye-encapsulating resin particles is a resin. Therefore, based on a protein quantification method such as the BCA method, a certain amount of fluorescent dye-encapsulated substance is used. The concentration of protein in the resin particle solution (all of which are bound to the surface of the fluorescent dye-containing resin particles) can be measured. The total weight of the protein can be calculated by multiplying the measured concentration by the volume of “a certain amount of solution”, and the total weight can be divided by the molecular weight of the protein (for example, the molecular weight of streptavidin is 52000). For example, the total number of the proteins (the first affinity molecules) used for the definition of B can be calculated.

  Also, the “number of fluorescent dye-containing resin particles” (b2) contained in a fixed amount of the solution, which is also included in the above-mentioned provision of B, is a particle counter (for example, “Liquid Particle Counter” manufactured by Lion Co., Ltd.) It is possible to measure with. By dividing the total number of first affinity molecules in the previous section by the number of fluorescent dye-containing resin particles, it is possible to calculate the number of first affinity molecules bound to one fluorescent dye-containing resin particle. .

If the values of b1 and b2 can be calculated, the number (average value) (B) of the first affinity molecules bound per fluorescent dye-containing resin particle can be obtained by b1 / b2.
The “emission intensity per particle of the fluorescent dye-containing resin particles” in C can be calculated as follows. That is, using a particle counter, a certain amount of fluorescent dye-containing resin particle solution (fluorescent dye-containing resin particle standard solution) having a constant concentration (for example, 0.01 nM, that is, 6.02 × 10 ⁹ particles / mL) is prepared. The fluorescence intensity at the emission peak wavelength is measured. By dividing the fluorescence intensity of the fluorescent dye-containing resin particle standard solution by the number of fluorescent dye-containing resin particles contained in the standard solution, the emission intensity per fluorescent dye-containing resin particle can be obtained.

The “emission intensity of the number of fluorescent dyes contained in one fluorescent dye-containing resin particle” in D can be calculated as follows. That is, a certain amount of a fluorescent dye solution (fluorescent dye standard) of a constant concentration (for example, 0.01 nM, that is, 6.02 × 10 ⁹ molecules / mL, and W × 10 ⁻¹⁴ g / mL if the fluorescent dye has a molecular weight of W). Solution) and the fluorescence intensity at the emission peak wavelength of the solution is measured. The fluorescence intensity of the fluorescent dye standard solution is expressed as “the number of molecules of the fluorescent dye contained in one fluorescent dye-containing resin particle (or its weight) / the number of molecules of the fluorescent dye in the fluorescent dye standard solution (or its weight)”. In addition, in order to correct the influence of concentration quenching as described above, by multiplying by “quantum yield of fluorescent dye alone / quantum yield of fluorescent dye-containing resin particles”, one fluorescent dye-containing resin particle The fluorescence intensity of a single fluorescent dye per unit number (or equivalent weight) contained in is obtained.

The method for measuring the emission intensity defined by C and D is not particularly limited, and the measurement may be performed under appropriate measurement conditions using a general fluorometer.
The fluorescent dye-containing resin particle solution whose signal value satisfies the above formula, in other words, the relative value of the signal value: S / {A × B × (C / D)} is in the range of 0.5 to 1.2. A certain fluorescent dye-containing resin particle solution is considered to have sufficient staining performance as a staining solution.

[Concentration adjustment process of fluorescent dye-containing resin particle solution]
The value (S / T) of the ratio of the signal value (S) measured in the above step to the total surface area (T) of the fluorescent dye-containing resin particles contained in the fluorescent dye-containing resin particle solution selected by the above method By diluting with an appropriate solvent so that the value becomes a predetermined value, it is possible to obtain a fluorescent dye-containing resin particle solution having a certain dyeing performance despite being derived from a plurality of lots. The solvent used here may be the same as the solvent used when preparing the solution of the fluorescent dye-containing resin particles bonded to the first affinity molecule prior to the selection step. For example, about 1% BSA-containing PBS is used. Particularly preferred.

  The total surface area of the fluorescent dye-containing resin particles defined by T is based on the average particle diameter of the fluorescent dye-containing resin particles, and the surface area per fluorescent dye-containing resin particle obtained by assuming that the particles are spheres. It can be calculated by multiplying the number of fluorescent dye-containing resin particles contained in the fixed amount of solution. Here, the method for measuring the average particle diameter is as described above.

  The use of the fluorescent dye-containing resin particles obtained as described above as a staining solution is not particularly limited. Here, as one embodiment of the invention, fluorescent immunostaining for quantifying a target antigen (membrane protein or the like) using fluorescent dye-containing resin particles will be described. However, for example, fluorescent dye-containing resin particles are used. Can be used for ISH (In Situ Hybridization), etc., for quantifying the target nucleic acid or ribonucleic acid molecule (gene).

-Use of fluorescent dye-containing resin particle solution-
The fluorescent dye-containing resin particle solution obtained by the production method of the present invention has the same use as a general fluorescent dye-containing resin particle solution, and can be used, for example, as a staining liquid for fluorescent immunostaining or FISH. . Specifically, for example, by preparing a preparation for a tissue section collected from a human body (patient) and observing a stained image reflecting the expression state of a specific protein or gene, the expression level and expression of the protein or gene Use as a staining solution for tissue immunostaining or FISH that can obtain information for pathological diagnosis based on the site and the like. Fluorescent immunostaining is not limited to tissue staining, but can also be applied to cell staining. The biomolecule to be detected is not particularly limited as long as a substance that specifically binds to it exists.

  Tissue immunostaining generally includes a specimen preparation process, a specimen pretreatment process, an immunostaining process, a specimen posttreatment process, and an observation process. Hereinafter, the immunostaining step, the specimen post-processing step, and the observation step will be described in this specification, but more detailed embodiments relating to these steps, and other embodiments and embodiments relating to fluorescent immunostaining (tissue immunostaining) in general. Furthermore, for embodiments relating to FISH, for example, International Publication WO2013 / 035703 Pamphlet, International Publication WO2013 / 147081 Pamphlet, International Publication WO2014 / 136776 Pamphlet and the like can be referred to.

[Immunostaining process]
In the immunostaining step, the procedure for performing fluorescent immunostaining for a specific antigen using the fluorescent dye-containing resin particles to which the first affinity molecule is bound is any one of the first to third methods described above as the first affinity molecule. It can be adjusted as appropriate depending on whether or not a group of molecules is used.

  A procedure in the case of using a first group of molecules, such as streptavidin, as the first affinity molecule will be described as an example. The immune complex to be formed in this case is (i) an antigen on a tissue section / a primary antibody in which biotin is bound as a second affinity molecule / a fluorescent dye-containing resin particle in which streptavidin is bound as a first affinity molecule. (Ii) Antigen on tissue section / primary antibody / secondary antibody bound with biotin as the second affinity molecule / fluorescent dye-encapsulated resin particle bound with streptavidin as the first affinity molecule, and the like. In the case of (i), the primary antibody bound to the second affinity molecule is reacted with the antigen on the tissue section, and then the fluorescent dye-containing resin particles bound to the first affinity molecule are reacted, Complexes can be formed and antigens can be fluorescently labeled. In the case of (ii), when the second affinity molecule is bound to the secondary antibody, the primary antibody is first reacted with the antigen on the tissue section, and then the secondary antibody to which the second affinity molecule is bound. By reacting and further reacting the fluorescent dye-containing resin particles to which the first affinity molecule is bound, the complex can be formed and the antigen can be fluorescently labeled. In addition, the preparation method of the fluorescent dye-containing resin particles to which the first affinity molecule is bound is as described above, and the primary antibody or the secondary antibody to which the second affinity molecule is bound is also according to a general method. Can be produced. Even when the second or third group of molecules is used as the first affinity molecule, it can be carried out according to the procedure described above.

  If necessary, a morphological observation staining step using a staining solution for morphological observation of cells or tissues may be performed before or after the immunostaining step or simultaneously with the immunostaining step. For example, when the morphology of a tissue specimen is observed, hematoxylin staining using hematoxylin (H staining) or hematoxylin-eosin staining using two dyes of hematoxylin and eosin (HE staining) is typically used. Morphological observation staining is not limited to these staining methods, and other morphological observation staining includes, for example, Papanicolaou staining (Pap staining) used for cytodiagnosis.

[Sample preparation process]
The sample post-treatment process includes a fixing process, a dehydration / penetration process, an encapsulating process, and a cleaning process as necessary.

  The immobilization treatment is a treatment for immobilizing the fluorescent dye-containing resin particles used for staining in the fluorescent immunostaining process on a biological material or an antibody bound to the biological material. By treatment with the immobilization treatment solution, the protein is crosslinked and denatured, and the fluorescent dye-encapsulated resin particles and the biological substance or the antibody bound to the biological substance are more firmly bound to each other chemically and physically, and stable. State and can. In the present invention, the fixation treatment can be performed by immersing the tissue section after the immunostaining step in the fixation treatment solution. For example, it can be performed by immersing the tissue section after the immunostaining step in a dilute paraformaldehyde aqueous solution for about several minutes to several hours.

  Examples of the fixing treatment solution include cross-linking agents such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, and methanol, and cell membrane permeants. Of these, formalin, paraformaldehyde, and glutaraldehyde are preferably used because they can be firmly fixed.

The dehydration / penetration treatment is performed by washing the stained tissue section with an aqueous washing solution such as PBS, followed by dehydration with EtOH (ethanol) and xylene replacement. Dehydration with EtOH involves substituting EtOH by sequentially immersing the tissue sections in EtOH with a reduced water content such as 50%, 30%, 10%, or 0%. Is done. Subsequently, the section substituted with EtOH is dipped in xylene to perform xylene substitution, and the section is penetrated.
The encapsulation process is performed by placing an oil-based encapsulant on the xylene-substituted section and placing a cover glass or the like.

[Observation process]
(I: Bright field observation process)
In the bright field observation process, when morphological observation staining is performed to identify the position of a biomolecule (antigen, etc.) to be stained in a cell or tissue, illumination light is applied to the stained tissue section, and the tissue This is a step of observing the coloring agent dye deposited on the section and acquiring an image relating to the shape of the cell membrane.

(Ii: fluorescence observation process)
In the fluorescence observation step, the fluorescent dye-containing resin particles in the immunostained tissue section are irradiated with excitation light to observe the fluorescence emitted by the included fluorescent dye, and the cells are stained in cells or tissues. This is a step of acquiring an image relating to the amount of biomolecules (such as the number of bright spots or emission intensity).

  The excitation light irradiation means is not particularly limited. For example, a stained tissue section may be irradiated with excitation light having an appropriate wavelength and output from a laser light source included in a fluorescence microscope using a filter that selectively transmits a predetermined wavelength as necessary.

  The excitation light is not particularly limited as long as the fluorescence emitted by the fluorescent dye can be identified in relation to the autofluorescence of the tissue section, but from the viewpoint of preventing the autofluorescence intensity from the tissue section from becoming too high. Excitation light having a wavelength of 700 nm is preferred. In addition, as the fluorescent substance constituting the fluorescent dye, a substance that emits fluorescence having a peak in the range of 480 nm or more, preferably in the range of 580 to 690 nm by the excitation light is used (thus, fluorescence having an emission wavelength in this region is measured). To do).

  In addition, in this step, biomolecule distribution information may be obtained from a (fluorescence) microscope tube so that rapid observation can be performed, or an image taken by a camera installed in the (fluorescence) microscope can be separately obtained. You may make it acquire by displaying on a display means (monitor etc.) and observing it. Depending on the fluorescent substance that constitutes the fluorescent dye used as the label, the biomolecule distribution information can be obtained from the image taken by the camera even if the biomolecule distribution information cannot be obtained sufficiently by visual observation from the microscope barrel. Sometimes it is possible to do.

  Obtaining the distribution information of the biomolecules includes, for example, measuring the number or density of biomolecules to be stained per cell based on the number of fluorescent bright spots or emission luminance. For excitation of the fluorescent dye, an excitation light source and a fluorescence detection optical filter corresponding to the absorption maximum wavelength and the fluorescence wavelength may be selected. For the measurement of the number of bright spots or emission luminance, it is preferable to use commercially available image analysis software (for example, all bright spot automatic measurement software “G-Count” (manufactured by Zeonstrom)), but the measuring means is particularly limited. Is not to be done.

  According to the following procedure, after preparing a fluorescent label (fluorescent dye-containing resin particle having a first affinity molecule bound to the surface, or a control object thereof) and preparing a fluorescent labeling solution (fluorescent label solution), A fluorescent labeling solution was tested using a plate on which the second affinity molecule was immobilized, and the performance as a staining solution for immunostaining was evaluated.

  The fluorescent dye-encapsulated resin particles used in each example are all “perylene diimide-encapsulated melamine resin particles”, and the particle size is determined by the ratio of the amount of resin in the raw material / the amount of dye in the synthesis process of the fluorescent dye-encapsulated resin particles. It is controlled by changing the amount of resin as constant (0.04).

  In Examples 1-4 and Comparative Examples 1-2, the molecule corresponding to the first affinity molecule is streptavidin (SA), and the molecule corresponding to the second affinity molecule is biotin. In immunostaining, a complex of antigen / primary antibody / biotin-conjugated secondary antibody / SA-conjugated fluorescent label is formed.

  In Examples 5 to 8 and Comparative Examples 3 to 4, the molecule corresponding to the first affinity molecule is a primary antibody (anti-HER2 rabbit monoclonal antibody), and the molecule corresponding to the second affinity molecule is a secondary antibody ( Anti-rabbit IgG antibody). In the immunostaining, a complex of an antigen / primary antibody-binding fluorescent label is formed.

  In Examples 9 to 12 and Comparative Examples 5 to 6, the molecule corresponding to the first affinity molecule is a secondary antibody (anti-rabbit IgG antibody), and the molecule corresponding to the second affinity molecule is a primary antibody (anti-antibody). HER2 rabbit antibody). In immunostaining, a complex of antigen / primary antibody / secondary antibody-binding fluorescent label is formed.

[1. Preparation of fluorescent label]
[Preparation Example 1] SA-bonded fluorescent dye-containing resin particles-1
(1-1) Step of synthesizing fluorescent dye-containing resin particles N, N′-bis (2,6-diisopropylphenyl) -1,6,7,12-tetraphenoxyperylene-3,4: 9,10-tetracarboxy Diimide was treated with concentrated sulfuric acid to produce a perylene diimide sulfonic acid derivative. This was converted to an acid chloride to obtain a perylene diimide sulfonic acid chloride derivative.

  After adding 17.3 mg of perylene diimide sulfonic acid chloride derivative to 22.5 mL of water, the mixture was heated on a hot stirrer at 70 ° C. for 20 minutes, and 0.78 g of melamine resin “Nicarac MX-035” (manufactured by Nippon Carbide Industries, Ltd.) The particle size was 150 nm), and the mixture was further heated and stirred for 5 minutes. After adding 100 μL of formic acid and heating and stirring at 60 ° C. for 20 minutes, the mixture was allowed to cool to room temperature. After cooling, the reaction mixture was placed in a centrifuge tube and placed in a centrifuge for 20 minutes at 12,000 rpm, and the supernatant was removed. The collected fluorescent dye-containing resin particles (perylene diimide-containing melamine resin particles) were washed with ethanol and water. The average particle size (average particle size of 100 particles counted in the SEM image) of the fluorescent dye-containing resin particles-1 was 200 nm.

(1-2) Modification Step with First Affinity Molecule Fluorescent dye-encapsulated resin particles-1 obtained in the synthesis step (1), 0.1 mg, are dispersed in 1.5 mL of EtOH (ethanol), and aminopropyltri 2 μL of methoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and reacted for 8 hours to carry out surface amination treatment.

  The surface-aminated fluorescent dye-encapsulating resin particles-1 were adjusted to 3 nM using PBS (phosphate buffered saline) containing EDTA (ethylenediaminetetraacetic acid) at a concentration of 2 mM. SM (PEG) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleimidopropionamide) -dodecaethylene glycol] ester) was mixed to a final concentration of 10 mM and reacted for 1 hour. The mixture was centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. Then, a PBS solution containing EDTA at a concentration of 2 mM was added, the precipitate was dispersed, and centrifuged again. By performing washing by the same procedure three times, fluorescent dye-containing resin particles-1 having a maleimide group added to the terminal were obtained.

  On the other hand, streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) can be bound to fluorescent dye-containing resin particles by performing thiol group addition treatment using succinimidyl S-acetylthioacetate (SATA) and then filtering through a gel filtration column. A streptavidin solution was obtained.

  1 mL of a PBS solution containing a fluorescent dye-containing resin particle-1 added with a maleimide group at a concentration of 13 nM, 1 mL of a PBS solution containing a streptavidin added with a thiol group at a concentration of 20 mM, and 2 mM of EDTA The mixture was mixed in a PBS solution containing a concentration and allowed to react for 1 hour. 10 mM mercaptoethanol was added to stop the reaction. After concentrating the obtained solution with a centrifugal filter, unreacted streptavidin and the like were removed using a gel filtration column for purification, and SA-binding fluorescent dye-encapsulating resin particles-1 were recovered. The recovered SA-binding fluorescent dye-containing resin particles-1 were stored in PBS containing 1% BSA.

[Production Example 2] SA-bonded fluorescent dye-containing resin particle-2
In the synthesis step (1) of fluorescent dye-containing resin particles, the amount of raw materials was changed to 14.4 mg of perylene diimide sulfonic acid chloride derivative, 0.65 g of melamine resin “Nicarac MX-035”, and 100 μL of formic acid, and otherwise In the same manner as in Example 1, fluorescent dye-containing resin particles-2 were obtained. The average particle size of the fluorescent dye-containing resin particles-2 was 150 nm. Subsequently, in the same manner as in the modification step (2) with the first affinity molecule of Example 1, SA-conjugated fluorescent dye-encapsulating resin particles-2 were obtained and stored.

[Production Example 3] SA-bonded fluorescent dye-containing resin particles-3
In the synthesis step (1) of fluorescent dye-containing resin particles, the amount of raw materials was changed to 12.8 mg of perylene diimide sulfonic acid chloride derivative, 0.58 g of melamine resin “Nicalac MX-035”, 100 μL of formic acid, and otherwise In the same manner as in Example 1, fluorescent dye-encapsulating resin particles-3 were obtained. The average particle size of the fluorescent dye-containing resin particles-3 was 100 nm. Subsequently, in the same manner as in the modification step (2) with the first affinity molecule in Example 1, SA-binding fluorescent dye-encapsulating resin particles-3 were obtained and stored.

[Production Example 4] SA-bonded fluorescent dye-containing resin particles-4
In the synthesis step (1) of fluorescent dye-containing resin particles, the amount of raw materials was changed to 9.3 mg of perylene diimide sulfonic acid chloride derivative, 0.42 g of melamine resin “Nicarac MX-035”, and 100 μL of formic acid, and the others were carried out In the same manner as in Example 1, fluorescent dye-containing resin particles -4 were obtained. The average particle size of the fluorescent dye-containing resin particles-4 was 50 nm. Subsequently, in the same manner as in the modification step (2) with the first affinity molecule in Example 1, SA-binding fluorescent dye-encapsulating resin particles -4 were obtained and stored.

[Preparation Example 5] Primary antibody-binding fluorescent dye-containing resin particles-1>
In the modification step (2) with the first affinity molecule, an anti-HER2 rabbit monoclonal antibody (4B5, manufactured by Ventana) is used as the primary antibody instead of streptavidin, and the primary antibody and maleimide group to which a thiol group has been added. In the same manner as in Production Example 1 except that the fluorescent dye-encapsulating resin particles to which was added were reacted, primary antibody-bound fluorescent dye-encapsulating resin particles-1 were obtained and stored.

[Production Example 6] Primary antibody-binding fluorescent dye-containing resin particle-2
In the modification step (2) with the first affinity molecule, an anti-HER2 rabbit monoclonal antibody (4B5) was used as the primary antibody instead of streptavidin, and the primary antibody to which the thiol group was added and the maleimide group were added. It changed so that fluorescent dye inclusion | inner_cover resin particle | grains might be made to react, and other than that was carried out similarly to the manufacture example 2, and obtained primary antibody coupling | bonding fluorescent dye inclusion resin particle-2, and preserve | saved.

[Preparation Example 7] Primary antibody-binding fluorescent dye-containing resin particle-3
In the modification step (2) with the first affinity molecule, an anti-HER2 rabbit monoclonal antibody (4B5) was used as the primary antibody instead of streptavidin, and the primary antibody to which the thiol group was added and the maleimide group were added. It changed so that fluorescent dye inclusion | inner_cover resin particle | grains might be made to react, and other than that was carried out similarly to the manufacture example 3, and obtained primary antibody coupling | bonding fluorescent dye inclusion | inner_resin particle | grains-3, and preserve | saved it.

[Preparation Example 8] Primary antibody-binding fluorescent dye-containing resin particle-4
In the modification step (2) with the first affinity molecule, an anti-HER2 rabbit monoclonal antibody (4B5) was used as the primary antibody instead of streptavidin, and the primary antibody to which the thiol group was added and the maleimide group were added. It changed so that fluorescent dye inclusion | inner_cover resin particle | grains might be made to react, and other than that was carried out similarly to preparation example 4, and obtained and preserve | saved primary antibody coupling | bonding fluorescent dye inclusion | inner_resin particle | grains-4.

[Preparation Example 9] Secondary antibody-binding fluorescent dye-containing resin particle-1
In the modification step (2) with the first affinity molecule, an anti-rabbit IgG antibody (LO-RG-1, manufactured by Funakoshi) is used as a secondary antibody instead of streptavidin, and the secondary antibody to which a thiol group is added And the fluorescent dye-encapsulating resin particles to which the maleimide group was added were reacted. Otherwise, the secondary antibody-binding fluorescent dye-encapsulating resin particles-2 were obtained and stored in the same manner as in Production Example 2.

[Production Example 10] Secondary antibody-binding fluorescent dye-containing resin particle-2
In the modification step (2) with the first affinity molecule, an anti-rabbit IgG antibody (LO-RG-1) is used as a secondary antibody instead of streptavidin, and the secondary antibody and maleimide group to which a thiol group has been added are A modification was made so that the added fluorescent dye-containing resin particles were reacted, and otherwise, in the same manner as in Production Example 2, secondary antibody-bound fluorescent dye-containing resin particles-2 were obtained and stored.

[Preparation Example 11] Secondary antibody-binding fluorescent dye-containing resin particle-3
In the modification step (2) with the first affinity molecule, an anti-rabbit IgG antibody (LO-RG-1) is used as a secondary antibody instead of streptavidin, and the secondary antibody and maleimide group to which a thiol group has been added are The added fluorescent dye-containing resin particles were changed to react, and other than that, the secondary antibody-binding fluorescent dye-containing resin particles-3 were obtained and stored in the same manner as in Production Example 3.

[Preparation Example 12] Secondary antibody-binding fluorescent dye-containing resin particle-4
In the modification step (2) with the first affinity molecule, an anti-rabbit IgG antibody (LO-RG-1) is used as a secondary antibody instead of streptavidin, and the secondary antibody and maleimide group to which a thiol group has been added are The added fluorescent dye-containing resin particles were changed to react, and other than that, secondary antibody-binding fluorescent dye-containing resin particles -4 were obtained and stored in the same manner as in Production Example 4.

[Comparative Preparation Example 1] SA-binding fluorescent dye N, N'-bis (2,6-diisopropylphenyl) -1,6,7,12-tetraphenoxyperylene-3,4: 9,10-tetracarboxydiimide and the like An amount of sulfonyl chloride was reacted to obtain a perylene diimide chloride derivative (reactive dye). Streptavidin (SA) was dissolved in 1 mL of 0.1 M borate buffer (pH 8.3) to 1 mg / mL. The reactive dye was dissolved in DMSO as 10 mg / mL. To the stirring SA solution, 100 μL of reactive dye was slowly added, stirred at room temperature, and reacted for 1 hour. By using Zeba Spin Desaling Columns and removing excess dye using PBS as a solvent, an SA-binding fluorescent dye having perylene diimide as a fluorescent dye was obtained.

[Comparative Production Example 2] SA-bonded resin particles When polystyrene particles are used as the resin particles, the SA-bonded resin particles can be produced by physical adsorption of polystyrene particles and SA. 1000 times equivalent amount of SA dissolved in 0.1 M borate buffer (pH 8.3) is added to commercially available COOH-terminated polystyrene particles (200 nm, Life Technologies) and stirred to have polystyrene particles as resin particles. SA-bonded resin particles were obtained.

[Comparative Production Example 3] Primary antibody-binding fluorescent dye The anti-HER2 rabbit monoclonal antibody (4B5) was used as the primary antibody instead of SA, and the reactive dye was changed to react with the primary antibody. Obtained a primary antibody-binding fluorescent dye in the same manner as in Comparative Example 1.

[Comparative Production Example 4] Primary antibody-binding resin particles An anti-HER2 rabbit monoclonal antibody (4B5) was used as the primary antibody instead of SA, and the primary antibody was modified to be adsorbed on polystyrene particles. In the same manner as in Production Example 2, primary antibody-bound resin particles were obtained.

[Comparative Preparation Example 5] Secondary antibody-binding fluorescent dye An anti-rabbit IgG antibody (LO-RG-1) was used as the secondary antibody instead of SA, and the reactive dye was changed to react with the secondary antibody. Other than that, a secondary antibody-binding fluorescent dye was obtained in the same manner as in Comparative Preparation Example 1.

[Comparative Production Example 6] Secondary antibody-binding resin particles An anti-rabbit IgG antibody (LO-RG-1) was used as the secondary antibody instead of SA, and the secondary antibody was modified to be adsorbed on polystyrene particles. Except that, secondary antibody-bound resin particles were obtained in the same manner as in Comparative Example 2.

[2. Test of fluorescent labeling solution]
[Examples 1 to 4 and Comparative Examples 1 to 2] Test of SA-binding fluorescent labeling solution SA-binding fluorescent dye-encapsulating resin particles of Preparation Examples 1 to 4 and SA-binding controls of Comparative Preparation Examples 1 to 2 (SA-binding fluorescent labeling) The solution (SA-binding fluorescent labeling solution) was prepared by the following procedure, and a test using a biotin plate was performed. Examples 1-4 and Comparative Examples 1-2 correspond to the tests on the SA-binding fluorescent labeling solutions of Production Examples 1-4 and Comparative Examples 1-2, respectively.

  A stock solution of the SA-conjugated fluorescent label was collected, the number of particles was measured with a particle counter “Liquid Particle Counter” (manufactured by Rion), and the concentration was adjusted to 0.05 nM. A commercially available biotinylated plate “Well-Coated Biotin, 96-well, Black” (manufactured by G-BIOSCIENCE) was washed 5 times with PBS. 100 μL each was added and reacted at 4 ° C. overnight. The plate after the reaction was washed with PBS, and then the signal value (S) of the fluorescence intensity was measured using a microplate reader “Plate Chameleon V” (manufactured by Hidex).

[Examples 5 to 8 and Comparative Examples 3 to 4] Test of primary antibody-binding fluorescent labeling solution Instead of the SA-binding fluorescent labels of Preparation Examples 1 to 4 and Comparative Preparation Examples 1 to 2, the preparation examples 5 to 8 The primary antibody-binding fluorescent dye-encapsulating resin particles and the primary antibody-binding control (primary antibody-binding fluorescent label) of Comparative Preparation Examples 3 to 4 were used, and the following procedure was used instead of the biotin plate 2 A primary antibody-binding fluorescent labeling solution was prepared in the same manner as in Examples 1 to 4 and Comparative Examples 1 and 2 except that a secondary antibody (anti-rabbit IgG antibody) plate was used. The fluorescence intensity signal value (S) was measured. Examples 5 to 8 and Comparative Examples 3 to 4 correspond to tests on the primary antibody-binding fluorescent labeling solutions of Production Examples 5 to 8 and Comparative Production Examples 3 to 4, respectively.

  100 μL per well of an anti-rabbit IgG antibody (LO-RG-1) solution prepared to a concentration of 20 μg / mL was added to a commercially available plate “Fluoronunk module plate F16” (manufactured by Maxisorp), and plate seal And left at 4 ° C. overnight. Subsequently, the plate was washed with 200 μL of PBS per well, 200 μL of blocking solution (PBS containing 1% BSA) was added per well, and the plate was sealed and stored at 4 ° C. In addition, the secondary antibody plate produced in this way can be used stably for about 6 months.

[Examples 9 to 12 and Comparative Examples 5 to 6] Test of secondary antibody-binding fluorescent labeling solution Instead of the SA-binding fluorescent labels of Preparation Examples 1 to 4 and Comparative Preparation Examples 1 to 2, the preparation examples 9 to 12 were used. The secondary antibody-binding fluorescent dye-containing resin particles and the secondary antibody-binding controls (secondary antibody-binding fluorescent labels) of Comparative Preparation Examples 5 to 6 were used, and the following procedure was used instead of the biotin plate 1 A secondary antibody-binding fluorescent labeling solution was prepared in the same procedure as in Examples 1 to 4 and Comparative Examples 1 and 2 except that a secondary antibody (anti-HER2 rabbit monoclonal antibody) plate) was used. The signal value (S) of the fluorescence intensity was measured by performing a test using. Examples 9 to 12 and Comparative Examples 5 to 6 correspond to the tests on the secondary antibody-binding fluorescent labeling solutions of Production Examples 9 to 12 and Comparative Production Examples 5 to 6, respectively.

  100 μL of anti-HER2 rabbit monoclonal antibody (4B5) solution prepared at a concentration of 20 μg / mL was added to a commercially available plate “Fluoronunk module plate F16” (manufactured by Maxisorp), and the plate was sealed. It was left overnight at 4 ° C. Subsequently, the plate was washed with 200 μL of PBS per well, 200 μL of blocking solution (PBS containing 1% BSA) was added per well, and the plate was sealed and stored at 4 ° C. The primary antibody plate prepared in this way can also be used stably for about 6 months.

  The signal value (S) obtained for each of Examples 1 to 12 and Comparative Examples 1 to 6 is combined with the signal value (A) of the fluorescent dye alone × one fluorescent dye-containing resin particle. Number of affinity molecules (B) × {Luminescence intensity per one fluorescent dye-containing resin particle (C) / (Luminescence intensity of a single fluorescent dye contained in one fluorescent dye-containing resin particle (D) Table 1 shows the relative values.

  For the signal value (A) of the fluorescent dye alone, first, a complex in which the first affinity molecule was bound to perylene diimide was prepared according to each Example and Comparative Example, and the complex was 0.05 nM. After preparing a solution containing the second affinity molecule and reacting with the plate on which the second affinity molecule was immobilized, the signal value of the fluorescence intensity was measured by the same method. Moreover, the quantum yield of perylene diimide and the quantum yield of the fluorescent dye-containing resin particles were measured by “Quantaurus-QY” (Hamamatsu Photonics Co., Ltd.). Using these measured values, the signal value (A) of the fluorescent dye alone was determined by the calculation method as described above.

  The number (B) of the first affinity molecules bound per fluorescent dye-containing resin particle is the total weight and the number of particles of all fluorescent dye-containing resin particles contained in a 0.05 nM, 100 μL solution. As well as the molecular weight of the first affinity molecule.

  The emission intensity (C) per fluorescent dye-containing resin particle was determined by the same method for the solution containing the fluorescent dye-containing resin particles used in each Example and Comparative Example at a concentration of 0.05 nM. It was determined by measuring the value.

  The emission intensity (D) of the number of fluorescent dyes contained in one fluorescent dye-containing resin particle was measured for a solution containing the complex used in (A) at a concentration of 0.05 nM. Intensity signal value, calculated value of the number of molecules of the fluorescent dye contained in the solution, calculated value of the number of molecules of the fluorescent dye contained in one fluorescent dye-containing resin particle, and fluorescence measured in the above (A) It was calculated from the quantum yield of the dye alone and the quantum yield of the fluorescent dye-containing resin particles.

[3. Staining of cultured cells]
<When SA-binding fluorescent labeling solution is used>
Using the SA-binding fluorescent dye-containing resin particle solution of Examples 1 to 4 and the SA-binding control solution (SA-binding fluorescent labeling solution) of Comparative Examples 1 and 2, the cultured cells are stained by the following procedure. Stained images were acquired.

  A cultured cell slide (HER2 score 3+; Pathology Laboratories) using SK-BR-3 cells was deparaffinized and then washed by immersing in water. The washed cultured cell slide was autoclaved at 121 ° C. for 5 minutes in a 10 mM citrate buffer (pH 6.0) to carry out antigen activation treatment. The cultured cell slide after the activation treatment was washed with PBS, followed by blocking treatment with 1% BSA-containing PBS for 1 hour in a wet box.

  After blocking treatment, an anti-HER2 rabbit monoclonal antibody (Bentana: 4B5) diluted to 0.05 nM with 1% BSA-containing PBS was reacted with the tissue section at room temperature for 2 hours. This was washed with PBS buffer, and reacted with a biotin-labeled anti-rabbit monoclonal antibody diluted to 2 μg / mL with 1% BSA-containing PBS at room temperature for 30 minutes.

Centrifugation of SA-binding fluorescent labeling solution, removal of supernatant, replacement with 0.6% α casein + 0.6% β casein + 3% BSA / TB 0.1% Tween20 0.015N NaN ₃ followed by filtration (Pore diameter 0.65 μm: manufactured by Millipore). Further, the cells were diluted to 0.2 nM with PBS containing 1% BSA and reacted with the above-mentioned reaction and neutral pH environment (pH 6.9 to 7.4) at room temperature for 3 hours, followed by culture. Cell slides were washed with PBS.

  After immunostaining, morphological observation staining was performed. The immunostained cultured cell slides were stained with Mayer's hematoxylin solution for 1 minute and stained with hematoxylin (H staining). The sections were then washed with running water at 45 ° C. for 3 minutes. Then, the operation which was immersed in pure ethanol for 5 minutes was performed 4 times, and washing | cleaning and dehydration were performed. Subsequently, the operation of immersing in xylene for 5 minutes was carried out 4 times to perform clearing. Finally, the tissue section was encapsulated using an encapsulant (“Enterlan New”, manufactured by Merck) to prepare a sample slide for observation.

  The immunostained and morphologically observed cultured cell slides were irradiated with predetermined excitation light to emit fluorescence, and observed and imaged with a fluorescence microscope (BX-53, Olympus). The excitation light was set to 575 to 600 nm by passing through an optical filter. Moreover, the range of the wavelength (nm) of the fluorescence to be observed was set to 612 to 682 nm by passing through an optical filter.

  Further, the number of bright spots and the emission luminance were measured using G-count, a bright spot measuring software manufactured by Zeon Angstrom. For bright spots, the average value is obtained by measuring the bright spots of 30 cells for each of the 8 spots in the slide, and the luminance is calculated by adding the fluorescence intensity of the entire field of view for each of the 8 spots. It was.

The excitation wavelength conditions for microscopic observation and imaging 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 imaging was arbitrarily set (for example, 4000 μsec) so that the luminance of the image is not saturated.
In addition, when observing an image by morphological observation staining, it was not necessary to irradiate the excitation light for exciting the fluorescent dye for immunostaining, and thus the observation was performed under the same observation conditions as the optical microscope.

<When primary antibody-binding fluorescent labeling solution is used>
Using the primary antibody-binding fluorescent dye-containing resin particle solution of Examples 5 to 8 and the primary antibody-binding control solution (primary antibody-binding fluorescent labeling solution) of Comparative Examples 3 to 4, the following procedure is used. The cultured cells were stained with to obtain a stained image.

  A cultured cell slide (HER2 score 3+; Pathology Laboratories) using SK-BR-3 cells was deparaffinized and then washed by immersing in water. The washed cultured cell slide was autoclaved at 121 ° C. for 5 minutes in a 10 mM citrate buffer (pH 6.0) to carry out antigen activation treatment. The cultured cell slide after the activation treatment was washed with PBS, followed by blocking treatment with 1% BSA-containing PBS for 1 hour in a wet box.

After centrifuging the primary antibody-bound fluorescent labeling solution, removing the supernatant, and replacing with 0.6% α casein + 0.6% β casein + 3% BSA / TB 0.1% Tween20 0.015N NaN ₃ , Filter processing (pore diameter 0.65 μm: manufactured by Millipore) was performed. Further, the cultured cell section diluted with PBS containing 1% BSA to 0.4 nM and subjected to the blocking reaction described above was reacted with neutral pH environment (pH 6.9 to 7.4) at room temperature all day and night. Cell slides were washed with PBS.

  After immunostaining, morphological observation staining was performed. The immunostained cultured cell slides were stained with Mayer's hematoxylin solution for 1 minute and stained with hematoxylin (H staining). The sections were then washed with running water at 45 ° C. for 3 minutes. Then, the operation which was immersed in pure ethanol for 5 minutes was performed 4 times, and washing | cleaning and dehydration were performed. Subsequently, the operation of immersing in xylene for 5 minutes was carried out 4 times to perform clearing. Finally, the tissue section was encapsulated using an encapsulant (“Enterlan New”, manufactured by Merck) to prepare a sample slide for observation.

<When a secondary antibody-binding fluorescent labeling solution is used>
Using the secondary antibody-binding fluorescent dye-encapsulating resin particles of Examples 9 to 12 and the secondary antibody-binding control substance (secondary antibody-binding fluorescent labeling solution) of Comparative Examples 5 to 6, cultured cells were treated by the following procedure. The stained image was obtained by staining.

  A cultured cell slide (HER2 score 3+; Pathology Laboratories) using SK-BR-3 cells was deparaffinized and then washed by immersing in water. The washed cultured cell slide was autoclaved at 121 ° C. for 5 minutes in a 10 mM citrate buffer (pH 6.0) to carry out antigen activation treatment. The cultured cell slide after the activation treatment was washed with PBS, followed by blocking treatment with 1% BSA-containing PBS for 1 hour in a wet box.

  After blocking treatment, an anti-HER2 rabbit monoclonal antibody (Bentana: 4B5) diluted to 0.05 nM with 1% BSA-containing PBS was reacted with the tissue section at room temperature for 2 hours. This was washed with a PBS buffer, and reacted with a biotin-labeled anti-rabbit monoclonal antibody that binds to the antibody diluted to 2 μg / mL with 1% BSA-containing PBS at room temperature for 30 minutes.

After centrifuging the secondary antibody-bound fluorescent labeling solution, removing the supernatant and replacing with 0.6% α casein + 0.6% β casein + 3% BSA / TB 0.1% Tween 20 0.015N NaN ₃ , Filter processing (pore diameter 0.65 μm: manufactured by Millipore) was performed. Furthermore, the cultured cell section diluted with PBS containing 1% BSA to 0.4 nM and subjected to the above-mentioned reaction was reacted with a neutral pH environment (pH 6.9 to 7.4) at room temperature all day and then cultured cells. Slides were washed with PBS.

  After immunostaining, morphological observation staining was performed. The immunostained cultured cell slides were stained with Mayer's hematoxylin solution for 1 minute and stained with hematoxylin (H staining). The sections were then washed with running water at 45 ° C. for 3 minutes. Then, the operation which was immersed in pure ethanol for 5 minutes was performed 4 times, and washing | cleaning and dehydration were performed. Subsequently, the operation of immersing in xylene for 5 minutes was carried out 4 times to perform clearing. Finally, the tissue section was encapsulated using an encapsulant (“Enterlan New”, manufactured by Merck) to prepare a sample slide for observation.

  The results of immunostaining for each of Examples 1 to 12 and Comparative Examples 1 to 6 are shown in Table 1. When the number of bright spots per SK-BR-3 cell is 10 or more, it is evaluated as ◯.

[4. Adjustment of staining solution concentration]
[Examples 13 to 16]
Immobilization of biotin at a constant density with respect to the total surface area (T) of fluorescent dye-containing resin particles (average particle diameters of 200 nm, 150 nm, 100 nm, and 50 nm, respectively) contained in the fluorescent dye-containing resin particle solutions of Examples 1 to 4 Diluted so that the ratio value (S / T) of the signal value (S) measured when the fluorescent dye-containing resin particle solution is sprayed on the prepared plate becomes a predetermined value (2 × 10 ¹² / nm ² ). As a result, a fluorescent dye-containing resin particle solution with an adjusted concentration was obtained.

[Comparative Examples 7 to 10]
By diluting the fluorescent dye-containing resin particle solutions of Examples 1 to 4 so that the concentration of the fluorescent dye-containing resin particles was 0.05 nM, fluorescent dye-containing resin particle solutions with adjusted concentrations were obtained.

[Comparative Examples 11-14]
For the fluorescent dye-containing resin particle solutions of Examples 1 to 4, first, the fluorescent dye-containing resin particle solution was diluted so that the concentration of the fluorescent dye-containing resin particles became a predetermined value (0.05 nM), and the fluorescence intensity was measured. By further diluting each diluted solution, a fluorescent dye-containing resin particle solution with adjusted brightness was obtained.

[5. Staining of cultured cells using a dye solution with adjusted concentration]
Using the staining solutions of Examples 13 to 16 and Comparative Examples 7 to 14, the above [3. The cells were stained in the same manner as in the method of [staining cultured cells] to obtain a stained image. For each example and comparative example, cell staining and staining image acquisition were repeated 9 times, and the number of bright spots after staining was measured.

  The results are shown in Table 2. When a solution having a constant S / T value was used as the staining solution, the variation in the number of bright spots was suppressed to ± 7% or less regardless of the average particle diameter of the fluorescent dye-containing resin particles (Examples 13 to 16). . On the other hand, when dyeing is performed using a solution in which the brightness of the fluorescent dye-containing resin particles is constant as a staining solution, the number of bright spots varies about ± 20%, and the concentration of the fluorescent dye-containing resin particles is made constant. It can be seen that even when the solution is used as a staining solution, a variation of about ± 15% occurs similarly (Comparative Examples 5 to 12).

Claims (8)

A fixed amount of a solution containing a fluorescent dye-containing resin particle having a first affinity molecule bound to the surface at a fixed concentration is fixed at a fixed density with a second affinity molecule that can specifically bind to the first affinity molecule. A method for producing a fluorescent dye-encapsulating resin particle solution, comprising a step of selecting a signal value (S) to be measured that satisfies the following formula when reacted with a plate that has been converted into a fluorescent plate.
0.5 × A × B × (C / D) ≦ S ≦ 1.2 × A × B × (C / D)
A: The same number of fluorescent dyes as those contained in all fluorescent dye-containing resin particles contained in a fixed amount of the fixed concentration solution bound with the first affinity molecule were reacted with the plate. Signal value measured at the time B: Divide the total number of first affinity molecules bound to the entire surface of all fluorescent dye-encapsulating resin particles contained in a fixed amount of the fixed solution by the number of fluorescent dye-encapsulating resin particles In addition, the number of first affinity molecules bonded per fluorescent dye-containing resin particle C: emission intensity per one fluorescent dye-containing resin particle D: included in one fluorescent dye-containing resin particle Luminescence intensity of a certain number of fluorescent dyes A fixed amount of a solution containing a fluorescent dye-containing resin particle having a first affinity molecule bound to the surface at a fixed concentration is fixed at a fixed density with a second affinity molecule that can specifically bind to the first affinity molecule. The total surface area of the fluorescent dye-containing resin particles contained in the fluorescent dye-containing resin particle solution with respect to the fluorescent dye-containing resin particle solution in which the measured signal value (S) satisfies the following formula when reacted with the plate The value (S / T) of the ratio of the signal value (S) measured when the fluorescent dye-containing resin particle solution is dispersed on a plate on which the second affinity molecule is fixed at a constant density with respect to (T). A method for producing a fluorescent dye-containing resin particle solution, the method comprising the step of diluting to a predetermined value and adjusting the concentration of the fluorescent dye-containing resin particle solution.
0.5 × A × B × (C / D) ≦ S ≦ 1.2 × A × B × (C / D)
A: The same number of fluorescent dyes as those contained in all fluorescent dye-containing resin particles contained in a fixed amount of the fixed concentration solution bound with the first affinity molecule were reacted with the plate. Signal value measured at the time B: Divide the total number of first affinity molecules bound to the entire surface of all fluorescent dye-encapsulating resin particles contained in a fixed amount of the fixed solution by the number of fluorescent dye-encapsulating resin particles In addition, the number of first affinity molecules bonded per fluorescent dye-containing resin particle C: emission intensity per one fluorescent dye-containing resin particle D: included in one fluorescent dye-containing resin particle Luminescence intensity of a certain number of fluorescent dyes   The production method according to claim 1 or 2, wherein the resin that is a base constituting the fluorescent dye-containing resin particles is a resin selected from the group consisting of a melamine resin and a styrene resin.   The production method according to any one of claims 1 to 3, wherein the fluorescent dye constituting the fluorescent dye-containing resin particles is an organic dye.   The production method according to any one of claims 1 to 4, wherein the fluorescent dye constituting the fluorescent dye-containing resin particles is selected from the group consisting of perylene diimide and fluorescein isothiocyanate.   The first affinity molecule bound to the fluorescent dye-containing resin particles is selected from the group consisting of biotin, avidin, streptavidin, neutravidin, anti-dinitrophenol antibody, and anti-digoxigenin antibody. The production method according to claim 1.   The production method according to any one of claims 1 to 6, wherein the first affinity molecule is bound to the fluorescent dye-containing resin particles via a polymer-derived spacer.   The method of claim 7, wherein the spacer is polyethylene glycol (PEG).