Control slide glass for pathological examination and its manufacturing method

Abstract

The purpose of the present disclosure is to provide a control slide glass comprising a control substance in a form capable of suppressing outflow of a dye produced by staining of the control substance for the purpose of accuracy management in pathological examination.　One aspect of the present embodiment is a control slide glass for pathological examination, said control slide glass being characterized in that a control spot which includes a control substance for the pathological examination and a dye-trapping material is provided on a slide glass.

Description

[Technical field]

[0001] The present invention relates to a control slide glass for pathological examination and a method for producing the same. [Background technology]

[0002] A pathological examination is a method of diagnosing the presence and type of disease by preparing a specimen by attaching cells and tissues collected from a patient to a slide glass and observing them, for example, at a microscope level. In a pathological examination, a staining method for the components of cells and tissues is essential to distinguish disease from colorless cells and tissues.

[0003] One example of the staining technique used in pathological testing is immunostaining. In immunostaining, antibody reagents are used to detect specific antigens expressed in cells and tissues. This is an extremely important staining technique in pathological testing because it allows visualization of the location of the antigen being tested.

[0004] Immunostaining is generally used for definitive diagnosis, so it is necessary to determine whether appropriate staining results are obtained. In order to ensure the reliability of staining results, it is strongly recommended in the medical field to perform contrast staining of a control that is known to be stained in addition to staining of the specimen.

[0005] As a slide glass capable of staining a specimen and a control substance simultaneously, a slide glass incorporating a control substance has been proposed (see, for example, Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2016 / 143717 [Patent Document 2] International Publication No. 2018 / 181483 Summary of the Invention [Problem to be solved by the invention]

[0007] In the slide glasses incorporating the control substance proposed in Patent Documents 1 and 2, the control substance is fixed to the outermost surface, so there was a risk that the dye generated from the control substance would leak into the specimen during immunostaining, or that the stability of the control substance would be low.

[0008] Therefore, one of the objectives of the present disclosure is to provide a control slide glass comprising a control substance in a form capable of suppressing the outflow of dye generated by staining the control substance, for the purpose of quality control in pathological testing.

[0009] Another object of the present disclosure is to provide a method for producing a control slide glass capable of achieving the above object. [Means for solving the problem]

[0010] An example aspect of this embodiment is as follows.

[0011] A control slide for a pathology test, comprising: A control slide glass comprising a control spot on the slide glass, the control spot containing a control substance in the pathological test and a dye-trapping material.

[0012] 1. A method for producing the control slide, comprising: preparing a mixture comprising a control substance and a dye-trapping material; and forming a control spot containing the mixture on a glass slide.

[0013] 1. A method for producing the control slide, comprising: Immobilizing the control material on a glass slide; and A method comprising the step of forming a control spot on a glass slide by applying a dye-trapping material onto the immobilized control material. This specification includes the disclosures of Japanese Patent Application No. 2021-095160, which is the priority basis of this application. Effect of the Invention

[0014] According to the present disclosure, it is possible to provide a control glass slide capable of simultaneously staining a specimen and a control substance on a single glass slide, and a method for manufacturing the control glass slide. The control glass slide of the present disclosure can suppress outflow of a dye generated by staining the control substance. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic cross-sectional view showing one embodiment of the control slide glass of the present embodiment. [Diagram 2] FIG. 2 is a schematic cross-sectional view showing one embodiment of the control slide glass of this embodiment. [Diagram 3] FIG. 3 shows the staining results of the control slides tested in Example 3. [Figure 4] FIG. 4 shows the staining results of the control slides tested in Example 4. [Diagram 5] FIG. 5 shows the staining results of the slide glass tested in Comparative Example 1. [Figure 6] FIG. 6 shows the staining results of the slide glass tested in Comparative Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] One aspect of this embodiment is a control slide glass for a pathological test, characterized in that the control slide glass has a control spot on the slide glass, the control spot containing a control substance in the pathological test and a dye-trapping material.

[0017] According to the above-mentioned embodiment, a control glass slide capable of simultaneously staining a specimen and a control substance on one slide glass can be provided. The control glass slide means a glass slide having a control spot and a portion for placing and staining a specimen. The control glass slide of the above-mentioned embodiment can suppress outflow of the dye generated by staining the control substance.

[0018] One aspect of this embodiment is a method for producing the control slide glass, the method comprising the steps of preparing a mixture containing a control substance and a dye-trapping material, and forming a control spot containing the mixture on the slide glass.

[0019] Moreover, one aspect of this embodiment is a method for producing the control slide glass, characterized in that it includes a step of fixing a control substance onto the slide glass, and a step of forming a control spot on the slide glass by applying a dye-trapping material onto the fixed control substance.

[0020] According to these embodiments, the control slide glass can be produced.

[0021] The present embodiment will be described in detail below. In the following description, the present embodiment will be described using the drawings as appropriate. The following description shows a specific example of the contents of the present embodiment, and the present embodiment is not limited to these descriptions. Various changes and modifications are possible by those skilled in the art within the scope of the technical ideas disclosed in this specification. In addition, in all the drawings for explaining the present embodiment, parts having the same functions are given the same reference numerals, and repeated explanations thereof may be omitted.

[0022] [Control slide] The control glass slide according to the present embodiment is a control glass slide for pathological examination, characterized in that it has a control spot on the glass slide, which contains a control substance in the pathological examination and a dye-trapping material. The control glass slide according to the present embodiment contains a dye-trapping material in the control spot, so that dye outflow from the control spot during staining can be suppressed. The control glass slide is usually used in pathological examinations involving staining, such as immunostaining. By providing the control glass slide according to the present embodiment, the diagnostic accuracy of the staining method in the pathological examination is improved. In addition, since the control substance is provided in a form that suppresses the outflow of the staining dye, it is possible to prevent the occurrence of false positives due to the outflow of the staining dye to the outside.

[0023] The control slide glass according to the present embodiment is used with a specimen placed on it. With the specimen placed on it, the specimen and the control spot are stained simultaneously, so that the specimen and the control substance can be stained under the same conditions. For example, in the enzyme antibody method in immunostaining, a specific antigen is detected using an antibody, but this staining method generally requires the progress of a multi-stage normal reaction, such as an antigen-antibody reaction between a primary antibody and an enzyme-labeled antibody, followed by an enzyme reaction of a color-developing dye. The multi-stage normal reaction is, for example, a reaction in which a primary antibody specifically binds to a specific antigen, an enzyme-labeled antibody specifically binds to the primary antibody, and an enzyme reaction of a color-developing dye proceeds.

[0024] By using the control glass slide according to this embodiment, it is possible to confirm the normal progress of the above-mentioned multi-stage reaction. For example, by using the control glass slide in an automatic staining device for pathological examination, it is possible to confirm that the automatic staining device is operating normally.

[0025] In general, a control glass slide for pathological staining should ideally have the following three characteristics; however, no control glass slide that satisfies all of these requirements has been developed to date. (1) The specimen and the control substance are stained simultaneously. Immunostaining is performed by sequentially adding, mixing, heating, etc., multiple staining reagents, but control tests are required to confirm under the same conditions that there are no abnormalities with each staining reagent and that each process has been carried out appropriately. (2) The dye must not leak out during staining. In a design in which the sample and control substance are stained simultaneously, if the dye leaks out from the control spot during staining of the control substance, there is a risk that the leaked dye will stain the sample that should not have been stained (causing a false positive). (3) The control substance has high storage stability. It is desirable to store the control slide glass until use, but the control substance introduced onto the slide glass will dry out and oxidize when exposed to the outside air, and there is a risk that the intended staining results will not be obtained.

[0026] The control slide glass according to this embodiment has the above properties (1) to (3), and is therefore extremely useful as a control slide glass for pathological examinations.

[0027] The control slide glass according to the present embodiment has a control spot on the slide glass, so that a control test can be performed under the same conditions as a test of a sample. In addition, the control slide glass according to the present embodiment contains a dye-trapping material in the control spot together with the control substance, so that it is possible to suppress the outflow of dye from the control spot to the outside when the control substance is stained. Furthermore, since the control substance is present together with the dye-trapping material in the control spot, it is considered that the contact with the outside air is reduced compared to when the control substance is immobilized on the outermost surface, and drying and oxidation can be suppressed.

[0028] Examples of the configuration of the slide glass according to this embodiment include an embodiment shown in FIG. 1 and an embodiment shown in FIG.

[0029] 1 (also referred to as the first embodiment), a control spot containing a control substance 2 and a dye-trapping material 3 is present on a slide glass 1. In the first embodiment, the control substance 2 is embedded in the dye-trapping material 3, and is preferably physically immobilized to the dye-trapping material 3.

[0030] 2 (also referred to as the second embodiment), a control spot containing a control substance 2 and a dye-trapping material 3 is present on a slide glass 1. In the second embodiment, the control substance 2 is immobilized on the slide glass 1 by a multifunctional linker molecule 4. Furthermore, the control substance 2 is usually embedded in the dye-trapping material 3.

[0031] In the control glass slide of the first embodiment, the control substance is preferably embedded and fixed in the dye-trapping material. The control glass slide of the first embodiment can be manufactured, for example, by mixing the control substance with the dye-trapping material in a solution or sol state, and then solidifying the dye-trapping material. The control substance can be introduced onto the glass slide as a control spot by being fixed in the dye-trapping material. Methods for solidifying the dye-trapping material include, for example, a method of chemical crosslinking using a chemical crosslinking agent, a method of polymerizing using a polymerization initiator, and a method of physical network formation using an ionic additive.

[0032] In the second embodiment of the control slide glass, the control substance is preferably immobilized on the slide glass and embedded in the dye trapping material. The second embodiment of the control slide glass can be manufactured, for example, by immobilizing the control substance on the slide glass, covering it with a dye trapping material in a solution or sol state, and solidifying the dye trapping material. In this case, the control substance is introduced onto the slide glass by being immobilized on the slide glass. The method of immobilizing the control substance on the slide glass can be a method of immobilization using a multifunctional linker molecule. As in the first embodiment, the method of solidifying the dye trapping material can be, for example, a method of chemical crosslinking using a chemical crosslinking agent, a method of polymer formation using a polymerization initiator, or a method of physical network formation using an ionic additive.

[0033] A control substance is, for example, a substance suspected of being expressed in a specimen, and is a substance to be detected in a pathological test. Another example of a control substance is a substance that behaves similarly to the substance to be detected in a pathological test. Examples of substances that behave similarly in a pathological test include a part or a substance having a part of the substance to be detected, or a modified form of the substance to be detected.

[0034] In one preferred embodiment, the control substance contains at least one substance selected from proteins, peptides, amino acid oligomers, and nucleic acids. These substances are often substances suspected of being expressed in specimens in pathological tests (substances to be detected), and therefore it is desirable to use these substances as control substances depending on the type of pathological test.

[0035] For example, in immunostaining, a marker protein for a particular disease can be used as a control substance.

[0036] The control slide glass is preferably used for quality control of immunostaining among stainings performed during pathological examination. In this case, the control substance preferably contains a protein, a peptide, or an amino acid oligomer. Specifically, a marker protein for a specific disease or a peptide having a partially identical sequence to a marker protein for a specific disease is particularly preferable. Examples of the marker protein for a specific disease include Ki-67, p53, CK, D2-40, CD20, CD3, ER, PGR, HER2, CD34, CD79, CD10, αSMA, S100, CD68, CK7, BCL2, CD56, p63, CD5, CK20, Synaptophysin, Vimentin, Chromogranin A, TTF-1, Desmin, CK-HMW, CD31, EMA, CD117, Cyclin D1, CD30, Myeloperoxidase, CD4, and CEA.

[0037] In the present disclosure, proteins, peptides, and amino acid oligomers are not strictly distinguished from one another; however, for example, a compound consisting of 50 or more amino acids bound by peptide bonds may be referred to as a protein, a compound consisting of 11 to 49 amino acids bound by peptide bonds, and a compound consisting of 2 to 10 amino acids bound by peptide bonds may be referred to as an amino acid oligomer.

[0038] The control slide glass may be used for quality control of staining tests other than immunostaining. Examples of staining tests other than immunostaining include in situ hybridization (ISH), which detects specific nucleic acids (DNA or RNA) in cells and tissues. In this case, nucleic acids (e.g., DNA or RNA, or fragments of these) can be used as the control substance.

[0039] The dye-trapping material is not particularly limited as long as it can suppress the outflow of the dye from the control spot when the control substance is stained. It is desirable for the dye-trapping material to have permeability to the staining reagent, low non-specific adsorption to the staining reagent, and furthermore, high transparency.

[0040] Marker proteins for specific diseases are stained due to the presence of antibody recognition sites, and therefore can be used as control substances as described above, but if left exposed to the outside air for a long period of time, they will dry out and oxidize, causing the antibody recognition sites to denature, and there is a possibility that normal staining results will not be obtained. The control slide glass according to the present disclosure has control substances such as specific disease marker proteins embedded in a dye-trapping material, which prevents drying and oxidation and ensures long-term storage stability.

[0041] For example, in immunostaining, diaminobenzidine (DAB) is generally used as a coloring dye. DAB undergoes polymerization by peroxidase produced by an enzyme-labeled antibody, becomes polymer DAB (pDAB), and is deposited as an insoluble precipitate, thereby staining the control substance or the periphery of the control substance. At this time, if there is no dye-trapping material, the pDAB produced by the control substance may reach the specimen present on the same slide glass, and normal staining results may not be obtained. On the other hand, since the precision control slide glass according to this embodiment has a dye-trapping material, pDAB is produced inside the dye-trapping material after the staining reagent or DAB has permeated the dye-trapping material, thereby suppressing the outflow of pDAB to the outside and obtaining normal staining results. In addition, the control slide glass according to this embodiment has a control spot on the slide glass, and the control spot contains a dye-trapping material, which prevents the control substance from drying, and can be expected to have long-term storage stability.

[0042] In one preferred embodiment, the dye-trapping material contains at least one material selected from amino acid polymers, sugar polymers, and synthetic polymers. The material may be used alone or in combination of two or more. If the dye-trapping material is composed of these materials, it is preferable because the dye-trapping material can be easily immobilized on a slide glass. There is no particular limit to the molecular weight of the dye-trapping material, but it is usually 10 kDa to 1000 kDa.

[0043] The amino acid polymer preferably contains at least one polymer selected from gelatin, collagen, albumin, polyglutamic acid, and polylysine, since the dye-trapping material containing such an amino acid polymer can be easily solidified by utilizing the functional group of the polymer.

[0044] The sugar polymer preferably contains at least one polymer selected from agarose, alginic acid, hyaluronic acid, carrageenan, pectin, methylcellulose, and hydroxyethylcellulose. When the dye-trapping material contains such a sugar polymer, it is preferable because the polymer has a functional group that makes it easy to solidify.

[0045] The synthetic polymer preferably contains at least one polymer selected from the group consisting of acrylic acid polymers, methacrylic acid polymers, acrylamide polymers, methacrylamide polymers, and ethylene glycol polymers. When the dye-trapping material contains such a synthetic polymer, it is preferable because solidification by polymer formation using a polymerization initiator is easy.

[0046] As the dye-trapping material, gelatin and collagen are particularly preferred because they exhibit a solid-liquid phase change that depends on temperature, and therefore can be molded simply by mixing with the control substance and leaving it to stand at a predetermined temperature.

[0047] In order to fix the dye-trapping material on the slide glass, it is preferable to solidify the dye-trapping material. That is, the control slide glass according to the present embodiment is preferable to have the dye-trapping material solidified. Examples of the solidification method include a method of forming a chemical crosslink using a chemical crosslinking agent, a method of forming a polymer using a polymerization initiator, and a method of forming a physical network using an ionic additive.

[0048] The chemical crosslinking agent may be at least one selected from DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride), formaldehyde, glutaraldehyde, paraformaldehyde, dimethyl suberiminate, genipin, (benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate, polyfunctional ethylene glycol-based polymers, and carbodiimide compounds.

[0049] The polyfunctional ethylene glycol polymer is not particularly limited in terms of molecular weight, and examples of the polyfunctional ethylene glycol polymer that can be used include poly(ethylene glycol) diamine, poly(ethylene glycol) di(3-aminopropyl), poly(ethylene glycol) diglycidyl ether, and poly(ethylene glycol) di(hydroxysuccinimide).

[0050] As the carbodiimide compound, for example, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate can be used.

[0051] As the polymerization initiator, for example, azo-based polymerization initiators such as 2,2'-azobis-2-amidinopropane dihydrochloride and azobisisobutyronitrile, and persulfates such as sodium persulfate, potassium persulfate and ammonium persulfate can be used.

[0052] As the ionic additive, for example, calcium salts such as calcium chloride, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium trihydrogen phosphate, calcium phosphate, calcium carbonate, calcium sulfate, calcium lactate, etc. can be used.

[0053] In the second embodiment, the control substance is immobilized on the slide glass by a multifunctional linker molecule. The multifunctional linker molecule preferably includes at least one linker molecule selected from glutaraldehyde, a silane coupling agent, and a multifunctional ethylene glycol-based polymer. These linker molecules are preferred because they can easily immobilize the control substance on the slide glass. The multifunctional linker molecule may be a bifunctional linker molecule or a linker molecule having three or more functional groups.

[0054] Examples of silane coupling agents that can be used include 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethyldimethoxysilane, 3-isocyanatepropyltriethoxysilane, 3-trimethoxysilylpropylsuccinic anhydride, 3-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, and 3-glycidoxypropyltriethoxysilane.

[0055] As the polyfunctional ethylene glycol polymer, a bifunctional ethylene glycol polymer is one of the preferred embodiments. As the polyfunctional ethylene glycol polymer, the molecular weight is not particularly limited, and for example, poly(ethylene glycol) diamine, poly(ethylene glycol) di(3-aminopropyl), poly(ethylene glycol) diglycidyl ether, poly(ethylene glycol) di(hydroxysuccinimide) can be used.

[0056] The control slide glass according to this embodiment contains a control substance and a dye-trapping material in the control spot, and may contain the above-mentioned components and their reactants, etc. Also, it may contain a solvent used in the process of forming the control spot, such as water, alcohol, etc.

[0057] The control slide glass according to this embodiment has a control spot area of ​​1 mm 2 More than 1000mm 2 In one preferred embodiment, the area of ​​the control spot is 4 mm or less. 2 More than 200mm 2 It is more preferable that the area of ​​the control spot is within the above range, since it is easy to observe in a pathological examination and is excellent in productivity.

[0058] The control slide glass according to the present embodiment may be a control slide glass having one control spot or a control slide glass having multiple control spots, the number of which can be appropriately determined depending on the type and purpose of the pathological test.

[0059] The control glass slide according to the present embodiment may be a microarray type control glass slide having a plurality of control spots. If the control glass slide is a microarray type control glass slide having a plurality of control spots each having a different type of control substance arranged on the glass slide, it is not necessary to manufacture a control glass slide for each specific control substance, and in immunostaining, it can also be used as a control when a cocktail antibody composed of a plurality of types of antibodies is used as a staining reagent, and it is also possible to check whether the tests of a plurality of cases have been completed normally in one assay.

[0060] In the microarray type control slide glass, a control spot having a control substance that is not the target of the test can be set as a negative control spot. By providing a negative control spot, when an error occurs in the staining operation such that all samples are stained, it is possible to accurately know that an error has occurred. When the control slide glass has multiple control spots, the dye trapping material and the control substance constituting the multiple control spots may be different for each control spot.

[0061] A control slide having multiple control spots, such as a microarray type control slide, has an area of ​​1 mm per control spot. 2 More than 200mm 2 Preferably less than 4 mm 2 More than 50mm 2 It is more preferable that the area of ​​the control spot is within the above range, since it is easy to observe in a pathological examination and is excellent in productivity.

[0062] In the control slide glass according to the present embodiment, the thickness of the control spot is preferably 0.1 μm to 1000 μm inclusive. In particular, in the first embodiment described above, since it is sufficient that the staining reagent penetrates into a part of the dye-trapping material, the thickness is preferably 0.1 μm to 1000 μm inclusive, more preferably 1 μm to 100 μm inclusive, in view of the fact that the tissue slice actually used in immunostaining is about 5 μm inclusive, the purpose of trapping the dye, and productivity.

[0063] The control slide glass according to this embodiment is one of the preferred embodiments in which the thickness of the control spot is 0.1 μm or more and 200 μm or less. In particular, in an embodiment in which the control substance is fixed on the slide glass by a polyfunctional linker molecule, such as the above-mentioned second embodiment, the thickness is preferably 0.1 μm or more and 200 μm or less, and more preferably 1 μm or more and 20 μm or less, since the staining reagent needs to penetrate to the bottom where the control substance is fixed.

[0064] The form or shape of the control spot of the control slide glass according to this embodiment is not particularly limited, but it is preferable that the control spot is porous, swollen gel, or xerogel because it is easy to perform a pathological test accurately. When the control spot is porous, it is particularly preferable because high sensitivity staining can be expected due to a high cleaning effect when staining is performed. As a method for making the control spot porous, for example, the method described in the Examples (a method having a dehydration step with ethanol) can be mentioned. The swollen gel control spot can be obtained, for example, by using an acrylic acid-based polymer as a dye trapping material in the presence of a solvent. In addition, the xerogel control spot can be obtained, for example, by freeze-drying the swollen gel control spot.

[0065] The ratio of the control substance and the dye-trapping material in the control spot of the slide glass according to the present embodiment is not particularly limited. In consideration of the staining intensity during staining and production costs, the mass ratio of the dye-trapping material and the control substance is preferably 1:0.00001 to 1:0.1, and more preferably 1:0.0001 to 1:0.01.

[0066] The amount of the control substance per unit area in the control spot of the control slide glass according to this embodiment is 0.01 to 1000 ng / mm in order to perform sufficient staining. 2 is preferably 0.1 to 100 ng / mm 2 It is more preferable that:

[0067] The control glass slide according to the present embodiment can quantify the amount of the control substance contained in the control spot by using a fluorescent reagent. For example, in immunostaining, a marker protein for a specific disease can be used as the control substance. In this case, the marker protein introduced into the control spot can be quantified by using a fluorescently labeled IgG that exhibits specific binding to the marker protein for a specific disease and quantifying the fluorescence intensity. This method can be used as a quality control method for the control glass slide. In this case, by selecting a fluorescently labeled IgG that does not affect the immunostaining, it is possible to perform a total inspection of the control glass slide. The type of the fluorescent reagent can be appropriately changed depending on the type of the control substance. The control substance bound to such a fluorescent reagent is also referred to as a fluorescently labeled control substance. That is, the control glass slide according to the present embodiment can contain a fluorescently labeled control substance as a control substance.

[0068] [Method for producing control slides] The method for producing a control slide glass according to this embodiment is the method for producing the above-mentioned control slide glass, and is roughly divided into two modes.

[0069] In a first embodiment, a method for producing the control glass slide includes the steps of preparing a mixture containing a control substance and a dye-trapping material, and forming a control spot containing the mixture on a glass slide, which can produce the control glass slide of the first embodiment.

[0070] In the first aspect, the mixture is a mixture solidified into a capillary shape, and this is one of the preferred aspects, which includes a step of cutting the mixture into a thin film and attaching the thin film of the mixture to a slide glass to form a control spot on the slide glass.

[0071] The second aspect is a method for producing the control glass slide, which comprises the steps of immobilizing a control substance on the glass slide and forming a control spot on the glass slide by applying a dye-trapping material onto the immobilized control substance. In this aspect, the control glass slide of the second aspect can be produced by using a multifunctional linker molecule when immobilizing the control substance on the glass slide.

[0072] A specific example of the first aspect is a method in which a mixture of gelatin and a control substance is added to a Teflon (registered trademark) mold, covered with a glass slide, the mixture is solidified under refrigeration, and the dye-trapping material is transferred onto the glass slide, thereby forming a control spot formed from the dye-trapping material and the control substance on the glass slide.

[0073] In one embodiment, for example, a method for mass-producing control glass slides may be employed in which a dye-trapping material and a control substance are encapsulated and mixed in a pipe-like capillary mold, the dye-trapping material is solidified into a capillary shape, a mixture solidified into a capillary shape is obtained, the mixture is cut into thin films, and then attached to glass slides.

[0074] A specific example of the second aspect is a method in which a control substance is immobilized on a slide glass with glutaraldehyde, and then collagen is applied to form a control spot composed of a dye trapping material and the control substance on the slide glass.

[0075] The above-mentioned mass production method can also be used to produce microarray-type control glass slides. For example, after different control substances are encapsulated and mixed in multiple dye-trapping materials, the dye-trapping materials are solidified into a thin tube shape to obtain a mixture solidified into multiple thin tube shapes, the multiple mixtures are bundled and cut into a thin film, and then attached to a glass slide, thereby making it possible to mass-produce microarray-type control glass slides.

[0076] In addition, when producing the control slide glass according to the present embodiment, a solvent such as water or alcohol may be used for the purpose of adjusting the viscosity of the dye-trapping material or the mixture of the control substance and the dye-trapping material, etc. The solvent used may remain in whole or in part on the control spot, or may be removed by drying or the like.

[0077] The method for producing the control slide glass according to this embodiment is not limited to the above example, and any production method can be adopted depending on the purpose. EXAMPLES

[0078] The present embodiment will be specifically described below with reference to examples, although the present embodiment is not limited to the following examples.

[0079] [Example 1] In Example 1, a control slide of the first embodiment was prepared.

[0080] Recombinant CD20 was used as a control substance, and gelatin and BSA were used as dye-trapping materials. Recombinant CD20, gelatin, BSA, and water were mixed and kept at 37°C to prepare a mixed aqueous solution of 5 wt% gelatin, 2.5 wt% BSA, and 100 μg / mL recombinant CD20 (balance: water).

[0081] 20 μL of the mixed solution was added to a Teflon mold, which was then covered with a glass slide and left to stand in a refrigerator for 1 hour. The glass slide was peeled off from the mold to confirm the transfer of the dye-trapping material containing the control substance onto the glass slide, and then the dye-trapping material was solidified by immersing in a 2 wt% DMT-MM / ethanol solution at 25°C for 5 hours. Next, the unreacted DMT-MM residues were blocked by immersing in 4% ethanolamine / ethanol at 25°C for 12 hours. The glass slide was washed to prepare a control glass slide having a control spot containing the control substance and the dye-trapping material.

[0082] The control spot produced by this method was porous, and the area of ​​the control spot was 240 mm 2 The thickness is 20 μm, and the amount of antigen contained per unit area of ​​the control spot is 8 ng / mm 2 , the amount of dye-trapping material per unit area is 4 μg / mm 2 It was.

[0083] [Example 2] In Example 2, a control slide of the second embodiment was prepared.

[0084] Recombinant CD20 was used as a control substance, gelatin and BSA as dye trapping materials, and glutaraldehyde as a bifunctional linker molecule. Recombinant CD20 was mixed with an aqueous glutaraldehyde solution to prepare a mixed aqueous solution of 100 μg / mL recombinant CD20 and 1 wt% glutaraldehyde (balance: water), and the solution was left to stand at 37°C for 1 hour. The mixed aqueous solution was added to an amino-coated slide glass and left to stand for 1 day to obtain a slide glass on which recombinant CD20 was immobilized.

[0085] Next, a mixed aqueous solution of 5 wt% gelatin and 2.5 wt% BSA (remainder: water) was prepared, and 20 μL was added to the Teflon mold, after which the slide glass on which recombinant CD20 was immobilized was placed and left to stand in a refrigerator for 1 hour. The slide glass was peeled off from the mold, and the transfer of the dye-trapping material onto the slide glass was confirmed, and then the dye-trapping material was solidified by immersing it in a 2 wt% DMT-MM / ethanol solution at 25°C for 5 hours. Next, the unreacted DMT-MM residue was blocked by immersing it in 4% ethanolamine / ethanol at 25°C for 12 hours. The slide glass was washed to prepare a control slide glass having a control spot containing the control substance and the dye-trapping material.

[0086] The control spot produced by this method was porous, and the area of ​​the control spot was 240 mm 2, thickness 20 μm, amount of antigen contained per unit area of ​​control spot 4 ng / mm 2 , the amount of dye-trapping material per unit area is 4 μg / mm 2 It was.

[0087] [Example 3] In Example 3, immunostaining was performed using the control slide glass prepared in Example 1.

[0088] 100 μL of 50 μg / mL anti-CD20 mouse monoclonal antibody was added to the control slide glass prepared in Example 1, and reacted at 37 ° C for 16 minutes. After washing the slide glass, 100 μL of 50 μg / mL peroxidase-labeled anti-mouse IgG goat polyclonal antibody was added, and reacted at 37 ° C for 8 minutes. After washing the slide glass, 100 μL each of 3,3-diaminobenzidine solution and hydrogen peroxide solution was added, and reacted at 37 ° C for 8 minutes. After washing the slide glass, 100 μL of copper sulfate aqueous solution was added, and reacted at 37 ° C for 4 minutes. The slide glass was washed, and the stained state was confirmed. It was possible to confirm that the control substance was stained. In addition, no outflow of the dye was confirmed. The control slide glass after staining is shown in FIG. 3.

[0089] [Example 4] In Example 4, immunostaining was performed using the control slide glass prepared in Example 2.

[0090] 100 μL of 50 μg / mL anti-CD20 mouse monoclonal antibody was added to the control slide glass prepared in Example 2, and reacted at 37 ° C for 16 minutes. After washing the slide glass, 100 μL of 50 μg / mL peroxidase-labeled anti-mouse IgG goat polyclonal antibody was added, and reacted at 37 ° C for 8 minutes. After washing the slide glass, 100 μL each of 3,3-diaminobenzidine solution and hydrogen peroxide solution was added, and reacted at 37 ° C for 8 minutes. After washing the slide glass, 100 μL of copper sulfate aqueous solution was added, and reacted at 37 ° C for 4 minutes. The slide glass was washed, and the stained state was confirmed. It was possible to confirm that the control substance was stained. In addition, no outflow of the dye was confirmed. The control slide glass after staining is shown in FIG. 4.

[0091] [Example 5] In Example 5, control slides of the first embodiment were produced in large quantities.

[0092] Recombinant p53 was used as a control substance, and gelatin and BSA were used as dye-trapping materials. Recombinant p53, gelatin, BSA, and water were mixed and kept at 37°C to prepare 10 mL of a mixed aqueous solution of 5 wt% gelatin, 2.5 wt% BSA, and 100 μg / mL recombinant p53 (balance: water).

[0093] The mixed aqueous solution was poured into a pipe-like Teflon mold (length 30 mm, outer diameter 20 mm, inner diameter 10 mm) and left to stand in a refrigerator for 24 hours. After removing from the mold, the dye-trapping material was solidified by immersing in a 2 wt% DMT-MM / ethanol solution at 25°C for 24 hours. Next, unreacted DMT-MM residues were blocked by immersing in 4% ethanolamine / ethanol at 25°C for 24 hours. The mixture of the prepared dye-trapping material and the control substance was cut into a thickness of 10 μm using a microtome and placed on a glass slide to prepare 25 control glass slides at a time.

[0094] The control spot created by this method was porous, and the area of ​​the control spot was 300 mm 2, thickness 10 μm, amount of antigen contained per unit area of ​​control spot 6 ng / mm 2 , the amount of dye-trapping material per unit area is 3 μg / mm 2 It was.

[0095] [Comparative Example 1] In Comparative Example 1, the same procedure as in Example 1 was followed except that recombinant CD20, the control substance, was not used, and slide glasses were prepared and subjected to immunostaining in the same manner as in Example 3. No staining was observed. The slide glasses after staining are shown in FIG.

[0096] [Comparative Example 2] In Comparative Example 2, the same procedure as in Example 2 was followed except that recombinant CD20, the control substance, was not used, and slide glasses were prepared and subjected to immunostaining in the same manner as in Example 4. No staining was observed. The slide glasses after staining are shown in FIG.

[0097] [Comparative Example 3] In Comparative Example 3, a control substance was immobilized on a slide glass using a bifunctional linker molecule without using a dye-trapping material, and immunostaining was performed using the resulting slide glass.

[0098] In Comparative Example 3, recombinant CD20 was used as a control substance, and glutaraldehyde was used as a bifunctional linker. Recombinant CD20 and an aqueous glutaraldehyde solution were mixed to prepare a mixed aqueous solution of 100 μg / mL recombinant CD20 and 1 wt% glutaraldehyde (balance: water), which was then left to stand at 37° C. for 1 hour. The mixed aqueous solution was added to an amino-coated slide glass and left to stand for 1 day to obtain a slide glass on which recombinant CD20 was immobilized.

[0099] Immunostaining was performed in the same manner as in Example 4, except that a slide glass on which recombinant CD20 was immobilized was used. Although staining of the control substance was confirmed, 20% of the colored dye was deposited on the control substance or its surroundings, and 80% was leaked to the outside. The deposited and leaked amounts were calculated by estimating the dye concentration by collecting all the leaked liquid and measuring the absorbance of the deposited part and the leaked liquid.

[0100] From the examples and comparative examples, it was suggested that the control slide glass of the example could be easily stained without dye leakage, and is useful for quality control in pathological examinations.

[0101] The upper limit and / or lower limit of the numerical ranges described in this specification can be arbitrarily combined to define a preferred range. For example, the upper limit and lower limit of the numerical range can be arbitrarily combined to define a preferred range, the upper limit of the numerical range can be arbitrarily combined to define a preferred range, and the lower limit of the numerical range can be arbitrarily combined to define a preferred range.

[0102] The claims following this written disclosure are expressly incorporated herein into this written disclosure, with each claim standing on its own as a separate embodiment. The present disclosure includes all instances in which an independent claim is replaced by its dependent claim. Furthermore, any additional embodiments derived from the independent claim and the subsequent dependent claims are also expressly incorporated into this written specification.

[0103] Those skilled in the art can use the above description to make the most of the present disclosure. The claims and embodiments disclosed herein are merely descriptive and exemplary, and should not be construed as limiting the scope of the present disclosure in any way. With the aid of this disclosure, changes can be made to the details of the above embodiments without departing from the basic principles of the present disclosure. In other words, various modifications and improvements of the embodiments specifically disclosed in the above specification are within the scope of the present disclosure.

[0104] Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. Further notes regarding the present invention are disclosed below.

[0105] (Additional Note) (Appendix 1) A control slide for a pathology test, comprising: A control slide glass comprising a control spot on the slide glass, the control spot containing a control substance in the pathological test and a dye-trapping material. (Appendix 2) 2. A control slide according to claim 1, The control slide glass, wherein the control substance comprises at least one substance selected from the group consisting of proteins, peptides, amino acid oligomers, and nucleic acids. (Appendix 3) 1. A control slide according to claim 1 or 2, The control slide glass, wherein the dye-trapping material contains at least one material selected from the group consisting of amino acid polymers, sugar polymers, and synthetic polymers. (Appendix 4) 1. A control slide according to claim 3, the amino acid polymer comprises at least one polymer selected from gelatin, collagen, albumin, polyglutamic acid, and polylysine; the sugar polymer comprises at least one polymer selected from agarose, alginic acid, hyaluronic acid, carrageenan, pectin, methylcellulose, and hydroxyethylcellulose; 1. The control slide glass, wherein the synthetic polymer comprises at least one polymer selected from the group consisting of acrylic acid-based polymers, methacrylic acid-based polymers, acrylamide-based polymers, methacrylamide-based polymers, and ethylene glycol-based polymers. (Appendix 5) A control slide glass according to any one of claims 1 to 4, The area of ​​the control spot is 1 mm 2 More than 1000mm 2 A control slide, characterized in that: (Appendix 6) A control slide glass according to any one of claims 1 to 5, A control slide glass, characterized in that the thickness of the control spot is 0.1 μm or more and 1000 μm or less. (Appendix 7) A control slide glass according to any one of claims 1 to 6, A control slide, wherein the control substance is immobilized onto the slide by a multifunctional linker molecule. (Appendix 8) 8. A control slide according to claim 7, The control slide glass, wherein the multifunctional linker molecule comprises at least one linker molecule selected from glutaraldehyde, a silane coupling agent, and a multifunctional ethylene glycol-based polymer. (Appendix 9) 9. A control slide according to claim 7 or 8, A control slide glass, characterized in that the thickness of the control spot is 0.1 μm or more and 200 μm or less. (Appendix 10) A control slide glass according to any one of claims 1 to 9, A control slide having a plurality of control spots. (Appendix 11) 11. A control slide according to claim 10, The area of ​​each control spot is 1 mm 2 More than 200mm 2 A control slide, characterized in that: (Appendix 12) A control slide glass according to any one of claims 1 to 11, A control slide, wherein the control material comprises a fluorescently labeled control material. (Appendix 13) A control slide glass according to any one of claims 1 to 12, A control slide glass, wherein the control spot is in the form of a porous material, a swollen gel, or a xerogel. (Appendix 14) A method for producing a control slide glass according to any one of claims 1 to 6 and claims 10 to 13, comprising: preparing a mixture comprising a control substance and a dye-trapping material; and forming a control spot containing the mixture on a glass slide. (Appendix 15) 15. The method of claim 14, comprising: The mixture is solidified into a tubular shape, The method further comprises the steps of cutting the mixture into a thin film and attaching the thin film of the mixture to a slide glass to form a control spot on the slide glass. (Appendix 16) A method for producing a control slide glass according to any one of claims 1 to 13, comprising: Immobilizing the control material on a glass slide; and A method comprising the step of forming a control spot on a glass slide by applying a dye-trapping material onto the immobilized control material. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Explanation of symbols]

[0106] 1: Slide glass 2: Control material 3: Dye-trapping materials 4: Multifunctional linker molecules

Claims

1. A control slide for a pathology test, comprising: A control spot is provided on a glass slide, the control spot including a control substance for the pathology test and a dye-trapping material; the control substance is immobilized on a glass slide by a multifunctional linker molecule; The control slide glass, wherein the multifunctional linker molecule comprises at least one linker molecule selected from glutaraldehyde, a silane coupling agent, and a multifunctional ethylene glycol-based polymer.

2. 2. The control slide of claim 1, The control slide glass, wherein the control substance comprises at least one substance selected from the group consisting of proteins, peptides, amino acid oligomers, and nucleic acids.

3. 2. The control slide of claim 1, The control slide glass, wherein the dye-trapping material comprises at least one material selected from the group consisting of amino acid polymers, sugar polymers, and synthetic polymers.

4. 4. The control slide of claim 3, the amino acid polymer comprises at least one polymer selected from gelatin, collagen, albumin, polyglutamic acid, and polylysine; the sugar polymer comprises at least one polymer selected from agarose, alginic acid, hyaluronic acid, carrageenan, pectin, methylcellulose, and hydroxyethylcellulose; The control slide glass, wherein the synthetic polymer comprises at least one polymer selected from the group consisting of acrylic acid-based polymers, methacrylic acid-based polymers, acrylamide-based polymers, methacrylamide-based polymers, and ethylene glycol-based polymers.

5. 2. The control slide of claim 1, The area of ​​the control spot is 1 mm ２ More than 1000mm ２ A control slide, characterized in that:

6. 2. The control slide of claim 1, A control slide glass, characterized in that the thickness of the control spot is 0.1 μm or more and 1000 μm or less.

7. 2. The control slide of claim 1, A control slide glass, characterized in that the thickness of the control spot is 0.1 μm or more and 200 μm or less.

8. 2. The control slide of claim 1, A control slide having a plurality of control spots.

9. 9. The control slide of claim 8, The area of ​​each control spot is 1 mm ２ More than 200 mm ２ A control slide, characterized in that:

10. 2. The control slide of claim 1, A control slide, wherein the control material comprises a fluorescently labeled control material.

11. A control slide according to any one of claims 1 to 10, A control slide glass, wherein the control spot is in the form of a porous material, a swollen gel, or a xerogel.

12. 13. A method for producing the control slide of claim 1, comprising the steps of: preparing a mixture comprising a control substance and a dye-trapping material; and forming a control spot containing the mixture on a glass slide.

13. 13. The method according to claim 12, further comprising: The mixture is solidified into a tubular shape, The method further comprises the steps of cutting the mixture into a thin film and attaching the thin film of the mixture to a slide glass to form a control spot on the slide glass.

14. 13. A method for producing the control slide of claim 1, comprising the steps of: Immobilizing the control material on a glass slide; and A method comprising the step of forming a control spot on a glass slide by applying a dye-trapping material onto the immobilized control material.

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