Control substance for dyeing inspection, slide glass prepared using the same, and manufacturing method of the same

Abstract

To provide a control substance which can be easily prepared and a slide glass prepared using the same, with regard to a control substance to be used in a control test for dyeing inspection, a slide glass prepared using the same, and a manufacturing method of the same.SOLUTION: A control substance for dyeing inspection includes: a target molecule which specifically binds to a substance contained in a dyeing reagent; a charging molecule which is negatively charged; and a binding substance for binding the target molecule and the charging molecule.SELECTED DRAWING: Figure 2A

Description

[Technical field]

[0001] The present invention relates to a control substance used in a control test in a staining inspection, a slide glass prepared using the same, and a manufacturing method thereof, and more particularly to a control substance that is not easily peeled off from the slide glass, a slide glass prepared using the same, and a manufacturing method thereof. [Background technology]

[0002] In pathology tests to determine whether a subject is affected by a tumor such as cancer, a method is used in which cell tissue taken from the subject is stained with a reagent that stains only the tumor, and if stained, the subject is diagnosed as having a tumor. In such cases, there is no room for error in the diagnosis by staining. For this reason, it is common to perform positive and negative control tests (control tests) using glass slides with tissue that definitely contains the tumor to be tested (positive) and tissue that does not contain the tumor (negative). Glass slides used for such control tests are commercially available.

[0003] Patent Document 1 discloses a slide glass for an immunostaining test in which a test substance in a test sample is detected using a labeled antibody, the slide glass for a control test having a sample fixing portion for fixing the test sample, and a thin film portion having a thin film of a resin composition on which a binding substance capable of directly or indirectly forming a complex with the labeled antibody and / or a non-binding substance incapable of forming a complex with the labeled antibody is fixed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2020-98097 A Summary of the Invention [Problem to be solved by the invention]

[0005] In the technology described in Patent Document 1, a binding substance (target molecule) is fixed to a thin film of a resin composition. Although paint is described as an example of the resin composition, it is expected that the preparation will be complicated, such as providing a thin film of the resin composition on a slide glass.

[0006] An object of the present invention is to provide a control substance that can be easily prepared and a slide glass prepared using the same. [Means for solving the problem]

[0007] The outline of the means of the present invention for achieving the above object is as follows.

[0008] A control substance for a staining test, comprising a target molecule that specifically binds to a substance contained in a staining reagent, a negatively charged molecule, and a binding substance that binds the target molecule and the charged molecule.

[0009] A staining test slide glass having the staining test control substance attached thereto.

[0010] A method for producing a control substance for a dyeing test, comprising the steps of agglomerating a protein solution, immersing the protein aggregates produced in the above step in a solution containing a target molecule to bind the target molecule, and chemically modifying the protein aggregates produced in the above step to which the target molecule is bound, to impart a negative charge. Effect of the Invention

[0011] According to the present invention, it is possible to provide a control substance that can be easily prepared, and a slide glass prepared using the same. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing a schematic diagram of a minimum configuration of a control substance of the present invention. [Figure 2A]FIG. 1 is a schematic diagram showing a preparation scheme for a control substance of the present invention (an example in which an amino acid is used as a quenching agent). [Figure 2B] FIG. 1 is a schematic diagram showing a preparation scheme for a control substance of the present invention (an example in which an amino acid having a thiol group of mercaptopropionic acid is used as a quenching agent). [Diagram 3] FIG. 1 is a diagram showing an example of a slide glass for a staining test according to the present invention. [Figure 4] 13 shows experimental results of Example 3. [Diagram 5] 13 shows experimental results for Comparative Example 2. [Figure 6A] 1 is a flow chart (part 1) showing a method for producing a control material for a dyeing test of the present invention. [Figure 6B] 2 is a flow chart (part 2) showing a method for producing a control material for a dyeing test of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The following description shows a specific example of the contents of the present invention, and the present invention 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 invention, parts having the same functions are given the same reference numerals, and repeated explanations thereof may be omitted.

[0014] A suitable use of the staining test slide glass provided with the control substance of the present invention (hereinafter sometimes referred to as "control substance") is to use it for quality control (checking whether the measurement is performed normally) of immunoassay (testing whether a sample taken from a human has an antibody to the target of the sample) and genetic testing (testing whether a sample has a gene to be tested).

[0015] In particular, analysis using an automatic staining device differs from manual methods in that the analysis cannot be adjusted by humans, and therefore stable quality control of control substances is required. If the substance is stained normally, it can be confirmed that the staining process has progressed normally, and if it is not stained normally, it can be confirmed that an abnormality has occurred in the staining process.

[0016] Conventionally, the target substance has generally been tissue taken from a human, but the control substance of the present invention can be realized based on the design guidelines described below.

[0017] The control substance of the present invention is characterized by comprising a target molecule that specifically binds to a substance contained in the staining reagent, a negatively charged molecule, and a binding substance that binds the target molecule and the charged molecule.

[0018] Any substance can be used as the binding substance as long as it can bind the target molecule and the charged molecule. Examples of the binding substance that can be used include polymers and copolymers containing units of acrylic acid, methacrylic acid, maleic acid, and maleic anhydride.

[0019] A more suitable binding substance is a protein. This is because the specimen material to be compared with the staining is derived from a living organism, and it is therefore preferable that the binding substance is also derived from the same organism. Furthermore, it is preferable that the protein is aggregated. The protein aggregate is bound to the target molecule to be detected, and the protein aggregate is characterized by being negatively charged.

[0020] The protein aggregate in the control substance of the present invention is not particularly limited, but it is preferable that the protein aggregate has aggregability, has low non-specific adsorption to antibody reagents, etc., and is easily available. Examples of the protein aggregate that satisfy these conditions include serum albumin and ovalbumin.

[0021] The target molecule in the control substance of the present invention can be appropriately changed depending on the application. For example, the most suitable example is to use a protein molecule or a peptide molecule as the target molecule and use it as a control substance for immunohistochemical staining (IHC). In addition, by using a nucleic acid as the target molecule, it can be used as a control substance for in situ hybridization (ISH).

[0022] Examples of protein molecules used as target molecules in the control substance of the present invention 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. Examples of peptide molecules include peptide molecules having a partially homologous sequence in the above protein molecules.

[0023] The method for binding the protein aggregates in the control substance of the present invention to the target molecule is not particularly limited, but an example is a method using a crosslinking agent.

[0024] From the viewpoint of giving a negative charge to the protein, the crosslinking agent preferably bonds between the amino group of the protein aggregate and the amino group or thiol group of the target molecule. Examples of crosslinking methods that satisfy such a bond include aldehyde-based or NHS-maleimide-based crosslinking methods. The choice of whether to use an aldehyde-based (e.g., glutaraldehyde) or NHS-maleimide depends on the application.

[0025] Since NHS-maleimide requires pH control during crosslinking, aldehyde-based crosslinkers are generally preferred, but since aldehyde-based crosslinkers increase background fluorescence, NHS-maleimide-based crosslinkers are preferred when using a fluorescence detection system such as FISH.

[0026] When a substance bearing only negative charges, rather than a protein aggregate, is used as the binding substance, it becomes necessary to bind the COOH of the binding substance to the NH2 of the target molecule.

[0027] In this case, since it is difficult to use aldehyde-based and NHS-maleimide-based crosslinking agents, it is particularly desirable to use the following crosslinking agents. In addition, in this case, it is desirable to use the following quenching agents in combination.

[0028] Crosslinking agent Triazine-based crosslinking agents, BOP-based crosslinking agents, and carbodiimide-based crosslinking agents can be used, such as DMT-MM, PyBOP, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. Quenching agent Substances containing amine compounds can be used, such as neutral amino acid molecules, acidic amino acid molecules, ethanolamine, etc.

[0029] When a protein or peptide molecule is used as the target molecule, the amino group derived from lysine or the thiol group derived from cysteine ​​can be used as the binding site. When a nucleic acid is used as the target molecule, an amino group or a thiol group can be introduced into the 3' or 5' end of the nucleic acid in advance to use it as the binding site.

[0030] The method of imparting a negative charge to a protein aggregate is not particularly limited, but may include a method using chemical modification. In particular, when a crosslinking agent is used in binding a protein aggregate to a target molecule, a quenching agent having a charge is reacted with the unreacted site of the crosslinking agent bound only to the protein aggregate, thereby imparting a negative charge to the protein aggregate.

[0031] Representative examples are given below. When an aldehyde-based crosslinking agent is used, a carboxyl group can be introduced into the protein aggregates to impart a negative charge by reacting with the amino group of a neutral or acidic amino acid molecule as a quenching agent. Considering that protein aggregates are composed of amino acids, the above example using an amino acid as a quenching agent is particularly preferred in order to avoid changing the properties of the protein aggregates (Figure 2A).

[0032] Although a peptide or an acidic peptide can be used as a quenching agent, chemical modifications with a high molecular weight or excessive charges may inhibit the reaction that is the target of the test.

[0033] When an NHS-maleimide system is used as a crosslinking agent, a carboxyl group can be introduced into the protein aggregates to impart a negative charge by reacting with the thiol group of mercaptopropionic acid as a quenching agent (Figure 2B).

[0034] Examples of crosslinking agents that can be used for binding to the target molecule of the present invention include aldehyde-based agents such as glyoxal, malondialdehyde, succinaldehyde, glutaraldehyde, and adipaldehyde. Glutaraldehyde, which is the most widely used, is particularly preferred.

[0035] Examples of crosslinking agents that can be used for binding to the target molecule of the present invention include, for example, NHS-maleimide-based crosslinkers, such as 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester, 3-(maleimido)propionic acid N-hydroxysuccinimide ester, 6-(maleimido)hexanoic acid N-hydroxysuccinimide ester, and maleimido-(PEG) n -succinimide ester, 3-maleimidobenzoic acid N-hydroxysuccinimide ester, or their sulfo group-introduced derivatives, 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysulfosuccinimide ester, 3-(maleimido)propionic acid N-hydroxysulfosuccinimide ester, 6-(maleimido)hexanoic acid N-hydroxysulfosuccinimide ester, maleimido-(PEG) n -N-hydroxysulfosuccinimide ester, 3-maleimidobenzoic acid N-hydroxysulfosuccinimide ester.

[0036] The most commonly used 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester is particularly preferred.

[0037] The crosslinking agent used for binding the target molecule of the present invention is not limited to the aldehyde type and NHS maleimide type shown above. In addition to these, anhydride type, isothiocyanate type, isocyanate type, sulfonyl chloride type, epoxide type, carbodiimide type, etc. can be used, and a negative charge can be imparted by treating with a quenching agent having a corresponding negative charge.

[0038] In addition, by using an acidic protein as the protein aggregate, a negatively charged protein aggregate can be obtained without relying on chemical modification. In this case, the quenching agent can be either charged or uncharged. Although there is no particular limitation, the acidic protein is preferably aggregative, has low non-specific adsorption to antibody reagents, and is easily available. Casein is an example that satisfies these conditions.

[0039] The strength of the negative charge of protein aggregates can be evaluated by their isoelectric point. For example, a control substance is crushed and suspended, and the zeta potential is measured while changing the pH, and the pH at which the zeta potential is 0 can be determined as the isoelectric point of the protein aggregates. The lower the isoelectric point, the greater the negative charge of the protein aggregates.

[0040] Considering that the pI of bovine serum albumin and ovalbumin is around 5, the isoelectric point pI of the protein aggregate in the control substance of the present invention is preferably pI<5, and more preferably pI<4.5.

[0041] The method for producing the control substance of the present invention is not particularly limited. For example, a method of agglutinating a protein solution in a mold, binding a target molecule, preparing a control substance to which a negative charge has been given by chemical modification, and slicing them to a required thickness, a method of thinly applying a protein solution to a fluorine plate-shaped mold, agglutinating the protein solution, binding a target molecule, preparing a control substance to which a negative charge has been given by chemical modification, and cutting out the protein solution to a required area, a method of spraying a protein solution onto a substrate such as a slide, agglutinating the protein solution, binding a target molecule, and preparing a control substance to which a negative charge has been given by chemical modification, as in inkjet printing, and the like, can be mentioned.

[0042] The method for aggregating the protein solution is not particularly limited, and examples thereof include a method of aggregating the protein by applying heat, a method of aggregating the protein by adding an acid or alkali, and a method of aggregating the protein by adding an organic substance.

[0043] The shape of the control substance of the present invention is not particularly limited, but is preferably cylindrical (circular thin film) in consideration of uniform heating and uniform application of the immunostaining reagent.

[0044] The thickness of the control substance of the present invention is not particularly limited. Since the specimen used for immunostaining is generally about 5 μm, and the height of the liquid surface of the staining reagent during immunostaining is generally about 100 μm, 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, from the viewpoints of productivity and prevention of peeling from the slide.

[0045] The area of ​​the control substance of the present invention is not particularly limited. From the viewpoint of productivity and visibility in the inspection, it is preferable that the area is within 1 mm 2 More than 1000mm 2 Preferably less than 5 mm 2 Above 100mm 2 It is more preferable that:

[0046] The target molecule of the control substance of the present invention can be subjected to histological fixation. Histological fixation is a chemical treatment for protecting a specimen from deterioration due to autolysis or putrefaction, and is often applied in immunohistochemical staining (IHC). One representative fixation method is fixation using formaldehyde. In this fixation method, the target molecule is crosslinked with tissue components in the vicinity of the target molecule using formaldehyde, thereby maintaining the target molecule in a state close to that in the living body for a long period of time.

[0047] However, this fixation method masks the reactive site of the antigen, so staining is only possible after activation (defixation) by heat treatment, etc. By subjecting the control material to the same histological fixation as the specimen, it becomes possible to confirm that both the activation (defixation) and staining processes were normal.

[0048] The present embodiment will be described in detail below with reference to examples, although the present embodiment is not limited to the following examples. EXAMPLES

[0049] An example of a slide glass for staining test according to the present invention is shown in Fig. 3. A positive control substance 13 and a negative control substance 14 are provided on a slide glass 12. Furthermore, a sample 15 for staining test is provided on the slide glass 12 with paraffin 16.

[0050] In Example 1, a control substance of the present invention was prepared.

[0051] A 10 wt% solution of bovine serum albumin was prepared and poured into a cylindrical mold with a diameter of 5 mm. The mold was heated at 90°C to produce aggregates of bovine serum albumin.

[0052] The aggregates were immersed in a 0.1 wt % glutaraldehyde solution and allowed to stand at room temperature for 3 hours.

[0053] The aggregates were immersed in a 100 μg / mL solution of recombinant human vimentin (pH 7.4) and allowed to stand at room temperature overnight.

[0054] The aggregates were immersed in a 4% formaldehyde solution (pH 7.4) and allowed to stand at room temperature for 8 hours.

[0055] The aggregates were immersed in a 2% aqueous alanine solution and allowed to stand at room temperature overnight.

[0056] The aggregate was immersed in ethanol / water = 70v / 30v% and left to stand overnight, then immersed in ethanol / water = 95v / 5v% and left to stand for 1 hour, and then immersed in ethanol and left to stand for 3 hours.

[0057] The aggregate was immersed in xylene / ethanol = 70v / 30v% and left to stand for 1 hour, then immersed in xylene / ethanol = 95v / 5v% and left to stand for 1 hour, and then immersed in xylene and left to stand for 3 hours.

[0058] The aggregate was immersed in a xylene solution of 50 wt% paraffin and allowed to stand for 1 hour at 60° C. The aggregate was immersed in paraffin, left at 60° C. for 3 hours, and then cooled to 4° C. to prepare a paraffin-embedded body of the aggregate.

[0059] Control material was prepared by microtome sectioning of paraffin-embedded aggregates. EXAMPLES

[0060] In Example 2, a control substance of the present invention was prepared by a method different from that in Example 1.

[0061] A 10 wt% solution of bovine serum albumin was prepared and poured into a fluorine plate mold to a height of 200 μm.

[0062] The mold was heated to 90°C to produce a thin film of aggregates of bovine serum albumin.

[0063] 0.1% 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (pH 8.5) was added dropwise to the template and allowed to stand at room temperature for 3 hours. After the reaction, the solution was removed from the template.

[0064] Mercaptoethylamine was added to a 1 mg / mL solution of recombinant human vimentin (pH 6.0) and the mixture was allowed to stand at 37° C. for 3 hours.

[0065] The protein components were purified by gel filtration, and a 100 μg / mL solution of reduced form of recombinant human vimentin (pH 6.0) was prepared.

[0066] A 100 μg / mL solution of reduced form of recombinant human vimentin (pH 6.0) was added dropwise to the template and allowed to stand at room temperature overnight. After the reaction, the solution was removed from the template.

[0067] The thin-film aggregate was immersed in a 4% formaldehyde solution (pH 7.4) and allowed to stand at room temperature for 8 hours. After the reaction, the solution was removed from the mold.

[0068] The thin-film aggregate was immersed in a 1% mercaptopropionic acid solution and allowed to stand overnight at room temperature. After the reaction, the solution was removed from the mold.

[0069] A control material was prepared by cutting the thin aggregate into a piece measuring 5 mm x 5 mm. EXAMPLES

[0070] In Example 3, quality control slides were prepared from the control materials prepared in Example 1 and immunostained using an automated immunostaining device. It was confirmed that all 16 control materials did not peel off from the slides. In addition, good staining properties were confirmed for all 16 control materials (Figure 5). <Comparative Example 1> In Comparative Example 1, a comparative substance was prepared without controlling the charge by chemical modification.

[0071] 10 wt% bovine serum albumin was prepared and poured into a cylindrical mold with a diameter of 5 mm.

[0072] The mold was heated at 90° C. to produce aggregates of bovine serum albumin.

[0073] The aggregates were immersed in a 0.1 wt % glutaraldehyde solution and allowed to stand at room temperature for 3 hours.

[0074] The aggregates were immersed in a 100 μg / mL solution of recombinant human vimentin (pH 7.4) and allowed to stand at room temperature overnight.

[0075] The aggregates were immersed in a 4% formaldehyde solution (pH 7.4) and allowed to stand at room temperature for 8 hours.

[0076] The aggregate was immersed in ethanol / water = 70v / 30v% and left to stand overnight, then immersed in ethanol / water = 95v / 5v% and left to stand for 1 hour, and then immersed in ethanol and left to stand for 3 hours.

[0077] The aggregate was immersed in xylene / ethanol = 70v / 30v% and left to stand for 1 hour, then immersed in xylene / ethanol = 95v / 5v% and left to stand for 1 hour, and then immersed in xylene and left to stand for 3 hours.

[0078] The aggregate was immersed in a xylene solution of 50 wt% paraffin and allowed to stand for 1 hour at 60° C. The aggregate was immersed in paraffin, left at 60° C. for 3 hours, and then cooled to 4° C. to prepare a paraffin-embedded body of the aggregate.

[0079] Paraffin-embedded aggregates were sliced ​​with a microtome to prepare control material.

[0080] FIG. 6 shows a flow chart for producing a control substance according to the present invention. <Comparative Example 2> In Comparative Example 2, as a control experiment for Example 2, comparative slides were prepared from the comparative material prepared in Comparative Example 1 and immunostained using an automated immunostaining device. Of 16 slides, it was confirmed that the comparative material on 6 slides peeled off from the slides (FIG. 5). <Additional Notes> A control substance is a substance that contains the target molecule to be detected in various tests, and is used for the purpose of quality control of the test. For example, in tests that use staining, the staining state of the control substance can be evaluated along with that of the specimen to confirm whether the staining process in the test proceeded normally or whether any abnormalities occurred. For example, quality control is particularly important for immunostaining in pathological tests, which is used for cancer diagnosis, etc.

[0081] In order to provide a stable supply of control materials at a lower cost, technology is being developed to replace positive human tissues taken from humans with artificial materials as control materials.

[0082] In addition, automated staining devices are becoming more and more common in clinical testing. With manual staining, it is possible to control the location of the reagent drop and the strength of the washing, but with an automated staining device, such control is difficult, and peeling of the control material from the slide can become an issue.

[0083] If the control material peels off, it becomes impossible to judge the validity of the staining process in the test. Therefore, it is expected that there will be an increasing demand for control materials that are difficult to peel off from slides.

[0084] According to the present invention, a solution can be provided which prevents the control substance from peeling off from the slide and allows a control test slide glass to be prepared relatively easily. [Explanation of symbols]

[0085] 1. Protein aggregates 2. Target molecule

Claims

1. A target molecule that specifically binds to a substance contained in the staining reagent, Negatively charged molecules and The substance comprises a binding material that binds the target molecule and the charged molecule, The aforementioned charged molecule is given a negative charge by chemical modification. The aforementioned binding substance is a protein, and a control substance for staining testing is characterized in that a negative charge is imparted to the unreacted sites of the crosslinking agent, which is bound only to the protein, by reacting a charged quenching agent with the crosslinking agent.

2. In the control substance for staining inspection according to claim 1, A control substance for staining tests, characterized in that the binding substance is a protein aggregate.

3. In the control substance for staining testing according to claim 2, The aforementioned protein is a control substance for staining tests, characterized by containing serum albumin or ovalbumin.

4. In the control substance for staining inspection according to claim 1, The control substance for staining tests is characterized in that the target molecule is a protein molecule or a peptide molecule.

5. In the control substance for staining testing according to claim 4, The aforementioned protein molecule is characterized by containing at least one of 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, or CEA, making it a control substance for staining tests.

6. In the control substance for staining testing according to claim 4, The control substance for staining tests is characterized in that the peptide molecule is a peptide molecule having a partially homologous sequence in at least one of the following molecules: 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, CyclinD1, CD30, Myeloperoxidase, CD4, or CEA.

7. In the control substance for staining testing according to claim 2, A control substance for staining inspection, characterized in that the target molecule and the binding substance are bound together by the crosslinking agent.

8. In the control substance for staining inspection according to claim 7, A control substance for staining testing, characterized in that the amino group or thiol group of the target molecule and the amino group of the protein aggregate, which is the binding substance, are crosslinked with an aldehyde or NHS maleimide.

9. In the control substance for staining inspection according to claim 1, The control substance for staining tests is characterized in that the target molecule is a nucleic acid.

10. In the control substance for staining inspection according to claim 9, A control substance for staining tests, characterized in that an amino group or thiol group, pre-introduced to the 3' or 5' end of the nucleic acid, is used as the binding site.

11. In the control substance for staining inspection according to claim 1, The control substance for staining tests is characterized in that the crosslinking agent is an aldehyde, the quenching agent is an amino group of a neutral amino acid molecule or an acidic amino acid molecule, and by reacting them, a carboxyl group is introduced into the protein aggregates, thereby imparting a negative charge.

12. In the control substance for staining inspection according to claim 1, The crosslinking agent is an NHS maleimide system, and the quenching agent is characterized by introducing a carboxyl group into protein aggregates and conferring a negative charge by reacting with the thiol group of mercapto fatty acid, thereby conferring a negative charge. This is a control substance for staining inspection.

13. In the control substance for staining inspection according to claim 1, The crosslinking agent is characterized by comprising at least one of the following: 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide, 3-(maleimido)propionic acid N-hydroxysuccinimide, 6-(maleimido)hexanoic acid N-hydroxysuccinimide, maleimido-(PEG)n-succinimide, 3-maleimidobenzoic acid N-hydroxysuccinimide, or sulfo group introduced therefrom: 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysulfosuccinimide, 3-(maleimido)propionic acid N-hydroxysulfosuccinimide, 6-(maleimido)hexanoic acid N-hydroxysulfosuccinimide, maleimido-(PEG)n-N-hydroxysulfosuccinimide, 3-maleimidobenzoic acid N-hydroxysulfosuccinimide.

14. In the control substance for staining inspection according to claim 1, A control substance for staining tests, characterized in that the binding substance is an acidic protein.

15. In the control substance for staining testing according to claim 2, A control substance for staining tests, characterized in that the isoelectric point of the protein aggregates is pI < 5.

16. In the control substance for staining testing according to claim 2, The aforementioned protein aggregates are in the form of a thin film, and the surface area of ​​the thin film is 1 mm². ２ Above 1000mm ２ A control substance for staining tests characterized by the following:

17. In the control substance for staining testing according to claim 2, The aforementioned protein aggregates are in the form of a thin film, and the thickness of the thin film is 0.5 μm or more and 500 μm or less, making it a control substance for staining inspection.

18. In the control substance for staining inspection according to claim 1, A control substance for staining tests, characterized in that the target molecule is histologically immobilized.

19. A staining test slide glass characterized by having the control substance for staining test described in claim 1 attached to it.

20. It includes a target molecule that specifically binds to a substance contained in the staining reagent, a negatively charged molecule, and a binding molecule that binds the target molecule and the charged molecule. The aforementioned charged molecule is given a negative charge by chemical modification. A kit for preparing a control substance for staining tests, characterized in that the binding molecule is a protein, and a negative charge is imparted to it by reacting a charged quenching agent with the unreacted site of the crosslinking agent that is bound only to the protein using a crosslinking agent.

21. A method for producing a control substance for staining tests, The first step is to coagulate the protein solution, The second step involves immersing the protein aggregate prepared in the first step in a solution containing the target molecule to bind the target molecule, A third step involves chemically modifying the protein aggregates to which the target molecule, prepared in the second step, is bound, to impart a negative charge. A method for producing a control substance for staining inspection, characterized by containing the following:

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