JP5993967B2 - Standard sample used for detection of intracellular biomolecule and method for detecting intracellular biomolecule - Google Patents

Description

  The present invention relates to a standard sample used when detecting biomolecules such as intracellular protein, nucleic acid, polysaccharide and the like. The present invention also relates to a method for detecting intracellular biomolecules by immunostaining, in situ hybridization, and the like using the standard sample.

  There are various methods for detecting biomolecules (proteins, DNA, RNA, polysaccharides, etc.) in cells and biological tissues and imaging their distribution. Detection and imaging using molecules that specifically bind to biomolecules, linking fluorescent proteins (tags) to expressed proteins, and using them as markers for detection and imaging, or enzyme activity And a method for detecting and imaging proteins. Among these, detection and imaging using molecules that specifically bind to biomolecules are the most widely performed, so-called immunostaining (immunohistochemistry, Immunohistochemistry, IHC) and in situ hybridization (ISH) using complementary binding between nucleic acids are common techniques.

  Using immunostaining (immunohistochemistry, IHC) or in situ hybridization (ISH), it is possible to microscopically observe the expression of specific proteins and genes related to specific diseases in tissues and cells collected from patients. These techniques are often used for pathological diagnosis.

For example, the HER2 gene is an oncogene with gene amplification and HER2 protein overexpression in 15 to 25% of human breast cancer cases, but breast cancer patients with HER2 gene amplification / HER2 protein overexpression have a poor prognosis. There are reports that it shows resistance to treatment with hormone therapy and CMF therapy. In addition, for breast cancer in which HER2 gene amplification / protein overexpression has been confirmed, significant improvement in survival time and survival rate has been observed by administration of Herceptin (registered trademark, generic name trastuzumab).
Therefore, for breast cancer patients, examination of HER2 gene / HER2 protein by immunostaining (immunohistochemistry, IHC) or in situ hybridization (ISH) is important in predicting prognosis and determining treatment policy. Become. However, this judgment is subjectively diagnosed by the pathologist by looking at the staining intensity, etc., and there is an opinion that about 20% of past HER2 tests were inaccurate, and the accuracy is still sufficient It's not a good thing.

  If the pathological diagnosis is mistaken, it leads to overlooking a serious disease, and the result of the pathological diagnosis greatly affects the treatment policy. Therefore, it is preferable to enhance the determination system by using not only a pathologist's subjective judgment but also an objective index. In order to increase the accuracy of determination by immunostaining (immunohistochemistry, IHC) or in situ hybridization (ISH), using a standard sample is one effective method. A standard sample is a sample containing a protein or nucleic acid to be detected at a predetermined concentration. By comparing the degree of staining of this standard sample with the degree of staining of a test sample, the target protein / gene It becomes possible to accurately determine the degree of expression.

For example, in JP-A-2-32260 (Patent Document 1), a plurality of depressions are provided in a block in which an agar solution is gelled, and gel pellets in which an antigen solution of a specific concentration is adsorbed in each depression. A standard sample characterized by is disclosed.
Also, WO91 / 05263 (Patent Document 2) discloses a standard sample obtained by embedding and slicing standard cells in gelatin or agar.
WO00 / 62064 (Patent Document 3) discloses that a split standard sample obtained by spotting a plurality of antigens having a specific concentration is attached to a slide glass for performing an immunostaining reaction.
Japanese Patent No. 4741486 (Patent Document 4) discloses a standard sample in which proteins are adsorbed on artificial beads.

  However, none of these standard samples was a standard sample having a size smaller than that of the cells. Further, in the beads described in Patent Document 4, the size and uniformity suitable for comparative observation cannot be controlled. Therefore, these standard samples have not been easy to compare with cells.

JP-A-2-32260 International publication WO91 / 05263 pamphlet International Publication WO00 / 62064 Pamphlet Japanese Patent No. 4741486

Conventional standard samples have not been easily observed in comparison with test samples containing cells.
Therefore, in view of the above-described conventional situation, the present invention facilitates comparison between a standard sample and a test sample containing cells, and a standard sample effective for accurately determining the presence of intracellular biomolecules and the standard. It is an object of the present invention to provide a method for detecting intracellular biomolecules using a sample.

  In order to solve the above-mentioned problems, the present inventors have intensively studied. As a result, hydrogel fine particles obtained by reacting a monomer comprising an alkene having a hydrophilic group with a crosslinking agent having two or more reactive sites are used. For example, it has been found that uniform microparticles can be produced by controlling the size to be smaller than that of cells, and if a biomolecule is attached thereto, a standard sample excellent in contrast with cells can be obtained. It came to complete.

  That is, this invention provides the following 1st invention which concerns on the standard sample used for the detection of an intracellular biomolecule, and the following 2nd invention which concerns on the detection method of an intracellular biomolecule.

The first invention provides a standard sample used for detection of intracellular biomolecules. This standard sample is adhered to the hydrogel fine particles obtained by reacting a plurality of types of compounds including a monomer comprising an alkene having a hydrophilic group and a crosslinking agent having two or more reaction sites. And biomolecules that have been prepared.
In the standard sample of the first invention, it is preferable to use a monomer having two or more carbon-carbon double bonds as the crosslinking agent having two or more reaction sites.
Further, in the above standard sample, as one of the plurality of kinds of compounds, a monomer composed of an alkene having a reactive group is used, and the reactive group and a biomolecule are bonded by a chemical reaction to form a hydrogel fine particle. It is preferable to attach a biomolecule to the surface.
Moreover, in said standard sample, it is preferable that hydrogel microparticles | fine-particles shall be 0.1-50 micrometers in average particle diameter.
In the standard sample, the hydrogel particles may be a plurality of types of hydrogel particles to which the biomolecules with different concentrations are attached.
As described above, when a plurality of types of hydrogel particles to which biomolecules with different concentrations are attached are used, the hydrogel particles may be hydrogel particles having different sizes depending on the concentration of the attached biomolecules.
Moreover, in the above-mentioned standard sample, when using a monomer comprising an alkene having a reactive group, p-nitrophenyl acrylate, N-acryloxy succinimide, or a combination thereof can be used. Then, acrylamide, N-isopropylacrylamide, methacrylic acid, or a combination thereof is used as the monomer composed of the alkene having the hydrophilic group, and methylenebisacrylamide is used as the crosslinking agent having two or more reaction sites. it can.
Furthermore, in the standard sample, the biomolecule can be a protein.

The second invention provides a method for detecting intracellular biomolecules. In this detection method, hydrogel fine particles obtained by reacting a plurality of types of compounds containing a monomer comprising an alkene having a hydrophilic group and a crosslinking agent having two or more reactive sites are used as biomolecules to be detected. A standard sample to which is attached is used. Then, as a first step, a test sample containing cells and the standard sample are mixed to create a specimen, and as a second step, the specimen prepared in the first step is stained, and a third step As described above, the degree of staining of the standard sample is compared with the degree of staining of cells of the test sample.
In the detection method of the second invention, in the first step, as the standard sample, a plurality of types of hydrogel fine particles to which biomolecules to be detected of different concentrations are attached are used. In the third step, The concentration of the biomolecule in the cell is measured by comparing the degree of staining of the plurality of types of hydrogel fine particles according to the concentration of the attached biomolecule and the degree of staining of the cell of the test sample. You can also.
Thus, when using multiple types of hydrogel microparticles | fine-particles, the said hydrogel microparticles | fine-particles shall differ in size according to the density | concentration of the said biomolecule which adhered.
In the detection method described above, in the first step, a hydrogel fine particle to which a biomolecule of a concentration detectable by staining is attached is used as a standard sample, and in the third step, staining of the standard sample is detected. If it is not possible, it can be determined that the detection experiment has failed.
In the detection method, in the first step, the standard sample has no biomolecule to be detected attached or a biomolecule different from the biomolecule to be detected attached. In the third step, if the negative control fine particles are sufficiently stained, it can be determined that the detection experiment has failed.
Furthermore, in the above detection method, the biomolecule may be a protein, and the staining may be immunostaining.

A standard sample used for detection of intracellular biomolecules of the first invention is obtained by reacting a plurality of types of compounds including a monomer comprising an alkene having a hydrophilic group and a crosslinking agent having two or more reaction sites. The hydrogel fine particles to be used are utilized. Therefore, the size of the hydrogel fine particles can be controlled to be smaller than that of the cell to be examined by appropriately selecting the type of monomer used as a raw material, the combination thereof, the blending amount, and the reaction conditions. Can be obtained. Therefore, the standard sample of the present invention in which biomolecules are attached to the hydrogel fine particles has an effect that it can be a standard sample having a size suitable for comparison with cells.
In addition, in the method for detecting intracellular biomolecules of the second invention, the standard sample of the present invention is mixed with a test sample (biological tissue or the like) containing cells to prepare a specimen, and this specimen is stained to give a living body. It detects molecules. Therefore, cells and standard samples can be compared in the same field of view in a microscope or on the same image, so the presence, localization, distribution, or concentration of biomolecules in cells can be determined more accurately. There is an effect that can be.

It is a figure which shows one Embodiment of the standard sample of this invention. It is a figure which shows other embodiment of the standard sample of this invention manufactured using the monomer which has a 2 or more carbon-carbon double bond. It is a figure which shows other embodiment of the standard sample of this invention manufactured using the monomer which consists of an alkene which has a reactive group. It is a figure which shows one Embodiment of the detection method of the intracellular biomolecule of this invention. It is the photograph replaced with drawing which shows the result of having observed the hydrogel fine particle by the electron microscope. It is the photograph replaced with drawing which shows the result of having performed immunostaining using the standard sample of this invention. It is a photograph replaced with drawing which shows the result of having performed immunostaining using the hydrogel microparticles | fine-particles from which the density | concentration of protein each differs.

The standard sample used for detection of intracellular biomolecules of the present invention is a hydrogel fine particle obtained by reacting a plurality of types of compounds including a monomer comprising an alkene having a hydrophilic group and a cross-linking agent having two or more reaction sites. And biomolecules are attached to the hydrogel fine particles.
Here, the biomolecule refers to a molecule existing in a living body such as protein, DNA, RNA, polysaccharide, lipid, and the like, and includes those in which these biomolecules are bound to each other like glycoprotein.

One embodiment of the standard sample of the present invention is shown in FIG.
A monomer composed of an alkene having a hydrophilic group is a compound having a carbon-carbon double bond (portion shown by a double line in FIG. 1) and a hydrophilic group. In FIG. 1, R represents a hydrogen atom or an arbitrary substituent. In this monomer, the carbon-carbon double bond portion of the alkene can be converted into a carbon radical by the radical initiator, and can be successively added to the carbon-carbon double bond portion of another monomer to form a polymer.

  Next, a cross-linking agent having two or more reactive sites has two or more reactive sites, for example, as shown in FIG. 1, the reactive sites are linked via an arbitrary substituent. It is a compound (in FIG. 1, R ′ represents an arbitrary substituent). Here, the reaction site means a site having a functional group capable of chemically reacting with a monomer comprising an alkene having a hydrophilic group or a polymer obtained by polymerizing the monomer. Through this cross-linking agent, two (or three or more) polymers are cross-linked. This crosslinking can be carried out by a one-step reaction in which a monomer composed of an alkene having a hydrophilic group and a crosslinking agent having two or more reaction sites are mixed and reacted. Alternatively, it is possible to carry out by a two-step reaction in which a monomer comprising an alkene having a hydrophilic group is first polymerized to form a polymer, and a polymer is further cross-linked by adding a cross-linking agent.

When a crosslinked polymer is formed in this way, polymer fine particles having a three-dimensional network structure are formed. And since this polymer microparticle has a hydrophilic group, it can become a hydrogel microparticle which included the aqueous solvent inside the network.
If a biomolecule is attached to the hydrogel fine particles, the standard sample of the present invention can be obtained.
The standard sample of the present invention can control the size of the hydrogel fine particles to be smaller than the cells to be examined by appropriately selecting the type of monomer used as a raw material, the combination thereof, the blending amount, and the reaction conditions. In addition, fine particles with high size uniformity can be obtained. Therefore, the standard sample of the present invention in which biomolecules are attached to the hydrogel fine particles has an effect of becoming a standard sample having a size suitable for comparison with cells.

  In the standard sample of the present invention, it is preferable to use a monomer having two or more carbon-carbon double bonds as a cross-linking agent having two or more reaction sites. This embodiment is shown in FIG. The cross-linking agent used here is a compound having two or more carbon-carbon double bonds and linked via an arbitrary substituent as shown in FIG. 2 (in FIG. 2, R ′ Represents an arbitrary substituent. This cross-linking agent can be added to the polymer at the carbon-carbon double bond moiety, so that two (or three or more) polymers will be cross-linked through this compound. The state in which the polymer is crosslinked is shown in the middle part of FIG. 2 (part of the polymer formed by polymerization).

  Since this crosslinking reaction is caused by a reaction between carbon-carbon double bonds, a monomer composed of an alkene having a hydrophilic group and a monomer having two or more carbon-carbon double bonds are mixed and polymerized. This can be done by a step reaction. Therefore, if a monomer having two or more carbon-carbon double bonds is used as a cross-linking agent, there is an effect that the reaction can be performed efficiently.

  The monomer composed of an alkene having a hydrophilic group used for producing the hydrogel fine particles of the standard sample of the present invention is an alkene having a carbon-carbon double bond in which at least one hydrogen is replaced by a hydrophilic group. Refers to the compound. Here, the alkene includes not only a straight-chain alkene but also a branched-chain alkene, and may have two or more double bonds, preferably a double bond. It is preferred for the polymerization to use one alkene. The hydrophilic group refers to a hydrophilic functional group or a substituent having a hydrophilic functional group. Here, the hydrophilic functional group includes an acidic functional group and a basic functional group, but is not limited thereto. For example, examples of the acidic functional group include a carboxylic acid group and a sulfonic acid group. Groups, phosphonic acid groups, sulfuric acid groups, formyl groups, thiocarboxylic acid groups, dithiocarboxylic acid groups, etc., and basic functional groups include amino groups, amide groups, hydroxyl groups, pyrrolidone groups, amine groups, etc. . Alkenes having a hydrophilic group may have other substituents in addition to the hydrophilic group.

Specific examples of the monomer comprising an alkene having a hydrophilic group include, but are not limited to, acrylamide, N-isopropylacrylamide, methacrylic acid, methacrylic acid-N, N-dimethylaminoethyl. N, N-dimethylaminopropylacrylamide, maleic acid, vinyl alcohol, allylamine, N, N-dimethylacrylamide, N- (2-hydroxylethyl) acrylamide, N-aminocarbonylmethacrylamide, N- (2-hydroxypropyl) Acrylamide, N- (acrylamidomethyl) pyrrolidone, 2-acryloylphenol, 3-hydroxybutyl methacrylate, methacrylamide, 2-aminoethyl methacrylate and the like can be used.
Among these, it is particularly preferable to use a monomer having acrylic acid or methacrylic acid as a base material in order to obtain hydrogel fine particles having a suitable size.

One kind of monomer composed of alkene having these hydrophilic groups may be used,
Two or more kinds may be used.
When two monomers, a monomer having a hydrophilic functional group capable of becoming a cation and a monomer having a hydrophilic functional group capable of becoming an anion, are used as a monomer comprising an alkene having a hydrophilic group, a cation The hydrogel fine particles can be made smaller by contraction due to an electric attractive force acting between the functional group of the anion and the functional group of the anion. On the other hand, when only one of the monomers having a functional group that can be a cation or an anion is used, the ions repel each other and swell to enlarge the hydrogel fine particles.
Thus, the size of the hydrogel fine particles can be controlled by appropriately selecting the monomer used for the polymerization.

  Examples of the cross-linking agent having two or more reactive sites used for producing the hydrogel fine particles of the standard sample of the present invention include, but are not limited to, two or more carbon-carbon double bonds. Or a monomer having two or more epoxy groups, a molecule having two or more hydroxyl groups, or the like can be used.

As described above, among these, it is preferable to use a monomer having two or more carbon-carbon double bonds as a crosslinking agent. The monomer having two or more carbon-carbon double bonds refers to a monomer having carbon-carbon double bonds in at least two positions of the molecule and causing an addition reaction at the carbon-carbon double bonds.
Examples of the monomer having two or more carbon-carbon double bonds include, but are not limited to, N, -N'-methylenebisacrylamide, N, -N'-methylenebismethacrylamide, N, -N'-ethylenebisacrylamide, N, -N'-ethylenebismethacrylamide, N, -N'-1,3-phenylenebisacrylamide, ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, divinylbenzene, trimethylolpropane tri Methacrylate or the like can be used.
As the monomer having two or more carbon-carbon double bonds, one type may be used, or two or more types may be used.

Examples of the molecule having two or more epoxy groups (crosslinking agent) include, but are not limited to, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, and the like. be able to.
These molecules having an epoxy group can react with a polymer having an amino group or a hydroxyl group as a hydrophilic group to crosslink the polymer. Therefore, it is preferable to produce hydrogel fine particles by a two-stage reaction in which a monomer comprising an alkene having a hydrophilic group is polymerized to form a polymer, and a polymer is further cross-linked by adding a cross-linking agent.

Moreover, as a molecule | numerator (crosslinking agent) which has two or more hydroxyl groups, although not necessarily limited, for example, diethylene glycol, triethylene glycol, pentaerythritol, etc. can be used.
These molecules having a hydroxyl group can react with a polymer having a carboxyl group as a hydrophilic group to crosslink the polymer. This reaction can be carried out in the presence of an oxo acid and an alcohol, and it is also preferable to produce hydrogel fine particles by a two-step reaction.

As shown in FIG. 1, the standard sample of the present invention is obtained by reacting a monomer comprising an alkene having a hydrophilic group with a cross-linking agent having two or more reaction sites to obtain hydrogel fine particles. It is obtained by attaching biomolecules to gel fine particles. Biomolecules can be attached to the hydrogel particles by combining the hydrophilic groups of the biomolecules and the functional groups of the hydrogel particles by chemical reaction, or the charges of the hydrophilic groups of the hydrogel particles and the charges of the biomolecules. There is a method of making it adhere by the Coulomb force between.
However, in order to easily and firmly attach biomolecules to the hydrogel fine particles, it is preferable to use a monomer composed of an alkene having a reactive group when producing the hydrogel fine particles.

One embodiment of the present invention using a monomer comprising an alkene having a reactive group is shown in FIG.
As shown in FIG. 3, in addition to a monomer comprising an alkene having a hydrophilic group and a monomer having two or more carbon double bonds, a monomer comprising an alkene having a reactive group is used for the polymerization.
When these plural types of monomers are polymerized, polymer fine particles having a three-dimensional network structure are formed, and hydrogel fine particles including an aqueous solvent inside the network are obtained by the hydrophilic group. And since this hydrogel fine particle has not only a hydrophilic group but a reactive group, as shown in FIG. 3, when mixed with a biomolecule, the reactive group reacts with a functional group of the biomolecule. Thus, the biomolecule can be firmly attached to the hydrogel fine particles by covalent bonding.
Further, by adjusting the blending amount of the monomer composed of the alkene having a reactive group, it is possible to control the amount of biomolecule attached to the hydrogel fine particles.

Here, the reactive group refers to a chemical group capable of forming a covalent bond by a chemical reaction with a biomolecule. For example, but not limited to, a p-nitrophenyloxy group, a succinimideoxycarbonyl group, Epoxy groups, formyl groups, maleimide groups, and the like can be used.
The monomer comprising an alkene having a reactive group is not limited to these. For example, p-nitrophenyl acrylate, N-acryloyloxysuccinimide, glycidyl methacrylate, N-formylacrylamide and the like can be used. .

  A monomer composed of an alkene having a reactive group may be added as a different kind of monomer from a monomer composed of an alkene having a hydrophilic group, or a alkene having both a hydrophilic group and a reactive group. You may use a kind of monomer as a monomer which has both functions.

  As a monomer used for the hydrogel fine particles, in addition to a monomer composed of an alkene having a hydrophilic group, a crosslinking agent having two or more reactive sites, and a monomer composed of an alkene having a reactive group, other monomers are used. You can also As such a monomer, a monomer having a carbon-carbon double bond capable of undergoing a polymerization reaction is preferable, for example, but not limited to, methyl methacrylate, vinyl acetate, ethylene, propylene, chloroethylene, Styrene, tetrafluoroethylene, acrylonitrile and the like can be used.

The blending ratio of the above monomers is preferably 20 to 99% by weight of a monomer composed of an alkene having a hydrophilic group, 1 to 20% of a crosslinking agent having two or more reactive sites, and an alkene having a reactive group. It is preferable to blend 0 to 70% by weight of the monomer and 0 to 30% of the other monomers.
The size of the hydrogel fine particles can be controlled by appropriately selecting the type of these monomers and adjusting the blending ratio.

In order to produce hydrogel fine particles by a one-step reaction, the monomers are polymerized by dissolving the above-mentioned multiple types of compounds in an aqueous solvent and stirring the mixture in a reaction vessel and further adding a polymerization initiator. Hydrogel fine particles can be obtained.
The reaction temperature during the polymerization is preferably 40 ° C. or higher and lower than the temperature at which the solvent volatilizes from the viewpoint of reaction efficiency, and in order to obtain uniform and substantially spherical fine particles, sufficient stirring may be performed. preferable.
Moreover, since oxygen and water contained in the solvent may inhibit the polymerization initiator, it is preferable to sufficiently replace the solvent with an inert gas.

As the solvent used in the reaction, a polar solvent in which the monomer used in the reaction dissolves can be used. For example, alcohols such as ethanol, isopropanol and t-butyl alcohol, and dioxane are not limited thereto. , Tetrahydrofuran and the like can be used.
In addition, the size of the hydrogel fine particles can be controlled by selecting a solvent. For example, the size of the hydrogel fine particles can be reduced by using a solvent having a smaller polarity.

  The polymerization initiator used for the polymerization of the monomer is not particularly limited as long as it generates radicals. For example, azobisisobutyronitrile, benzoyl peroxide, lauroyl peroxide, cumene hydroperoxide, di-tert-butyl A peroxide or the like can be used.

In attaching the biomolecule to the hydrogel fine particles, it is preferable to perform polymerization using a monomer composed of an alkene having a reactive group as described above to obtain hydrogel fine particles having a reactive group on the surface.
In order to attach biomolecules to the hydrogel fine particles, first, the hydrogel fine particles are washed and dispersed in a solvent, and then a predetermined amount of biomolecule is added, or a biomolecule of a known concentration is added. By adding the solution, hydrogel fine particles and biomolecules can be reacted.

  After the reaction between the hydrogel fine particles and the biomolecule, it is preferable to perform a treatment to block the unreacted reactive group from being bonded to other biomolecules. If unreacted reactive groups remain on the surface of the hydrogel microparticles, for example, when biomolecules are detected by antibodies, the antibodies adhere to the hydrogel microparticles, which decreases the accuracy as a standard sample. Because it ends up. In order to block a reactive group, it is not necessarily limited to these, For example, it is preferable to use the low molecular weight compound for a block which has a functional group which can react with a reactive group. Unwashed reactive groups can be blocked by washing the hydrogel fine particles to which biomolecules are bound, and then dispersing them in a solvent and adding a blocking low-molecular compound and reacting for a sufficient time.

  As a low molecular weight compound for blocking, for example, when a p-nitrophenyloxy group is used as a reactive group, a low molecular weight compound having an amino group that reacts with the reactive group, such as ethylenediamine, ethylamine, 1,3-propane Diamine, propylamine and the like can be used.

  In order to attach biomolecules to the hydrogel fine particles, there is a method in which a monomer composed of an alkene having a reactive group is not used. For example, but not limited to these, the functional group of the biomolecule and the functional group of the hydrogel fine particles can be combined under specific conditions using a catalyst or the like. For example, an amino group of a biomolecule and a carboxyl group of a hydrogel fine particle can be amide-bonded using a coupling catalyst (for example, 4-dimethylaminopyridine). Alternatively, a hydrophobic monomer can be added to polymerize the hydrogel fine particles, and the hydrophobic portion formed by this monomer and the hydrophobic portion of the biomolecule can be adsorbed by hydrophobic interaction. In addition, the biomolecule can be adsorbed to the hydrogel fine particles by the Coulomb force between the charge of the hydrophilic group of the hydrogel fine particles and the charge of the biomolecule. Further, in the process of polymerizing the hydrogel fine particles, the biomolecules can be attached to the hydrogel fine particles by incorporating the biomolecules into the network structure of the hydrogel fine particles.

  Examples of biomolecules attached to the hydrogel particles include, but are not limited to, purification from biological samples, production of recombinant proteins using genetic engineering, chemical synthesis, and any method using a nucleic acid synthesizer It can be obtained by synthesis of nucleic acid of sequence.

  After producing the hydrogel fine particles as described above, the hydrogel fine particles may be further modified by a chemical reaction. For example, but not limited thereto, fluorescent dyes, fluorescent proteins, hydrophilic groups, reactive groups, and the like can be modified.

  In addition to the hydrogel fine particles to which biomolecules are attached to the standard sample of the present invention, for example, but not limited to, solvents, pH adjusters, buffers, enzyme inhibitors, osmotic pressure adjusters, etc. Can also be included.

The size of the standard sample of the present invention can be controlled by adjusting the type and blending amount of monomers used in the synthesis.
When a specific biomolecule is detected, when a cell is stained, a part of the cell is stained. Therefore, it is preferable that the standard sample to be compared with this is a size smaller than the cell. When a standard sample having a size larger than the cells is used, the standard sample occupies most of the visual field when the cells are compared with the standard sample and observed with a microscope.
As a preferable size of the standard sample of the present invention, the average particle diameter is preferably 0.1 to 50 μm, and more preferably 0.5 to 3 μm.
The “average particle size” used in the present invention refers to Otsuka Electronics FPAR-1000F, and a gel fine particle dispersion diluted to about 0.01% is placed in a cell, and at room temperature by dynamic light scattering / photon correlation spectroscopy. It refers to the measured average particle size (scattering intensity average particle size).

In addition, the shape of the standard sample may be any shape, but it is preferable to use substantially spherical fine particles because they can be easily distinguished from distorted elliptical cells.
In the present invention, if the reaction is carried out while stirring in a reaction vessel, the shape becomes spherical. However, if the reaction is carried out in a special environment such as a fine tube, other shapes can be used.

In another embodiment of the present invention, a set of a plurality of types of hydrogel particles having different concentrations of biomolecules attached to the hydrogel particles can be used as the standard sample.
When the sets of hydrogel fine particles having different biomolecule concentrations are stained, the intensity of staining differs depending on the concentration of each biomolecule. Therefore, by comparing the degree of staining in the cells of the test sample with the degree of stepwise staining of the standard sample of the present invention, the degree of expression of biomolecules in the cells of the test sample can be increased with higher accuracy. There is an effect that it can be determined.
Furthermore, the concentration of staining is digitized, and the relationship between the staining intensity of the standard sample and the concentration of the biomolecule is plotted, and a calibration curve is created to measure the concentration of the biomolecule in the cells of the test sample. It is also possible.

  In order to obtain a set of a plurality of types of hydrogel particles with different concentrations of biomolecules attached to the hydrogel particles, for example, but not limited to, a solution containing a high concentration of biomolecules, a medium concentration It can be obtained by preparing solutions of a plurality of types of biomolecules having different concentrations, such as a solution containing a biomolecule and a solution containing a low-concentration biomolecule, and treating the hydrogel fine particles with these solutions, respectively. Moreover, if the compounding quantity of the monomer which consists of an alkene which has a reactive group is changed in steps, and it superposes | polymerizes, the set of hydrogel microparticles | fine-particles from which the number of the reactive groups of a surface each differs will be obtained. Therefore, by treating the set of hydrogel particles with a biomolecule solution having the same concentration, a set of hydrogel particles having different concentrations of attached biomolecules can be obtained.

  As another method for producing a set of a plurality of types of hydrogel fine particles having different concentrations of biomolecules attached to the hydrogel fine particles, the following method can also be used. First, hydrogel fine particles having reactive groups are manufactured, and before binding biomolecules to the reactive groups, a block treatment is performed using a low molecular weight compound for blocking to reduce the amount of active reactive groups. deep. By changing the amount of the low molecular weight compound for blocking or the block treatment time stepwise, a plurality of types of hydrogel fine particles having different amounts of active reactive groups can be obtained. If biomolecules are reacted with these plural types of hydrogel fine particles, a set of hydrogel fine particles having different concentrations of bound biomolecules can be obtained.

In another embodiment of the present invention, the hydrogel microparticles having different sizes depending on the concentration of the biomolecules in a set of a plurality of types of hydrogel microparticles having different biomolecule concentrations attached to the hydrogel microparticles. The set can be a standard sample.
In order to change the size of the hydrogel fine particles, as described above, for example, by changing the type of monomer used for polymerization, hydrogel fine particles having different sizes can be obtained. In this case, since it can be understood that the concentration is different if the size of the fine particles is different, there is an effect that the comparative observation becomes easy.

Next, a method for detecting intracellular biomolecules using the standard sample of the present invention will be described.
One embodiment of the detection method of the present invention is shown in FIG.
In the method for detecting intracellular biomolecules of the present invention, hydrogel fine particles obtained by reacting a plurality of types of compounds containing a monomer comprising an alkene having a hydrophilic group and a crosslinking agent having two or more reaction sites are used. Use.
As a first step of the present invention, first, a standard sample in which a biomolecule to be detected is attached to a hydrogel fine particle and a test sample containing cells (for example, a biological tissue sampled from a patient) As shown in the upper part of FIG.
Next, as a second step, the created specimen is stained. The state where the specimen is stained is shown in the middle of FIG.
Then, as a third step, the degree of staining of the standard sample is compared with the degree of staining of the cells of the test sample. As shown in the middle part of FIG. 4, when the cells are sufficiently intensely stained as compared with the standard sample, it can be determined that the biomolecule to be detected is sufficiently expressed.

  According to the detection method of the present invention, as shown in FIG. 4, the cells and the standard sample can be compared in the same field of view in the microscope observation or on the same image. It is possible to determine the presence, localization, distribution, or concentration of biomolecules in.

Hereinafter, the method for detecting intracellular biomolecules of the present invention will be described in detail step by step.
The first step of the detection method of the present invention is a process for preparing a specimen by mixing the standard sample of the present invention with a test sample containing cells. Here, the test sample containing cells may be any sample as long as it contains cells, and can be obtained as a part of human or non-human animal or plant organs or biological tissues or cells. Moreover, you may use microorganisms and a cultured cell as a test sample containing a cell. Furthermore, as long as observation is possible, you may include the fragment of these cells.

As the test sample, a chemically or physically fixed sample may be used, or a non-fixed sample may be used. Examples of chemical fixation include, but are not limited to, for example, formaldehyde, glutaraldehyde, dimethyl suberimidate dihydrochloride, osmium tetroxide, and the like infiltrated into living tissue or cells. There is a method of immobilization by cross-linking the polymer substance inside. In addition, the physical fixation is not limited to these, but there is, for example, a method of immobilizing a macromolecular substance in a living tissue or cell by boiling or thermal coagulation by microwave irradiation or freezing. In addition, a combination of these may be used, for example, a sample to be tested may be rapidly frozen and replaced and fixed in formalin / acetone. In addition, the test sample may be subjected to a treatment such as antigen activation.
In addition, a test sample that has not been subjected to the above-described chemical / physical fixation or antigen activation may be used. Alternatively, the above-described fixation or treatment may be performed after mixing a test sample containing cells and the standard sample of the present invention.

Usually, when a part of an organ or a living tissue is used as a test sample, it is difficult to make a microscopic observation with the same size, so a slice with a certain thickness is prepared. The section preparation method is not limited to these. For example, a section (specimen) of a standard sample and a test sample is embedded in a frozen tissue embedding agent such as an OCT compound and then frozen. A method of slicing with a freezing microtome, a method of slicing a test sample as it is with a vibrating microtome without freezing, or a method of slicing with a microtome after the test sample is embedded in paraffin and cooled and hardened and so on.
When cultured cells or microorganisms are used as test samples, they are embedded in an embedding agent to form blocks, sliced with embedding agents, or fixed to a glass substrate. However, it is also possible to proceed to the next step of staining by mixing it with the standard sample as it is.

The mixing of the standard sample of the present invention with a test sample containing cells means that both are combined and at least a part of both is mixed. For example, a specimen can be prepared by mixing a test sample (such as a biological tissue) containing cells and a standard sample in gelatin. This specimen can be further embedded with OCT compound, paraffin or the like to prepare a section.
Moreover, both the test sample and the standard sample can be added to an embedding agent such as OCT compound or paraffin to prepare a specimen in which both are mixed in the embedding agent.
Furthermore, a capsule in which the standard sample of the present invention is embedded in a gel or the like is prepared, and the sample is embedded in a block prepared by embedding the test sample in OCT compound or paraffin, thereby mixing both samples. May be produced.

Next, the second step of the detection method of the present invention is a step of staining a test sample mixed with the standard sample of the present invention.
Here, the staining in the present invention does not mean “staining” in a narrow sense to attach a dye, but enables imaging of the presence, localization, distribution or concentration of a biomolecule in a cell. Say. For example, but not limited thereto, a label such as a dye, a fluorescent dye, a colloidal gold particle, a fluorescent protein, a radioactive isotope, or a labeling enzyme is directly or indirectly specific to the biomolecule to be detected. It can be stained by binding. Then, biomolecules can be imaged (visualized or imaged) by coloring with these labels, luminescence, radiation, or coloring with a dye generated by an enzyme reaction.

  Methods for binding these labels to specific biomolecules are not limited to these. For example, a method using an antibody that specifically binds to a biomolecule to be detected, complementary to a target nucleic acid. A method of hybridizing nucleic acids having specific sequences, a method of using low molecular weight compounds and toxins having affinity for specific biomolecules, and a method of linking a label such as a fluorescent protein to a protein expressed using genetic engineering techniques And a method using a lectin which is a protein having an ability to bind to a sugar chain.

As a method of staining a protein as a biomolecule, immunostaining (immunohistochemistry, Immunohistochemistry, IHC) using an antibody specific for the protein is often used. This immunostaining is a technique in which two pillars are an enzyme antibody method using an antibody in which an enzyme such as peroxidase or alkaline phosphatase is linked as a label, and a fluorescent antibody method using an antibody in which a fluorescent dye is linked as a label. is there.
Of these two methods, the enzyme antibody method is suitable for detecting biomolecules in tissue sections for pathological diagnosis and the like, while the fluorescent antibody method is suitable for detecting biomolecules in cultured cells.

The enzyme antibody method suitable for protein detection in pathological diagnosis will be described in detail. Examples of the enzyme used as a label include, but are not limited to, peroxidase (horseradish peroxidase: HRP), alkali Enzymes such as phosphatase (alkaline phosphatase: AP) and glucose oxidase (β-D galactosidase: GAL) can be used.
As a substrate for these enzymes, for example, 3,3'-diaminobenzidine (DAB) can be used to develop a brown color, or 3-amino-9-ethylcarbazole (AEC) can be used as a red color for peroxidase. Can develop color.

  Enzyme antibody methods are roughly classified into direct methods and indirect methods. In the direct method, an enzyme is directly linked to an antibody and reacted with an antigen. On the other hand, the indirect method is a method in which the antibody (primary antibody) against the antigen to be detected is not enzyme-labeled but the antibody against the antibody (secondary antibody) is enzyme-labeled and detected. The indirect method has a larger number of reactions than the direct method, but if the primary antibody is prepared from the same species of animal, all kinds of immunostaining can be performed with one type of secondary antibody, and the sensitivity is also excellent. Yes. Therefore, indirect methods are often used in enzyme antibody methods.

  In performing immunostaining, an immunostaining method using non-contact stirring by a variable electric field disclosed in JP 2012-13598 A can be used. In this method, a solution containing an antibody is dropped onto a tissue where protein is detected by immunostaining, and the droplet is stirred by a varying electric field to promote an antigen-antibody reaction. Specifically, an electrode is disposed below the slide glass on which the tissue is placed, an electrode is disposed on the side facing the electrode so as not to contact the droplet, and a varying electric field is generated between the two electrodes. generate. This fluctuating electric field is a superposition of a rectangular wave and a frequency signal of 10 to 300 Hz, and the droplet is stirred by this fluctuating electric field, and the antigen-antibody reaction is promoted. If this rapid immunostaining method using a varying electric field is used, immunostaining that normally requires a time of about 70 to 200 minutes can be rapidly performed in about 15 to 30 minutes. Since the standard sample of the present invention facilitates determination in immunostaining and enables accurate determination in a short time, by using this rapid immunostaining method in combination with the standard sample of the present invention, rapid Pathological diagnosis becomes possible. For example, a rapid pathological diagnosis during surgery in which a surgical strategy is determined by rapidly pathologically diagnosing tissue collected from a patient during surgery.

As another method for staining proteins as biomolecules, there is a method in which a tag such as a fluorescent protein is linked to a protein to be detected using a genetic engineering technique. As the fluorescent protein, EGFP (green fluorescence), DsRed (red fluorescence), ECFP (blue fluorescence), or the like can be used.
Other methods include, but are not limited to, for example, a method of staining a glycoprotein sugar chain with a lectin, a method of staining a protein called F-actin with a toxin called phalloidin, and a protein binding to a nucleic acid. And a method of staining with a nucleic acid labeled with a fluorescent dye (Southwestern).

As a method for staining a nucleic acid as a biomolecule, in situ hybridization (ISH) in which a nucleic acid having a sequence complementary to a target nucleic acid is hybridized is generally used.
In ISH, a nucleic acid having a sequence complementary to a nucleic acid to be detected is synthesized, and a probe labeled with the complementary nucleic acid is prepared. Then, a probe is added to the test sample as a section, and the nucleic acid to be detected and the probe are hybridized. When observed with a fluorescence microscope after hybridization, the nucleic acid to be detected can be detected. In the method of detecting cell DNA using ISH, the copy number of a specific gene in the cell chromosome can be examined.

In performing in situ hybridization, hybridization using non-contact stirring described in JP 2010-119388 A can be used. This method promotes a hybridization reaction by agitating a droplet in which a nucleic acid and its complementary nucleic acid are mixed in a non-contact manner by a varying electric field in which a frequency signal of 0.1 to 800 Hz is superimposed on a square wave. is there. In this case as well, rapid pathological diagnosis is possible by using the standard sample of the present invention and the hybridization using this non-contact stirring together.
Other methods for staining nucleic acids as biomolecules include, but are not limited to, for example, staining using antibodies specific for nucleic acids.

Methods for staining lipids as biomolecules are not limited to these, but include, for example, methods that use antibodies specific for lipids, and substances that bind to lipids such as Filipin and Lysenin. There is a method of using.
The method for staining a polysaccharide as a biomolecule is not limited to these methods. For example, a method using an antibody specific for a polysaccharide, a staining using Alcian Blue that binds to a polysaccharide, There is a method using a lectin.

The third step of the present invention is a step of comparing the degree of staining of the standard sample of the present invention with the degree of staining of the biomolecule present in the cells of the test sample.
The degree of these dyeings can be grasped by the intensity of color development, light emission, radiation and the like. The intensity of color development / light emission can be visually recognized with a microscope or the like, and the intensity of radiation can be imaged using a film that is sensitive to radiation.

  Conventionally, the degree of staining has been subjectively determined by researchers and pathologists by visually recognizing the intensity of coloring. However, the degree of staining is affected by, for example, the contact time with the antibody, the antibody concentration, the color development time, and the like, so that even if the amount of the same biomolecule is used, a certain result is not always obtained. Also, if the staining reaction fails even though the biomolecule to be detected exists, it is determined that there is no biomolecule to be detected because it is not stained without being aware of the failure. There is also a risk of being.

On the other hand, when the standard sample of the present invention is used, the amount of biomolecules contained in the standard sample can be set to a predetermined value at the time of production, and this standard sample is subjected to exactly the same staining treatment as the test sample. Therefore, referring to the intensity of staining of this standard sample, there is an effect that the amount of biomolecule in the cell can be grasped more accurately. In addition, if the staining reaction fails, even if the standard sample contains a sufficiently detectable amount of biomolecules, the standard sample will not be sufficiently stained. You can see what has failed. That is, the standard sample of the present invention also functions as a positive control.
The standard sample of the present invention can be mixed with a test sample containing cells, and imaging by staining or the like can be performed in a state where both are mixed. Therefore, the test sample and the standard sample can be compared in the same field of view in the microscopic observation or on the same image, so that the presence, localization, distribution or concentration of the biomolecule in the cell can be more accurately determined. There is an effect that it can be determined.

  For example, the determination of HER2 protein overexpression by current immunostaining is in four stages of 0, 1+, 2+, 3+, and it is observed that “over 30% of cancer cells have strong complete cell membrane positive staining”. In this case, it was determined to be 3+ and treated as Herceptin (registered trademark) (HER2 Examination Guidebook Third Edition, September 2009, created by Trastuzumab Pathology Committee). However, since “a strong complete positive staining of cell membrane” is subjectively determined by a pathologist, the determination accuracy is not sufficient. By using the standard sample of the present invention, it is possible to grasp the degree of staining of “strong complete cell membrane positive staining” by the degree of staining of the standard sample, and the degree of staining of cancer cells by microscopic observation By comparing in the same field of view, it is possible to improve the accuracy of determination.

In another embodiment of the present invention, the concentration of biomolecules attached to the hydrogel fine particles is a predetermined concentration, a plurality of different concentrations are prepared, and the set of hydegel fine particles is mixed with the test sample. You can also.
In this embodiment, since the hydrogel fine particles have different concentrations of biomolecules contained therein, the staining intensity is stepwise. Therefore, it is also possible to grasp the approximate concentration of biomolecules at the site stained in the cells of the test sample by comparing the degree of stepwise staining of a set of a plurality of hydrogel fine particles.
In addition, a calibration curve can be created by plotting the staining intensity quantified by image processing on the X axis and the concentration of biomolecules contained in the fine particles having the staining intensity on the Y axis.
And the density | concentration of the biomolecule in the location dye | stained in the cell can be measured by comparing the intracellular dyeing | staining of a test sample with the said calibration curve.

In another embodiment of the present invention, a plurality of hydrogel fine particles having different concentrations of biomolecules contained therein can be produced, and the size of the hydrogel fine particles can be varied depending on the concentration.
In this case, since it can be understood that the concentration is different if the size of the fine particles is different, there is an effect that observation with a microscope or the like becomes easy.

  In another embodiment of the present invention, negative control fine particles containing a biomolecule different from the biomolecule to be detected may be included in the standard sample of the present invention. Since the negative control fine particles are molecules different from the biomolecule to be detected, they should not be stained originally. However, when this negative control fine particle is stained, it is a result of a non-specific staining reaction or the like, so that it is possible to grasp that the detection experiment has failed. Here, it is more preferable that the negative control fine particles have a size different from that of other fine particles because the negative control fine particles can be easily identified.

(Manufacture of hydrogel fine particles)
A solution of acrylamide 10 mmol (0.71 g), methacrylic acid 10 mmol (0.82 g), nitrophenyl acrylate 10 mmol (2.70 g), and methylenebisacrylamide 5 mmol (0.77 g) in 40 mL ethanol. Prepared. This solution was poured into a flask equipped with a stirring bar equipped with a blade, a condenser, a nitrogen introduction tube, and a thermometer. The four-necked flask was immersed in a constant temperature bath at 60 ° C., and nitrogen gas was bubbled through the solution for 30 minutes, and then a solution of 1.5 g of the polymerization initiator azobisisobutyronitrile in ethanol was injected. The reaction system became cloudy in about 30 minutes, and the formation of particles was confirmed. The reaction was stopped in 6 hours, and the produced hydrogel fine particles were collected. Using Otsuka Electronics' FPAR-1000F, a hydrogel fine particle dispersion diluted to about 0.01% was placed in a cell and measured by dynamic light scattering / photon correlation spectroscopy at room temperature. The average particle size was 1.2 μm. It was.

(Adsorption of biomolecules)
The hydrogel fine particles were subjected to centrifugal purification, and then an aqueous dispersion containing 0.1% hydrogel fine particles was obtained. The dispersion was divided into four, and 0, 0.001, 0.005, and 0.010% of cytokeratin 19, which is a protein specifically expressed in lung cancer, was added to each. The mixture was lightly stirred at room temperature for 30 minutes to attach cytokeratin 19 to the reactive group (p-nitrophenyloxy group) of the hydrogel fine particles. Next, 1 mmol (0.061 g) of ethanolamine was added to each and stirred again for 30 minutes to block unreacted reactive groups. Thereafter, the mixture was centrifuged at 1000 rpm for 10 minutes, and the supernatant was exchanged with pure water. Storage was performed in a refrigerator.

(Electron microscope observation)
The hydrogel fine particles obtained as described above were observed with a scanning electron microscope. The electron microscope image is shown in FIG. As shown in the electron microscope image, the standard sample of the present invention was monodisperse hydrogel fine particles having a substantially spherical shape and uniform size.

(Preparation of sections for staining)
Lung cancer cultured cells H358 were fixed with a fixative (50 mM Dimethyl suberimidate dihydrochloride), then suspended in 6% gelatin and solidified (cell block). Hydrogel fine particles with cytokeratin 19 attached in this gelatin (positive control) and hydrogel fine particles without cytokeratin 19 attached (negative control) were suspended as a standard sample at the same time.
The cell block containing the standard sample was embedded in OCT compound (Sakura Finetek Japan CO., Ltd., Japan) and frozen with cold acetone at −80 ° C. using Histo-Tek Pino (Sakura Finetek Japan). This specimen was sliced into a thickness of 5 μm with a cryostat (CM1900 Leica, Wetzlar, Germany) and pasted on a slide glass. The obtained sections were fixed with acetone for 2 minutes.

(Immunostaining)
The section prepared above was washed 3 times with phosphate buffer solution (5 seconds each), and anti-cytokeratin antibody cocktail (AE1 / AE3; Progen Biotechnik GmbH, Heiderberg) was added 1: 200 times (5 μg / ml) And allowed to react for 1 hour. Then, after washing 3 times with a phosphate buffer, it was reacted with a secondary antibody (EnVison + System / HRP Mouse (DAB +) Dako) for 30 minutes. Then, it was washed 3 times with a phosphate buffer and developed with DAB (3,3 ′ diaminobenzidine substrate). The nucleus was stained with a hematoxylin solution, covered with a cover glass, and observed with a microscope. The result is shown in FIG.
FIG. 6A shows the negative control and lung cancer cell H358 tissues stained with anti-cytokeratin antibody cocktail AE1 / AE3. FIG. 6B shows the positive control and lung cancer cell H358 tissues stained with anti-cytokeratin antibody cocktail AE1 / AE3. In FIG. 6A, lung cancer cell H358 (indicated by the arrow B) is stained, but the negative control is not stained and is not shown in the photograph. On the other hand, in FIG. 6B, lung cancer cells H358 (indicated by the arrow B) and positive control (indicated by the arrow A) are both stained. As shown in FIG. 6, lung cancer cells are stained with anti-cytokeratin antibody cocktail AE1 / AE3. On the other hand, the positive control is stained with the anti-cytokeratin antibody cocktail AE1 / AE3, while the negative control is not stained.

(Manufacture of hydrogel fine particles)
Next, a set of hydrogel microparticles with different bound protein concentrations was produced.
First, as a monomer solution for synthesizing hydrogel particles, acrylamide 10 mmol (0.710 g), methacrylic acid 10 mmol (0.860 g), nitrophenyl acrylate 4 mmol (0.756 g), methylenebisacrylamide 4 mmol (0.616 g) was prepared by dissolving in 40 mL of ethanol: methanol = 4: 1 in the flask. The mixture was heated to 60 ° C. with stirring, and 0.5 g of a polymerization initiator azobisisobutyronitrile dissolved in ethanol was injected. The polymerization was carried out by maintaining the temperature at 60 ° C. and stirring uniformly at 200 rpm with a winged stirring rod for 6 hours. At this time, in order to accelerate polymerization, the air in the flask was replaced with nitrogen, the solution was bubbled for about 20 minutes, and the gas that might be present in the liquid was replaced with nitrogen. The reaction system became cloudy in about 15 minutes, and the formation of particles was confirmed. The reaction was stopped in 6 hours, and the generated hydrogel fine particles were collected by centrifugation.

(blocking)
The hydrogel particles created this time have a reactive group that binds to the protein, so that the target protein can be bound. And the hydrogel fine particle which the target protein couple | bonded can be dye | stained by the immuno-staining by the enzyme antibody method using the antibody with respect to a target protein. On the other hand, in the tissue where the target protein is actually expressed, the intensity of immunostaining changes depending on the amount of the protein. Similarly, the staining intensity can be changed by increasing or decreasing the amount of protein bound to the hydrogel fine particles. However, the primary antibody used in immunostaining binds to the reactive group that has not reacted with the protein, and the staining is demonstrated, so that the hydrogel fine particles not bound to the protein may be stained, or only a small amount of protein may be stained. There was a concern that hydrogel fine particles that were not bound (which should originally have low staining intensity) would be strongly stained. Thus, glycine (1 mol / L) was added to the hydrogel fine particle dispersion and allowed to react for 1 hour to block unreactive groups to which no protein was bound. As a result, nonspecific reaction was suppressed.

(Protein binding)
Hydrogel fine particles produced by polymerization at 60 ° C. and 200 rpm for 6 hours were removed by centrifugation, and the alcohol as a solvent was replaced with distilled water. This hydrogel fine particle dispersion was divided into three, and 0, 0.001, and 0.010% of Cytokeratin 19 protein was added. For blocking as described above, 1 mol glycine solution was added to each and allowed to stand at 4 ° C. for 1 hour to react.

(Implementation of antigen-antibody reaction)
Three types of hydrogel fine particle dispersions after the reaction were removed by centrifugation, dispersed in sodium alginate, and solidified by adding CaCl ₂ . The sample was embedded in an OCT compound (Sakura Finetek Japan CO., Ltd., Japan) and frozen with -80 ° C. cold acetone using Histo-Tek Pino (Sakura Finetek Japan). This specimen was sliced into a thickness of 4 μm with a cryostat (CM1900 Leica, Wetzlar, Germany) and pasted on a slide glass. The obtained sections were fixed with acetone for 2 minutes.
After the section prepared above was washed 3 times with phosphate buffer solution (5 seconds each), anti-cytokeratin antibody cocktail (AE1 / AE3; Progen Biotechnik GmbH, Heiderberg) added 1: 200 times concentration (5μg / ml) And allowed to react for 10 minutes. Then, after washing 3 times with a phosphate buffer, it was reacted with a secondary antibody (EnVison + System / HRP Mouse (DAB +) Dako) for 20 minutes. Then, it was washed 3 times with a phosphate buffer and developed with DAB (3,3 ′ diaminobenzidine substrate). The result is shown in FIG. FIGS. 7A to 7C show the results of immunostaining of hydrogel fine particles to which proteins were bound under the conditions where Cytokeratin 19 was added at 0%, 0.001% and 0.010%, respectively. As apparent from FIG. 7, the staining intensity increased as the amount of protein added increased.

Claims (14)

In a standard sample used for detection of intracellular biomolecules,
Hydrogel fine particles obtained by reacting a plurality of types of compounds including a monomer comprising an alkene having a hydrophilic group and a crosslinking agent having two or more reactive sites;
A standard sample comprising: a biomolecule to be detected attached to the hydrogel fine particles.   The standard sample according to claim 1, wherein the crosslinking agent having two or more reactive sites is a monomer having two or more carbon-carbon double bonds. The plurality of types of compounds include a monomer composed of an alkene having a reactive group, and the biomolecule to be detected is attached to the hydrogel fine particles by chemically reacting with and binding to the reactive group. The standard sample according to claim 1 or 2, wherein   The standard sample according to any one of claims 1 to 3, wherein the hydrogel fine particles are hydrogel fine particles having an average particle diameter of 0.1 to 50 µm. The standard sample according to any one of claims 1 to 4, wherein the hydrogel fine particles are a plurality of types of hydrogel fine particles to which biomolecules to be detected of the same type and different concentrations are attached. The standard sample according to claim 5, wherein the hydrogel fine particles are hydrogel fine particles having different sizes depending on the concentration of the biomolecule to be detected attached thereto.   As the monomer comprising an alkene having a hydrophilic group, acrylamide, N-isopropylacrylamide, methacrylic acid or a combination thereof is used, and as the cross-linking agent having two or more reactive sites, methylene bisacrylamide is used. The standard sample according to any one of claims 3 to 5, wherein p-nitrophenyl acrylate, N-acryloyloxysuccinimide, or a combination thereof is used as a monomer comprising an alkene having a group. The standard sample according to claim 1, wherein the biomolecule to be detected is a protein. In a method for detecting intracellular biomolecules,
A standard sample in which biomolecules to be detected are attached to hydrogel fine particles obtained by reacting a plurality of types of compounds including a monomer comprising an alkene having a hydrophilic group and a crosslinking agent having two or more reaction sites. The
A first step of preparing a specimen by mixing with a test sample containing cells;
A second step of staining the specimen;
A detection method comprising a third step of comparing the degree of staining of the standard sample and the degree of staining of cells of the test sample. The first step uses, as the standard sample, a plurality of types of hydrogel fine particles to which biomolecules to be detected of the same type and different concentrations are attached,
The third step comprises comparing the degree of staining of the plurality of types of hydrogel fine particles according to the concentration of attached biomolecules with the degree of staining of the cells of the test sample. The detection method according to claim 9, wherein the concentration of the biomolecule is measured.   The detection method according to claim 10, wherein the hydrogel fine particles are hydrogel fine particles having different sizes depending on the concentration of attached biomolecules. In the first step, a hydrogel fine particle to which a biomolecule at a concentration detectable by staining is attached is used as a standard sample,
The detection method according to claim 9, wherein in the third step, if the staining of the standard sample cannot be detected, it is determined that the detection experiment has failed. In the first step, the standard sample is negative consisting of the hydrogel fine particles to which the biomolecule to be detected is not attached or a biomolecule different from the biomolecule to be detected is attached. Further comprising control particulates,
The detection method according to claim 9, wherein in the third step, when the negative control fine particles are sufficiently stained, it is determined that the detection experiment has failed. The detection method according to claim 9, wherein the biomolecule to be detected is a protein, and the staining is immunostaining.