Control material for pathological examination, control slide glass, and method of producing control material

Abstract

To provide a control material that can be immunostained only upon proper completion of activation and staining processes without using cells and tissues, a control slide glass with the control material installed thereon, and a method of producing the control material.SOLUTION: An aspect of the present embodiment is a control material for pathological examination, containing an antigen and amine-containing polymer compound, where the antigen is histologically fixed.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a control material for pathology testing, a control slide, and a method for producing the control material. [Background technology]

[0002] A pathological examination is a method to diagnose the presence and type of lesions by preparing specimens by attaching samples such as cells and tissues taken from a patient to a glass slide and observing them (for example, at a microscope level). In a pathological examination, staining of the components of cells and tissues is essential to detect lesions from colorless specimens.

[0003] Immunostaining is one of the staining techniques used in pathological testing. In immunostaining, antibody reagents are used to detect specific antigens in a specimen. This is an extremely important staining technique in pathological testing because it allows visualization of the location of the antigen being tested.

[0004] Generally, specimens for immunostaining are histologically fixed. Histological fixation is a chemical treatment to protect specimens from deterioration due to autolysis or putrefaction, and one of the most representative fixation methods is fixation using an aldehyde compound. In this fixation method, antigens and nearby tissue components are crosslinked with an aldehyde compound, so that the antigens are maintained in a state close to that in the living body for a long period of time. However, this fixation method masks the reactive sites of the antigens, so that staining is only possible after activation (defixation) by heating or other methods.

[0005] In other words, two processes, activation and staining, are required for immunostaining in pathological examinations. In order to confirm whether immunostaining has been completed normally, it is necessary to confirm whether each process has been completed normally. In response to such requirements, control glass slides that can confirm whether activation and staining processes have been completed normally are commercially available. A control glass slide is a glass slide that has a control substance and a specimen support section on the same surface. The specimen and the control substance placed on the specimen support section are activated and stained at the same time, and the staining of the specified range of the control substance is confirmed, thereby confirming that immunostaining has been completed normally. However, these glass slides use cells and tissues in which the expression of antigens has been confirmed in advance as control substances, so they are expensive and standardization is difficult due to differences between lots.

[0006] In response to this, as shown in Patent Documents 1 and 2, a control glass slide has been proposed in which an antigen serving as a control substance is directly immobilized on a glass slide. [Prior art documents] [Patent documents]

[0007] [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]

[0008] The control glass slides proposed in Patent Documents 1 and 2 do not use cells or tissues, and therefore are inexpensive and can reduce the difference between lots. However, in these control glass slides, the antigen is immobilized as a single molecule, so that the histological fixation of the antigen and its neighboring components cannot be reproduced, and they cannot be used as a control for activation. In other words, the present inventors thought that there is still room for improvement in the control glass slides proposed in Patent Documents 1 and 2, because the control substance is stained even if activation does not proceed normally.

[0009] Therefore, one of the objectives of the present disclosure is to provide a control material that is immunostained only when the activation and staining processes are completed normally, without using cells or tissues, which tend to be expensive and have large variations between lots.

[0010] Another object of the present disclosure is to provide a control slide glass on which the control substance is placed and a method for producing the control substance. [Means for solving the problem]

[0011] An example aspect of this embodiment is as follows.

[0012] A control material for a pathology test, comprising: A control substance comprising an antigen and an amine-containing polymer compound, wherein the antigen is histologically fixed. A control slide on which the control substance is placed. A method for producing the control substance, comprising the steps of: A method for producing a control substance selected from the following (1) to (3). (1) A method for producing a control substance, comprising histologically fixing a solid mixture of an antigen and an amine-containing polymer compound with an aldehyde compound and cutting the solid mixture into a thin film using a microtome; (2) A method for producing a control substance, which comprises applying a mixed solution of an antigen and an amine-containing polymer compound with a bar coater, solidifying the mixture, and then histologically fixing the mixture with an aldehyde compound; (3) A method for producing a control substance, in which a mixed solution of an antigen and an amine-containing polymer compound is injected into a template, solidified, and histologically fixed with an aldehyde compound. Effect of the Invention

[0013] The present disclosure provides a control material that is immunostained only when activation and staining processes are completed successfully, a control slide glass on which the control material is placed, and a method for manufacturing the control material. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a conceptual diagram showing one embodiment of the process for producing a control substance according to the present embodiment. The control substance is produced from an antigen 1 and an amine-containing polymer compound 2, and is histologically fixed using an aldehyde compound. (The left side of FIG. 1 shows the control substance before histological fixation, and the right side shows the control substance after histological fixation.) [Diagram 2] 2 is a diagram showing the constitution of the control substance of this embodiment according to the first solid-phase immobilization method, which is composed of an antigen 1 and an amine-containing polymer compound 2, and is histologically fixed (preferably using an aldehyde compound). [Diagram 3] 3 is a diagram showing the constitution of the control substance of this embodiment according to the second solid-phase method, which is composed of an antigen 1, an amine-containing polymer compound 2, and an antigen-binding substance 3, and is histologically fixed (preferably using an aldehyde compound). [Figure 4] 4 shows a control slide glass using the control substance of this embodiment, which is composed of an antigen 1, an amine-containing polymer compound 2, a slide glass 4, and a specimen support portion 5. [Diagram 5]5 shows a control slide glass using the control substance of this embodiment, which is composed of an antigen 1, an amine-containing polymer compound 2, an antigen-binding substance 3, a slide glass 4, and a specimen support portion 5. [Figure 6] FIG. 6 shows the staining results of the control slides tested in Example 5. [Figure 7] FIG. 7 shows the staining results of the control slide glass tested in Comparative Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] One aspect of this embodiment is a control substance for pathological testing, which contains an antigen and an amine-containing polymer compound, and is characterized in that the antigen is histologically fixed.

[0016] In the control substance of the above embodiment, the antigen is histologically fixed, and therefore sufficient staining cannot be performed unless activation is performed. Therefore, the control substance of the above embodiment is a control substance that is immunostained only when activation and staining processes are normally completed. Since the control substance of the above embodiment does not require the use of cells or tissues, it is inexpensive and can reduce lot-to-lot differences. Therefore, it can also contribute to improving the diagnostic accuracy of pathological tests.

[0017] One aspect of this embodiment is a control slide glass in which the control substance is placed on a slide glass.

[0018] Since the control slide glass of the above embodiment has the above-mentioned control substance placed thereon, it is possible to perform immunostaining of a specimen while checking whether activation and staining in immunostaining have progressed normally, which can contribute to improving the diagnostic accuracy of pathological tests.

[0019] One aspect of this embodiment is a method for producing the control substance, which is a method for producing a control substance selected from the following (1) to (3). (1) A method for producing a control substance, comprising histologically fixing a solid mixture of an antigen and an amine-containing polymer compound with an aldehyde compound and cutting the solid mixture into a thin film using a microtome; (2) A method for producing a control substance, which comprises applying a mixed solution of an antigen and an amine-containing polymer compound with a bar coater, solidifying the mixture, and then histologically fixing the mixture with an aldehyde compound; (3) A method for producing a control substance, in which a mixed solution of an antigen and an amine-containing polymer compound is injected into a mold template, solidified, and histologically fixed with an aldehyde compound.

[0020] The control substance can be produced by the method for producing a control substance of the above aspect.

[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 substance and method of producing the control substance] The control substance of this embodiment is a control substance for pathological examination, and is characterized in that it contains an antigen and an amine-containing polymer compound, and the antigen is histologically fixed. The control substance of this embodiment can confirm that the activation and staining processes of the specimen in immunostaining have been completed normally. The control substance of this embodiment is used by being placed on a substrate on which immunostaining is performed. The most suitable use is to place it on a slide glass to use it as a control slide glass.

[0023] The control substance preferably has the following two characteristics. (1) Abnormal staining is exhibited when activation is not normally completed. The ideal control substance for immunostaining is to function as a control for activation. For this reason, it is necessary to exhibit abnormal staining when activation is not normally completed. In this disclosure, abnormal staining is a concept that includes no staining, staining that is significantly lighter than when stained after activation, or staining that is clearly distinguishable from normal staining after activation, such as a different color (hue) when stained. (2) The control material is composed without using cells or tissues. If cells or tissues in which the expression of the antigen of interest in the test has been confirmed in advance are used as the control material, the control glass slide itself will be expensive, and there will be a risk of differences between lots, making standardization difficult.

[0024] An antigen is a substance suspected of being expressed in a specimen, and is the substance to be detected in a pathological test. Another example of an antigen 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.

[0025] The antigen may be, for example, at least one antigen selected from an antigen protein, a peptide having a sequence partially homologous to the antigen protein, and an oligomer of amino acids having a sequence partially homologous to the antigen protein. The antigen protein preferably includes at least one antigen protein selected from 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. The antigen protein may be natural or recombinant. The antigen may be used alone or in combination of two or more. Furthermore, when a control slide glass described below has a plurality of control substances placed thereon, the antigens contained in each control substance may be the same type of antigen or different antigens.

[0026] The control substance may be appropriately set as a negative control substance, which contains an antigen that is not the target of the test. By using the negative control substance in combination, a more accurate test can be performed. The negative control substance is preferably used, for example, in a control slide glass on which a plurality of control substances are placed.

[0027] The amine-containing polymer compound is not particularly limited as long as it is a polymer compound having an amino group, but is a compound having at least the following two functions. The first feature is the function of distributing amino groups in the vicinity of the antigen. This allows the antigen to be histologically fixed with the amino groups in the vicinity thereof, preferably by histologically fixing the antigen with an aldehyde compound (aldehyde cross-linking), thereby imitating histological fixation in an actual specimen. The second feature is the function of immobilizing at least one of the antigen and the antigen-binding substance. For example, at least one of the antigen and the antigen-binding substance can be immobilized by mixing at least one of the antigen and the antigen-binding substance with an amine-containing polymer compound in a solution or sol state, and then solidifying the amine-containing polymer compound. At least one of the antigen and the antigen-binding substance can also be allowed to act on the immobilized amine-containing polymer compound.

[0028] Histological fixation of antigens in immunostaining specimens is a method of crosslinking, for example, aldehyde crosslinking, between the amino group of the antigen and the tissue components in the vicinity thereof, and is performed to maintain the antigen contained in the specimen in a state close to that in the living body. This fixation method requires activation of the antigen by heat treatment or the like before staining in order to shield the reactive site of the antigen and reduce the activity of the antigen. Here, the control substance of this embodiment is prepared, for example, as shown in FIG. 1. It is possible to realize histological fixation that mimics a specimen in a pathological examination by using an amine-containing polymer compound 2 that immobilizes the antigen 1 while providing an amino group in the vicinity of the antigen.

[0029] The amine-containing polymeric compound that can be used in this embodiment is usually required to have a plurality of amino groups to realize sufficient histological fixation of the antigen, and to be able to be solidified by some method to physically solidify the antigen or antigen-binding substance. From the viewpoint of satisfying these conditions, it is preferable that the amine-containing polymeric compound contains at least one amine-containing polymeric compound selected from proteins, peptides, polysaccharides, vinyl synthetic polymers, allyl synthetic polymers, and compounds having these as partial structures. Examples of compounds having these as partial structures include copolymers having these as partial structures. The amino group of the amine-containing polymeric compound is preferably a primary amino group (-NH2).

[0030] It is preferable that the protein comprises at least one protein selected from serum albumin, ovalbumin, gelatin, collagen, and casein, the peptide comprises at least one peptide selected from polylysine and polyornithine, the polysaccharide comprises at least one polysaccharide selected from chitosan and chitin, the vinyl-based synthetic polymer comprises polyvinylamine, and the allyl-based synthetic polymer comprises polyallylamine.

[0031] As the amine-containing polymer compound, serum albumin is particularly preferable from the viewpoint of availability and suppression of non-specific adsorption of staining reagents, etc., but is not limited thereto. When a biological material such as a protein is used, it may be either natural or recombinant.

[0032] The control substance may contain an antigen-binding substance. Examples of the antigen-binding substance include antibodies and receptors that exhibit binding only to specific antigens. Examples of the antibodies include IgG, IgA, and IgM. Examples of the receptors include peptide receptors having the Fc region of the antibodies. The control substance containing an antigen-binding substance can be obtained, for example, by indirectly immobilizing Vimentin by acting Vimentin, an antigen, on an amine-containing polymer compound to which Rabbit anti Vimentin IgG, an antigen-binding substance, has been immobilized in advance.

[0033] Another example of the antigen-binding substance is a polyfunctional linker molecule. Examples of the polyfunctional linker molecule include glutaraldehyde, a chemical condensation agent, a silane coupling agent, and a bifunctional ethylene glycol-based polymer. A control substance containing an antigen-binding substance can be obtained by, for example, reacting a film of an amine-containing polymer compound with glutaraldehyde, washing the film, and then reacting the film with Vimentin, which is an antigen, to indirectly immobilize Vimentin.

[0034] That is, in one preferred embodiment, the antigen-binding substance contains at least one antigen-binding substance selected from IgG, IgA, IgM, peptide receptors having the Fc regions of these antibodies, and polyfunctional linker molecules. In another preferred embodiment, the polyfunctional linker molecule contains at least one polyfunctional linker molecule selected from glutaraldehyde, a chemical condensation agent, a silane coupling agent, and a bifunctional ethylene glycol-based polymer.

[0035] The control substance is an antigen that has been histologically fixed, and it is preferable that the histological fixation is performed using an aldehyde compound. That is, it is preferable that the antigen is fixed by aldehyde crosslinking. Examples of the aldehyde compound include formaldehyde and glutaraldehyde, which are widely used as histological fixatives. The aldehyde compound may be used alone or in combination of two or more kinds. Other aldehyde compounds may also be used.

[0036] The control substances are histologically fixed antigens, which can be broadly classified into two types, the first solid-phase type and the second solid-phase type, according to the method of fixing the antigen (solid-phase method). The solid-phase type can be selected according to the characteristics and application of the antigen.

[0037] The control substance in the first solid-phase mode is composed of at least a mixture of an antigen and an amine-containing polymer, and the antigen is histologically fixed, preferably with an aldehyde compound. In the control substance in the first solid-phase mode, the antigen is directly immobilized in the amine-containing polymer by being physically embedded therein, as shown in FIG. 2.

[0038] The control substance in the second solid-phase mode is composed of at least a mixture of an antigen, an amine-containing polymer, and an antigen-binding substance, and the antigen is histologically fixed, preferably with an aldehyde compound. The control substance in the second solid-phase mode is an amine-containing polymer physically embedded with an antigen-binding substance, and the antigen is indirectly immobilized thereon, as shown in Figure 3.

[0039] A control substance can be used as a control for activation and staining in immunostaining by evaluating its staining property. The degree of staining can be quantified using a colorimeter, spectrophotometer, etc. For example, with a colorimeter, the RGB values ​​before and after staining can be placed in an RGB spatial coordinate system, and the distance between the two points can be quantified as the degree of staining. Also, with a spectrophotometer, the maximum peak intensity corresponding to the dye used can be quantified as the degree of staining.

[0040] The control substance contains an antigen and an amine-containing polymer, and when the staining intensity of a substance in which the antigen is not histologically fixed is defined as 100, the staining intensity of the control substance is preferably less than 50, and the staining intensity of the control substance after activation is preferably 50 or more. The substance containing an antigen and an amine-containing polymer, in which the antigen is not histologically fixed, is usually a substance containing the same antigen and the same amine-containing polymer as the control substance in the same amount. That is, a substance containing an antigen and an amine-containing polymer, in which the antigen is not histologically fixed, for a certain control substance is a substance prepared by the same method except that histological fixation is not performed. The control substance contains an antigen and an amine-containing polymer, in which the antigen is not histologically fixed, is defined as 100, and when the staining intensity of the control substance is preferably less than 50, and more preferably 30 or less. The staining intensity of the control substance after activation is preferably 50 or more, and more preferably 70 or more.

[0041] Histological fixation in immunostaining improves the preservation of antigens, but reduces the activity of the antigens. Therefore, it is necessary to improve the activity of the antigens by releasing the histological fixation through activation immediately before use. For example, if the control substance has a staining degree of 30 or less and is activated to a staining degree of 70 or more, the staining degree of 70 can be set as the line at which normal immunostaining (activation and staining) can be performed, and the control can be used as a control that can confirm whether the immunostaining was performed normally. In addition, the improvement in staining degree before and after activation can confirm that the antigen was histologically fixed at the time point before activation.

[0042] The amount of amino groups per mass of the control substance is preferably 0.001 mmol / g or more and 10 mmol / g or less, more preferably 0.01 mmol / g or more and 1 mmol / g or less, in order to realize sufficient histological fixation of the antigen and to take into consideration the electrostatic non-specific adsorption of the staining reagent and the electrostatic repulsion with the cationic dye. If the amount of amino groups is too small, the antigen contained in the control substance may not be sufficiently histologically fixed, and it may not function as an activation control. On the other hand, if the amount of amino groups is too large, the staining reagent may be non-specifically adsorbed to the control substance, or the control substance may be electrostatically repelled by the cationic dye, and the original staining result may not be obtained. For example, in immunostaining, 3,3-diaminobenzidine solution (DAB) is generally used as a coloring dye, but DAB is a cationic dye, and if the amount of amino groups in the control substance is too large, DAB may not be deposited due to repulsion between cations.

[0043] Considering the securing of sufficient antigen-antibody reaction and the ease of histological fixation of antigen, the amount of antigen per mass of the control substance is preferably 0.1 μg / g or more and 10 mg / g or less, more preferably 10 μg / g or more and 1 mg / g or less. If the amount of antigen is too small, even if a staining reaction is performed, a sufficient amount of staining reagent cannot be reacted, and the staining property is impaired, so that it may not function sufficiently as a control. On the other hand, if the amount of antigen is too large, all of the antigen contained cannot be sufficiently fixed histologically, so that it may not function sufficiently as a control for activation.

[0044] It is desirable for the control substance to be in a solid form, and the control substance is usually solidified by solidifying the amine-containing polymer compound. The method for solidifying the amine-containing polymer compound is not particularly limited, and a solidification method according to the characteristics of the compound used can be adopted. For example, in the case of serum albumin, a gel-like solid can be easily obtained by applying strong heat of about 55°C to 100°C to an aqueous solution of serum albumin or by adding ethanol. As another example, in the case of gelatin or collagen, a gel-like solid can be easily obtained by using a chemical crosslinking agent.

[0045] The chemical crosslinking agent that can be used for the amine-containing polymer compound is not particularly limited and can be selected according to the compound to be used. For example, in the case of gelatin or collagen, DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride), glutaraldehyde, dimethyl suberiminate, genipin, (benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate, polyfunctional ethylene glycol-based polymers, carbodiimide compounds, etc. can be used. The chemical crosslinking agent may be used alone or in combination of two or more.

[0046] Regardless of whether the control substance is the first solidification type or the second solidification type, in addition to the above-mentioned components, the control substance may contain a gelation promoter within a range that does not impair the effects of the present invention. The gelation promoter can be selected from compounds that have the property of gelling themselves or compounds that are expected to have a thickening effect. The gelation promoter can be gelatin, collagen, agar, curdlan, gellan gum, alginic acid, carrageenan, pectin, hyaluronic acid, etc.

[0047] The control substance is preferably porous or has a finely uneven surface. This is because the contact area between the control substance and the staining reagent or washing solution can be increased, enabling more sensitive immunostaining. For example, a control substance having a porous shape or finely uneven surface can be obtained by dehydrating a hydrous gel-like control substance with ethanol or the like, or by freeze-drying the control substance.

[0048] The control substance can be used for quality inspection before shipment and immediately before use. The control substance can quantify the amount of antigen introduced by using a fluorescent reagent. For example, if a control substance is prepared using a fluorescently labeled antigen, the amount of antigen contained therein can be quantified by fluorescent observation before use. At this time, if the amount of antigen introduced is more or less than the amount within a specified range, it can be determined that the control substance is not suitable. In addition, if it is not possible to prepare a fluorescently labeled antigen, the antigen can be indirectly fluorescently labeled by using a fluorescently labeled antibody that does not affect immunostaining, and the amount of antigen contained therein can be quantified in the same manner as described above.

[0049] The method for producing the control substance is not particularly limited. For example, the method for producing the control substance may be a method using a microtome, a method using a bar coater, or a method using a template (preferably a thin film template). The method for producing the control substance may be a method for producing the control substance selected from the following (1) to (3). (1) A method for producing a control substance, in which a solid mixture of an antigen and an amine-containing polymer is histologically fixed with an aldehyde compound and cut into a thin film using a microtome. (2) A method for producing a control substance, in which a mixed solution of an antigen and an amine-containing polymer compound is applied with a bar coater, solidified, and histologically fixed with an aldehyde compound. (3) A method for producing a control substance, in which a mixed solution of an antigen and an amine-containing polymer compound is injected into a template, solidified, and histologically fixed with an aldehyde compound.

[0050] An example of a method for producing the control substance (1) above is a method in which a mixed solution of an antigen and an amine-containing polymer compound is solidified into a capillary shape, immersed in an aqueous aldehyde solution for histological fixation, and thinly cut with a microtome to produce the control substance.

[0051] An example of a method for producing the control substance (2) above is a method in which a mixed solution of an antigen and an amine-containing polymer compound is thinly applied with a bar coater, solidified, immersed in an aqueous aldehyde solution for histological fixation, and cut into spots to produce the control substance.

[0052] An example of a method for producing the control substance (3) above is a method for producing the control substance by injecting a mixed solution of an antigen and an amine-containing polymer compound into a thin film template, solidifying the mixture, and immersing the mixture in an aqueous aldehyde solution for histological fixation.

[0053] [Control slide glass and method of manufacturing the control slide glass] The control slide glass of this embodiment is a control slide glass on which a control substance is placed.

[0054] A control slide glass is used when immunostaining a specimen. When a control slide glass is used to activate and stain a specimen and a control substance simultaneously on the same slide glass, the specimen and the control are processed under the same conditions. If the activation and staining processes are normal, the control substance will be stained normally, and if there is an abnormality in either or both, the control substance will show abnormal staining properties. Therefore, the reliability of the staining state of the specimen can be evaluated from the staining state of the control substance.

[0055] In the enzyme antibody method for immunostaining, specific antigens are detected using antibodies, but this staining method generally requires the normal processing of multiple-step reactions using multiple types of reagents, including antigen activation in the specimen, specific reactions between the antigen in the specimen and the primary antibody, specific reactions between the primary antibody and labeled antibody, and enzyme reactions with color-developing dyes. The control slide glass is equipped with the control substances described above, so it can be confirmed that there are no abnormalities such as inactivation or aggregation in the multiple types of reagents described above, and that the multiple-step reactions are proceeding normally.

[0056] The control slide preferably has the following three characteristics: The first point is to have a control substance that shows abnormal staining when activation is not completed normally. The ideal immunostaining control should also function as a control for activation. In other words, it should show abnormal staining when activation is not completed normally. The second point is that the slide must have a sample support area for placing the sample. In order to activate and stain the sample and the control substance under the same conditions, the sample must be placed on the same slide on which the control substance is placed. The third point is that the control material is composed without using cells or tissues. If cells or tissues in which the expression of the antigen of interest in the test has been confirmed in advance are used as the control material, the control glass slide itself will be expensive, and there will be a risk of differences between lots, making standardization difficult.

[0057] When preparing a control glass slide using a control substance, the position of the control substance on the slide is not limited. However, from the viewpoint of securing a specimen support portion, the control substance may be placed at the edge of the slide. The control glass slide allows the specimen and the control substance to be activated and immunostained under the same conditions on the same slide. The structure of the control glass slide on which the control substance is placed by the first solid-phase method is shown in Figure 4. The structure of the control glass slide on which the control substance is placed by the second solid-phase method is shown in Figure 5.

[0058] The area of ​​the control material placed is 1 mm 2 More than 1000mm 2 From the viewpoint of both ease of observation during pathological examination and productivity of the control material, it is preferable that the 2 More than 1000mm 2 It is preferable that the thickness is less than 10 mm. 2 More than 100mm 2 It is more preferable that:

[0059] The shape of the control substance placed is not particularly limited, and may be circular, elliptical, polygonal, rectangular, linear, or the like, depending on the manufacturing method and the purpose of the test. Considering the uniform application of heat and the uniform application of the immunostaining reagent, a circular shape is preferable, but is not limited thereto.

[0060] In one preferred embodiment, the thickness of the applied control substance is 0.5 μm or more and 500 μm or less. Since the specimen used in immunostaining is generally about 5 μm, and the height of the liquid surface of the staining reagent during immunostaining is generally about 100 μm, from the viewpoint of productivity, the thickness is preferably 0.5 μm or more and 500 μm or less, and more preferably 1 μm or more and 100 μm or less.

[0061] The control glass slide has a control substance placed on it, and the control substance may be placed at one location on the slide, or at two or more locations. The control glass slide may be a microarray type control glass slide having a plurality of control substances placed on the slide.

[0062] In the case of a control glass slide in which one control substance is placed on a glass slide, a separate control glass slide needs to be manufactured for each specific antigen. However, in the case of a microarray-type control glass slide in which multiple control substances containing different types of antigens are placed on a glass slide, there is no need to manufacture a control glass slide for each specific antigen. In addition, a control substance that is not the subject of the test can be set as a negative control substance, allowing for more accurate testing.

[0063] Furthermore, a microarray-type control slide can also be used as a control when using a cocktail antibody composed of multiple types of antibodies as a staining reagent, making it possible to check whether tests on multiple cases were completed normally with a single assay.

[0064] The number of control substances in the microarray-type control slide glass can be appropriately set depending on the purpose of the test, but since the immunostaining results of adjacent control substances may affect the results, it is preferable that each control substance is spaced apart.

[0065] In the case of a microarray type control glass slide, a control substance containing an amine-containing polymer compound but not containing an antigen may be prepared and placed on the glass slide to provide a site that can be used as a negative control. By using such a site, more accurate testing can be performed.

[0066] The method of introducing the control substance onto the slide glass is not particularly limited, and examples thereof include a method in which the above-mentioned solidification is performed directly on the slide glass, and a method in which the control substance embedded in paraffin is floated in a liquid tank and scooped up with the slide glass to remove water, etc. EXAMPLES

[0067] The present embodiment will be specifically described below with reference to examples, although the present embodiment is not limited to the following examples.

[0068] [Example 1] In Example 1, the control substance was prepared according to the first solid phase mode by a method using a microtome.

[0069] Vimentin was used as an antigen, and bovine serum albumin was used as an amine-containing polymer. A mixed solution containing 100 μg / mL vimentin and 10 wt% bovine serum albumin (the remainder is PBS buffer: 8.00 g / L NaCl, 0.20 g / L KCl, 1.15 g / L Na2HPO4, 0.20 g / L KH2PO4, pH 7.4) was prepared by mixing a vimentin solution with a bovine serum albumin solution.

[0070] 1 mL of the mixed solution was taken and added to an Eppendorf tube, and then heated at 90°C for 30 minutes using a thermoshaker to gel the solution. The gel was removed and immersed in 4% formaldehyde PBS buffer for one day to histologically fix the Vimentin contained in the gel. The gel was then dehydrated by immersing it in pure water for one day, ethanol for one day, and xylene for one day. The dehydrated gel was immersed in paraffin solution at 60°C for one day, and then cooled at 4°C to prepare a paraffin-embedded gel block. The gel block was mounted on a microtome and sliced ​​to a thickness of 4 μm to prepare a paraffin-embedded control material.

[0071] [Example 2] In Example 2, the control substance was prepared according to the first solid phase mode using a bar coater.

[0072] Vimentin was used as an antigen, and bovine serum albumin was used as an amine-containing polymer. A mixed solution containing 100 μg / mL vimentin and 20 wt% bovine serum albumin (the remainder is PBS buffer: 8.00 g / L NaCl, 0.20 g / L KCl, 1.15 g / L Na2HPO4, 0.20 g / L KH2PO4, pH 7.4) was prepared by mixing a vimentin solution with a bovine serum albumin solution.

[0073] 20 μL of the mixed solution was dropped onto a slide glass and spread to a thickness of 20 μm using a bar coater. The spread solution was heated at 95°C for 1 minute to gel it. The gel was then immersed in formaldehyde PBS buffer for 1 day to histologically fix the Vimentin contained in the gel, and a control substance was prepared.

[0074] [Example 3] In Example 3, the control substance was prepared according to the first solid-phase format, using a thin film template.

[0075] Vimentin was used as an antigen, and gelatin was used as an amine-containing polymer compound. A mixed solution containing 100 μg / mL Vimentin and 5 wt% gelatin (the remainder was PBS buffer: 8.00 g / L NaCl, 0.20 g / L KCl, 1.15 g / L Na2HPO4, 0.20 g / L KH2PO4, pH 7.4) was prepared by mixing a Vimentin solution with a gelatin solution.

[0076] A Teflon (registered trademark) mold was used as the thin film template. 20 μL of the mixed solution was dropped onto the thin film template and left to stand in a refrigerator for 1 hour. The antigen-gelatin mixed solution was gelled by immersing in a 2 wt% DMT-MM / ethanol solution at 25°C for 5 hours. Next, the gel was immersed in a formaldehyde PBS buffer for 1 day to histologically fix the Vimentin contained in the gel, producing a control substance. Unreacted DMT-MM residues were blocked by immersing in 4% ethanolamine / ethanol at 25°C for 12 hours.

[0077] [Example 4] In Example 4, the control substance was prepared according to the second solid phase mode by a method using a microtome.

[0078] Vimentin was used as an antigen, bovine serum albumin as an amine-containing polymer compound, and anti-Vimentin goat polyclonal antibody (anti-Vimentin) as an antigen-binding substance. A mixed solution containing 100 μg / mL anti-Vimentin and 10 wt% bovine serum albumin (remainder PBS buffer: 8.00 g / L NaCl, 0.20 g / L KCl, 1.15 g / L Na2HPO4, 0.20 g / L KH2PO4, pH 7.4) was prepared by mixing an anti-Vimentin solution with a bovine serum albumin solution.

[0079] 1 mL of the mixed solution was added to an Eppendorf tube, which was then heated at 90°C for 30 minutes using a thermoshaker to gel the solution. The gel was dehydrated by immersing it in ethanol for 1 day and in xylene for 1 day. The dehydrated gel was immersed in paraffin solution at 60°C for 1 day, and then cooled at 4°C to prepare a paraffin-embedded gel block. The gel block was mounted on a microtome and sliced ​​to a thickness of 4 μm. The sliced ​​paraffin-embedded gel was immersed in xylene for 3 minutes, repeated immersion three times while exchanging xylene, immersed in ethanol for 3 minutes, repeated immersion three times while exchanging ethanol, and then immersed in pure water to deparaffinize. The sliced ​​gel was immersed in a 10 μg / mL Vimentin solution to indirectly solidify Vimentin in the sliced ​​gel. The gel was immersed in formaldehyde-PBS buffer for one day to histologically fix the Vimentin contained in the gel, thereby preparing a control substance.

[0080] [Example 5] In Example 5, the slide glass on which the control substance prepared in Example 1 was placed was subjected to immunostaining.

[0081] A paraffin-embedded control substance was floated in a liquid bath and scooped up with a glass slide to prepare a control glass slide containing the control substance.

[0082] A control slide was deparaffinized by immersing it in xylene for 3 minutes, repeating the immersion three times while changing the xylene, immersing it in ethanol for 3 minutes, repeating the immersion three times while changing the ethanol, and then immersing it in pure water.

[0083] A commercially available CC1 activation buffer was added to the deparaffinized control slide glass, and then the slide was heated at 95°C for 60 minutes to activate the histologically fixed Vimentin.

[0084] 1 mL of 5 μg / mL anti-Vimentin rabbit polyclonal antibody was added to the slide glass and reacted at 37°C for 16 minutes. After washing the slide glass, 1 mL of 5 μg / mL peroxidase-labeled anti-rabbit IgG goat polyclonal antibody was added and reacted at 37°C for 8 minutes. After washing the slide glass, 100 μL each of DAB 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 staining state was confirmed. Staining derived from Vimentin could be confirmed (Figure 6).

[0085] [Example 6] In Example 6, the staining intensity when the control substance prepared in Example 1 was immunostained was quantified from the RGB values ​​before and after staining using a colorimeter.

[0086] First, a substance consisting of Vimentin and bovine serum albumin prepared by the method described in Example 1 except that histological fixation was not performed was attached to a slide glass and stained by the method described in Example 5, and the staining degree of the stained substance was defined as 100. That is, the RGB values ​​before and after staining were placed in an RGB spatial coordinate system, and the distance between the two points was defined as a staining degree of 100.

[0087] A slide glass was produced using the control material prepared in Example 1 by the method described in Example 5. Staining was performed without activation by the method described in Example 5. The staining intensity when immunostaining was performed without activation was 20.

[0088] On the other hand, the staining intensity when activated and immunostained (Example 5) was 80. It was confirmed that the control substance can be used as a control.

[0089] [Comparative Example 1] In Comparative Example 1, as a control experiment for Example 5, a substance prepared using only bovine serum albumin was attached to a slide glass and subjected to activation and immunostaining. No staining was observed, and it was confirmed that the substance can be used as a negative control substance (FIG. 7). The staining degree was 0.

[0090] [Comparative Example 2] In Comparative Example 2, as a control experiment of Example 5, a substance consisting of Vimentin and bovine serum albumin was attached to a slide glass without histological fixation, and was subjected to abnormal activation treatment, that is, treatment under conditions that would not result in activation if fixed, after heating at 40°C for 60 minutes, and then immunostaining. Staining due to Vimentin could be confirmed. The staining degree was 100. It was confirmed that it could not be used as a control because it was stained despite abnormal activation.

[0091] 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 illustrative 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. The following additional notes are disclosed regarding the present invention.

[0092] (Additional Note) (Appendix 1) A control material for a pathology test, comprising: A control substance comprising an antigen and an amine-containing polymer compound, wherein the antigen is histologically fixed. (Appendix 2) A control substance as described in Appendix 1, A control substance comprising an antigen and an amine-containing polymer compound, the control substance having a staining intensity of less than 50 when the antigen is stained on a substance that is not histologically fixed, the control substance having a staining intensity of 50 or more when the control substance is stained after activation, when the staining intensity is defined as 100. (Appendix 3) A control substance as described in appendix 1 or 2, A control substance comprising an antigen-binding substance. (Appendix 4) A control substance according to any one of appendices 1 to 3, The antigen comprises at least one antigen selected from an antigen protein, a peptide having a partially homologous sequence with the antigen protein, and an oligomer of amino acids having a partially homologous sequence with the antigen protein; The control substance is characterized in that the antigen protein comprises at least one antigen protein selected from 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. (Appendix 5) A control substance as described in Appendix 3, A control substance characterized in that the antigen-binding substance contains at least one type of antigen-binding substance selected from IgG, IgA, IgM, peptide receptors having the Fc regions of these antibodies, and multifunctional linker molecules. (Appendix 6) A control substance as described in Appendix 5, A control substance, characterized in that the polyfunctional linker molecule comprises at least one type of polyfunctional linker molecule selected from glutaraldehyde, a chemical condensation agent, a silane coupling agent, and a bifunctional ethylene glycol-based polymer. (Appendix 7) A control substance according to any one of appendices 1 to 6, The control substance is characterized in that the amine-containing polymer compound contains at least one amine-containing polymer compound selected from the group consisting of proteins, peptides, polysaccharides, vinyl-based synthetic polymers, allyl-based synthetic polymers, and compounds having these as partial structures. (Appendix 8) A control substance as described in Appendix 7, The protein comprises at least one protein selected from serum albumin, ovalbumin, gelatin, collagen, and casein; The peptide comprises at least one peptide selected from polylysine and polyornithine, The polysaccharide comprises at least one polysaccharide selected from chitosan and chitin; The vinyl synthetic polymer includes polyvinylamine, A control substance, wherein the synthetic allyl polymer contains polyallylamine. (Appendix 9) A control substance according to any one of appendices 1 to 8, A control substance, characterized in that the amount of amino groups per mass of the control substance is 0.001 mmol / g or more and 10 mmol / g or less. (Appendix 10) A control substance according to any one of appendices 1 to 9, the histological fixation is carried out using an aldehyde compound; The control substance, wherein the aldehyde compound is at least one aldehyde compound selected from the group consisting of formaldehyde and glutaraldehyde. (Appendix 11) A control slide having a control substance described in any one of appendices 1 to 10 placed on the slide. (Appendix 12) 12. A control slide according to claim 11, The area of ​​the control substance placed is 1 mm 2 More than 1000mm 2 A control slide, characterized in that: (Appendix 13) 13. A control slide according to claim 11 or 12, A control slide glass, characterized in that the thickness of the control substance placed thereon is 0.5 μm or more and 500 μm or less. (Appendix 14) A control slide glass according to any one of claims 11 to 13, A control glass slide, which is a microarray type control glass slide having a plurality of control substances arranged on the glass slide. (Appendix 15) A method for producing a control substance according to any one of appendices 1 to 10, A method for producing a control substance selected from the following (1) to (3). (1) A method for producing a control substance, comprising histologically fixing a solid mixture of an antigen and an amine-containing polymer compound with an aldehyde compound and cutting the solid mixture into a thin film using a microtome; (2) A method for producing a control substance, which comprises applying a mixed solution of an antigen and an amine-containing polymer compound with a bar coater, solidifying the mixture, and then histologically fixing the mixture with an aldehyde compound; (3) A method for producing a control substance, in which a mixed solution of an antigen and an amine-containing polymer compound is injected into a template, solidified, and histologically fixed with an aldehyde compound. [Explanation of symbols]

[0093] 1: Antigen 2: Amine-containing polymer compounds 3: Antigen-binding substance 4: Slide glass 5: Sample holder

Claims

1. A control substance for use in controlling antigen retrieval and immunostaining in a pathological examination, comprising: an antigen and an amine-containing polymer compound, the antigen is histologically fixed; the amount of antigen per mass of the control substance is 0.1 μg / g or more and 10 mg / g or less; A control substance characterized in that the amount of amino groups per mass of the control substance is 0.001 mmol / g or more and 10 mmol / g or less.

2. 2. The control substance according to claim 1, A control substance comprising an antigen and an amine-containing polymer compound, wherein the staining intensity when the antigen is stained on a substance that is not histologically fixed is defined as 100, and the staining intensity when the control substance is stained is less than 50, and the staining intensity when the control substance is stained after activation is 50 or more.

3. 2. The control substance according to claim 1, A control substance comprising an antigen-binding substance.

4. 2. The control substance according to claim 1, the antigen comprises at least one antigen selected from an antigen protein, a peptide having a sequence partially homologous to the antigen protein, and an amino acid oligomer having a sequence partially homologous to the antigen protein; The control substance is characterized in that the antigen protein comprises at least one antigen protein selected from 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.

5. 4. The control substance according to claim 3, A control substance characterized in that the antigen-binding substance contains at least one antigen-binding substance selected from IgG, IgA, IgM, peptide receptors having the Fc region of these antibodies, and multifunctional linker molecules.

6. 6. The control substance according to claim 5, A control substance, characterized in that the polyfunctional linker molecule contains at least one polyfunctional linker molecule selected from glutaraldehyde, a chemical condensation agent, a silane coupling agent, and a bifunctional ethylene glycol-based polymer.

7. 2. The control substance according to claim 1, The control substance is characterized in that the amine-containing polymer compound contains at least one amine-containing polymer compound selected from proteins, peptides, polysaccharides, vinyl-based synthetic polymers, allylic synthetic polymers, and compounds having any of these as a partial structure.

8. 8. The control substance according to claim 7, the protein comprises at least one protein selected from serum albumin, ovalbumin, gelatin, collagen, and casein; the peptide comprises at least one peptide selected from polylysine and polyornithine, the polysaccharide comprises at least one polysaccharide selected from chitosan and chitin; the vinyl synthetic polymer contains polyvinylamine, A control substance characterized in that the allyl-based synthetic polymer contains polyallylamine.

9. 2. The control substance according to claim 1, the histological fixation is carried out using an aldehyde compound; A control substance, wherein the aldehyde compound is at least one aldehyde compound selected from formaldehyde and glutaraldehyde.

10. A control slide glass comprising the control substance according to claim 1 placed on the slide glass.

11. 11. The control slide of claim 10, The area of ​​the control substance placed is 1 mm ２ More than 1000mm ２ A control slide characterized in that:

12. 11. The control slide of claim 10, A control slide glass, characterized in that the thickness of the control substance placed thereon is 0.5 μm or more and 500 μm or less.

13. 11. The control slide of claim 10, A control glass slide, characterized in that it is a microarray type control glass slide in which a plurality of control substances are placed on the glass slide.

14. A method for producing a control substance according to any one of claims 1 to 9, A method for producing a control substance selected from the following (1) to (3): (1) A method for producing a control substance, which comprises histologically fixing a solid mixture of an antigen and an amine-containing polymer compound with an aldehyde compound and cutting the solid mixture into a thin film using a microtome; (2) A method for producing a control substance, which comprises applying a mixed solution of an antigen and an amine-containing polymer compound with a bar coater, solidifying the mixture, and then histologically fixing the mixture with an aldehyde compound; (3) A method for producing a control substance, in which a mixed solution of an antigen and an amine-containing polymer compound is poured into a template, solidified, and histologically fixed with an aldehyde compound.

Images