Method for detecting cell membrane protein

Abstract

The present invention addresses the problem of providing a method for staining a cell membrane protein with high accuracy. Provided is a method for detecting a cell membrane protein in an embedded immobilized tissue, the method comprising an embedding medium removal step for removing an embedding medium by bringing the immobilized tissue, which contains an embedding medium in a solid state, into contact with a solution for removal without baking.

Description

Technical Field

[0001] The present invention relates to a method for detecting cell membrane proteins in embedded fixed tissue and a method for determining a treatment for an individual from which the fixed tissue is derived using the method. Background Art

[0002] Specific cell membrane proteins on cancer cells, due to their cancer specificity, can not only be used as disease markers for cancer detection, but also various antibody drugs using these proteins as target antigens are being used in cancer treatment as cancer therapeutics with few side effects. For example, the cytoplasmic-activation and proliferation-associated protein 1 (CAPRIN-1) protein is known to be expressed on the cell membrane surface of many cancer cells and is promising as a cancer disease marker (Patent Document 1). In addition, antibodies against the CAPRIN-1 protein are known to have promising uses as therapeutic and / or preventive drugs for cancer (Patent Document 2).

[0003] In recent years, research on enhancing the efficacy of these antibody drugs has been promoted, and in particular, the development of antibody-drug complexes (ADCs) formed by conjugating drugs that have a strong ability to kill cells directly to antibodies has been actively promoted (Non-Patent Documents 1 and 2). In this way, antibody drugs have been actively developed and various types of drugs have been launched on the market.

[0004] However, in the treatment of molecular targeted drugs including these antibody drugs, the significant differences in drug efficacy between individuals are considered to be one of the major technical issues. Among them, the presence ratio and type of cell membrane proteins that serve as antigens for antibody drugs in diseased cells such as cancer cells vary from individual to individual, which is closely related to this. Therefore, in recent years, companion diagnostics that select a treatment method that is particularly effective for a patient based on information obtained from samples obtained from the patient have gained attention. In particular, since cell membrane proteins are often used as antigens for antibody drugs, it is important to accurately grasp the presence of cell membrane proteins in the sample.

[0005] Immunohistochemical staining is often used in the analysis of protein expression in samples. In general immunohistochemical staining, first, the embedding medium (paraffin, etc.) is melted by a method such as baking (also known as calcining) in which the sample is heated under an atmosphere, and then the embedding medium is removed by dissolving it in an organic solvent, etc. (Patent Documents 3 and 4). Further, the sample with the embedding medium removed is incubated with an antibody to perform staining. However, a highly accurate staining method for cell membrane proteins has not yet been established.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: WO2010 / 016527

[0009] Patent Document 2: WO2010 / 016526

[0010] Patent Document 3: WO2011 / 025442

[0011] Patent Document 4: WO2004 / 077057

[0012] Non-patent literature

[0013] Non-patent literature 1: Lancet Oncol 2016;17:e256-62

[0014] Non-patent literature 2: Pharm Res. 2015 Nov; 32(11): 3526-40 Summary of the Invention

[0015] Problems to be solved by the invention

[0016] An object of the present invention is to provide a method for staining cell membrane proteins with high precision.

[0017] Means for solving problems

[0018] The present inventors conducted intensive studies and found that the accuracy of cell membrane protein staining is significantly improved by eliminating the baking operation, which has not been considered to have an adverse effect on the results, thereby completing the present invention.

[0019] Specifically, the present invention has the following features [1] to

[21] .

[0020] [1-1] A method for detecting cell membrane proteins is a method for detecting cell membrane proteins in embedded fixed tissue, the method comprising: an embedding medium removal step of contacting the fixed tissue containing a solid embedding medium with a removal solution to remove the embedding medium; an antigen activation step of activating the cell membrane proteins in the fixed tissue after the removal step; a primary antibody reaction step of reacting the cell membrane proteins of the fixed tissue with a primary antibody, the primary antibody having immunoreactivity with the cell membrane proteins; a secondary antibody reaction step of reacting a labeled secondary antibody with the fixed tissue after the primary antibody reaction step, the labeled secondary antibody having immunoreactivity with the primary antibody; and a label detection step of detecting the labeled secondary antibody bound to the cell membrane protein via the primary antibody after the secondary antibody reaction step.

[0021] [1-2] A method for detecting cell membrane proteins is a method for detecting cell membrane proteins by removing an embedding medium from an embedded fixed tissue, the method comprising: an embedding medium removal step of contacting the fixed tissue containing a solid embedding medium with a removal solution to remove the embedding medium; a labeling step of binding the cell membrane proteins of the fixed tissue after the embedding medium removal step to a labeled antibody; and a label detection step of detecting the labeled antibody bound to the cell membrane protein after the labeling step.

[0022] [1-3] The method according to [1-2], further comprising, after the embedding medium removal step, an antigen activation step of activating the cell membrane protein in the fixed tissue.

[0023] [1-4] The method according to [1-2] or [1-3], wherein the labeling step comprises a primary antibody reaction step and a secondary antibody reaction step, wherein in the primary antibody reaction step, the cell membrane protein of the fixed tissue after the embedding medium removal step is reacted with the primary antibody, and the primary antibody is immunoreactive with the cell membrane protein, and in the secondary antibody reaction step, the labeled secondary antibody is reacted with the fixed tissue after the primary antibody reaction step, and the labeled secondary antibody is immunoreactive with the primary antibody.

[0024] [2-1] The method according to any one of [1-1] to [1-4], wherein the embedding medium removal step is a step of removing the embedding medium by bringing the fixed tissue into contact with the removal solution without baking.

[0025] [2-2] The method according to any one of [1-1] to [2-1], wherein the embedding medium removal step is a step of removing the embedding medium by bringing the fixed tissue into contact with the removal solution at a temperature lower than the melting point of the embedding medium.

[0026] [3] The method according to any one of [1-1] to [2-2], wherein the removal solution is a solution containing a surfactant and / or an organic solvent.

[0027] [4] The method according to any one of [1] to [3], wherein the contact in the embedding medium removal step is immersion.

[0028] [5] The method according to any one of [1] to [4], wherein the antigen activation step includes a heating step at 90 to 130°C.

[0029] [6] The method according to any one of [1] to [5], comprising a cooling step of cooling the fixed tissue after the antigen activation step.

[0030] [7] The method according to any one of [1] to [6], wherein the labeled secondary antibody is a complex of an antibody immunoreactive with the primary antibody and peroxidase bound to a polymer carrier.

[0031] [8] The method according to [7], wherein the label detection step is a step of detecting the cell membrane protein that has developed color using a color developing reagent reactive with the peroxidase.

[0032] [9] The method according to [8], wherein the color developing reagent is 3,3'-diaminobenzidine (DAB).

[0033] [10-1] The method according to any one of [1] to [9], wherein the embedding medium is paraffin.

[0034] [10-2] The method according to [10-1], wherein the embedding medium removal step is a step of removing the embedding medium by contacting the fixed tissue with the removal solution while maintaining the fixed tissue at a temperature lower than 45°C.

[0035] [11-1] The method according to any one of [1] to [10-2], wherein the cell membrane protein is a cell membrane protein expressed on the surface of cancer cells.

[0036] [11-2] The method according to any one of [1] to [11-1], wherein the cell membrane protein is a disease marker.

[0037]

[12] The method according to [11-2], wherein the disease is cancer.

[0038]

[13] The method according to [11-2] or

[12] , wherein the disease marker is CAPRIN-1 protein.

[0039]

[14] A method for determining a treatment for an individual from which a fixed tissue is derived, the method comprising: a step of detecting a disease marker in the embedded fixed tissue using any one of the methods described in

[11] to

[13] ; and a treatment determination step of determining a treatment for the individual based on the disease marker detected by the step.

[0040]

[15] A device for detecting cell membrane proteins, which is a device for detecting cell membrane proteins in embedded fixed tissue, comprising: an embedding medium removal section for removing the embedding medium by bringing the fixed tissue containing a solid embedding medium into contact with a removal solution; a labeling reaction section for binding the cell membrane proteins of the fixed tissue that has passed through the embedding medium removal section to a labeled antibody; and a labeling detection section for detecting the labeled antibody bound to the cell membrane proteins in the fixed tissue that has passed through the labeling reaction section.

[0041]

[16] According to the device described in

[15] , the embedding medium removing section includes a temperature regulating unit that makes the temperature of the fixed tissue lower than the melting point of the embedding medium.

[0042]

[17] The device according to

[15] or

[16] further comprises an antigen activation reaction section for activating the cell membrane protein in the fixed tissue that has passed through the embedding medium removal section.

[0043]

[18] The device according to any one of

[15] to

[17] , wherein the cell membrane protein is a cell membrane protein expressed on the surface of cancer cells.

[0044]

[19] The device according to any one of

[15] to

[18] , wherein the cell membrane protein is a disease marker.

[0045]

[20] The device according to

[19] , wherein the disease is cancer.

[0046]

[21] The device according to

[19] or

[20] , wherein the disease marker is CAPRIN-1 protein.

[0047] This specification incorporates the disclosure of Japanese Patent Application No. 2023-050776, upon which the present application claims priority.

[0048] Effects of the Invention

[0049] According to the detection method of the present invention, cell membrane proteins in embedded fixed tissue can be detected with high accuracy.

[0050] According to the method for determining a therapeutic method of the present invention, an appropriate therapeutic method can be determined based on the expression information of the cell membrane protein detected with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Shown are exemplary images of ovarian cancer tissue stained using the staining method of Example 1. A shows an image stained for cell nuclei and CAPRIN-1 protein. B shows an image stained with the nuclear signal excluded. Arrows indicate cells with appropriately stained cell membranes, and the scale bar represents 50 μm.

[0052] Figure 2 Shown are exemplary images of ovarian cancer tissue stained using the staining method of Comparative Example 1. A shows an image stained for cell nuclei and CAPRIN-1 protein. B shows an image stained without nuclear signals. Arrows indicate cells inappropriately stained in the cytoplasm. The scale bar represents 50 μm.

[0053] Figure 3Shown are stained images of gastric cancer tissues judged to be score 0 (CAPRIN-1 negative). A shows a stained image of cell nuclei and CAPRIN-1 protein. B shows a stained image with the cell nucleus signal excluded. The scale bar in the figure represents 50 μm.

[0054] Figure 4 Shown are stained images of gastric cancer tissue judged to be score 1 (CAPRIN-1 negative). A shows the staining image of cell nuclei and CAPRIN-1 protein. B shows the staining image with the cell nucleus signal excluded. The scale bar in the figure represents 50 μm.

[0055] Figure 5 Shown are stained images of gastric cancer tissues judged to be score 2 (CAPRIN-1 positive). A shows the staining image of cell nuclei and CAPRIN-1 protein. B shows the staining image with the cell nucleus signal excluded. The scale bar in the figure represents 50 μm.

[0056] Figure 6 Shown are stained images of gastric cancer tissues scored as 3 (CAPRIN-1 positive). A shows the staining of cell nuclei and CAPRIN-1 protein. B shows the staining image with the cell nucleus signal excluded. The scale bar in the figure represents 50 μm.

[0057] Figure 7 Shown are stained images of renal cancer tissue judged to be score 0 (CAPRIN-1 negative). A shows the staining image of cell nuclei and CAPRIN-1 protein. B shows the staining image with the cell nucleus signal excluded. The scale bar in the figure represents 50 μm.

[0058] Figure 8 Shown are stained images of renal cancer tissue judged to be score 1 (CAPRIN-1 negative). A shows the staining image of cell nuclei and CAPRIN-1 protein. B shows the staining image with the cell nucleus signal excluded. The scale bar in the figure represents 50 μm.

[0059] Figure 9 Shown are stained images of renal cancer tissue judged to be score 2 (CAPRIN-1 positive). A shows the staining image of cell nuclei and CAPRIN-1 protein. B shows the staining image with the cell nucleus signal excluded. The scale bar in the figure represents 50 μm.

[0060] Figure 10 Shown are stained images of renal cancer tissue judged to be score 3 (CAPRIN-1 positive). A shows the staining image of cell nuclei and CAPRIN-1 protein. B shows the staining image with the cell nucleus signal excluded. The scale bar in the figure represents 50 μm.

[0061] Figure 11 Shown are exemplary images of ovarian cancer tissue stained using the staining method of Example 1. A shows an image stained for cell nuclei and CAPRIN-1 protein. B shows an image stained with the cell nucleus signal excluded. In the figures, the scale bar represents 50 μm.

[0062] Figure 12 Shown are exemplary images of ovarian cancer tissue stained using the staining method of Example 3. A shows a staining image of cell nuclei and CAPRIN-1 protein. B shows a staining image in which the cell nucleus signal is excluded. In the figures, the scale bar represents 50 μm.

[0063] Figure 13 Shown are exemplary images of ovarian cancer tissue stained using the staining method of Example 4. A shows a staining image of cell nuclei and CAPRIN-1 protein. B shows a staining image in which the cell nucleus signal is excluded. In the figures, the scale bar represents 50 μm.

[0064] Figure 14 Shown are exemplary images of ovarian cancer tissue stained using the staining method of Example 5. A shows a staining image of cell nuclei and CAPRIN-1 protein. B shows a staining image in which the cell nucleus signal is excluded. In the figures, the scale bar represents 50 μm.

[0065] Figure 15 This is a flow chart showing an exemplary embodiment of a method for detecting cell membrane proteins.

[0066] Figure 16 This is a functional block diagram illustrating an example of the cell membrane protein detection device described in the third embodiment.

[0067] Figure 17 This figure shows an example of the hardware configuration of a control unit involved in a cell membrane protein detection device. DETAILED DESCRIPTION

[0068] 1. Detection methods of cell membrane proteins

[0069] 1-1. Overview

[0070] The first embodiment of the present invention is a method for detecting cell membrane proteins. This method comprises an embedding medium removal step, a labeling step, and a label detection step as essential steps, and an antigen activation step, a cooling step, a nonspecific reaction inhibition step, and a color development step as optional steps. This method enables highly specific detection of cell membrane proteins.

[0071] 1-2. Definition

[0072] The term "embedding medium" refers to the reagent used for embedding. "Embedding" refers to the process of allowing the reagent to penetrate into the biological sample and solidify the biological sample.

[0073] "Fixed tissue" refers to a biological specimen containing fixed tissue. "Fixation" refers to a process performed to protect the biological specimen from degradation due to autolysis and putrefaction by denaturing and / or precipitating proteins within the specimen. As used herein, "fixation" includes any fixation process utilizing a cross-linking agent, a coagulant-precipitating agent, or a combination thereof.

[0074] The term "biological sample" refers to a sample composed of cells from an organism. Specifically, "biological sample" as used in this specification includes both samples isolated from an individual organism and samples containing cells prepared in vitro. The term "biological sample" is not particularly limited as long as it is a eukaryotic organism, and includes, for example, mammals such as primates, pets, livestock, and sport animals. Preferred organisms include humans, horses, pigs, cattle, sheep, goats, dogs, and cats.

[0075] The term "cell membrane protein" refers to a protein present on the cell membrane. The term "cell membrane protein" as used herein includes any of transmembrane proteins, peripheral membrane proteins, and lipid-modified proteins. Both peripheral membrane proteins and lipid-modified proteins do not have a transmembrane domain.

[0076] A "transmembrane domain" is a protein domain that has an affinity for the lipid bilayer that constitutes the cell membrane and penetrates the lipid bilayer. In contrast, protein domains within cell membrane proteins that are exposed to the outside of the cell are called extracellular domains, while protein domains that are exposed to the inside of the cell are called intracellular domains. For example, lipid-modified proteins and portions of proteins surrounding the cell membrane, which are not entirely embedded in the cell membrane, can be referred to as either extracellular or intracellular domains.

[0077] The term "solid state" refers to a state without fluidity. The solid state herein preferably does not include a semi-solid state such as a gel.

[0078] As used herein, "immunoreactivity" refers to the property of an antibody to bind to an antigen or a partial polypeptide thereof in vivo. For example, it refers to the ability to bind to an antigen through an antigen-antibody reaction. Binding to an antigen herein includes both specific and nonspecific binding.

[0079] The term "antibody" refers to an immunoglobulin or an antigen-binding fragment thereof that specifically binds to another molecule. The term "antibody" as used herein includes both monoclonal and polyclonal antibodies and antigen-binding fragments thereof.

[0080] The term "label" refers to a substance whose presence can be detected. In this specification, "labeling" a substance means linking the substance to a label via a covalent bond.

[0081] "Baking" refers to a process of heating under an atmosphere. As used herein, "baking" specifically refers to heating fixed tissue under an atmosphere to a temperature above the melting point of the embedding medium. This process is typically performed to partially melt the embedding medium and allow the tissue to adhere to a slide, etc.

[0082] The term "disease marker" refers to a biomolecule (e.g., a nucleic acid molecule, a protein molecule) that serves as an indicator for determining the risk of developing a disease or onset. In this specification, it particularly refers to a protein that serves as an indicator for determining the risk of developing a disease or onset.

[0083] The term "cancer" refers to a malignant neoplasm and is used interchangeably with "tumor." In this specification, "cancer" includes primary cancer, metastatic cancer, metastatic cancer, and recurrent cancer.

[0084] The term "CAPRIN-1 (Cytopasmic-activation and proliferation-associated protein 1) protein" refers to a cell membrane protein primarily expressed on the cell membrane of cancer cells. The term "CAPRIN-1" as used herein also includes proteins with biological functions equivalent to those of human CAPRIN-1, such as homologs (i.e., homologs or orthologs), mutants resulting from genetic polymorphisms, and derivatives.

[0085] "Treatment" refers to treatment aimed at completely or partially curing or alleviating a disease or condition or its associated symptoms, or preventing or delaying its progression, in an individual already experiencing symptoms. "Prevention" refers to treatment aimed at preventing or delaying the onset of a disease or condition in an individual at risk of developing the disease or condition. "Therapeutic methods" or "preventative methods" refer to methods used in therapeutic or preventative treatment.

[0086] 1-3. Methods

[0087] The method of this embodiment includes the embedding medium removal step, labeling step, and label detection step as essential steps, and includes the antigen activation step, cooling step, nonspecific reaction inhibition step, and color development step as optional steps. Each step is described in detail below.

[0088] 1-3-1. Embedding medium removal step (S0101)

[0089] The "embedding medium removal step ( S0101 )" is an essential step, and is a step of bringing the fixed tissue containing the embedding medium in a solid state into contact with a removal solution to remove the embedding medium.

[0090] Embedding medium

[0091] The embedding medium is not particularly limited as long as it can embed and solidify the tissue, and can be appropriately selected according to, for example, the type of tissue to be fixed, the embedding temperature conditions, the detection method used in the label detection step, and the like.

[0092] For example, when embedding at a temperature exceeding 0° C., specific embedding media include wax, paraffin (e.g., Paraplast, Broloid, and Tissuecan), paraffin wax, acrylic resin, methacrylic resin (e.g., ethylene glycol dimethacrylate, glycol methacrylate, butyl methacrylate, hydroxypropyl methacrylate, methyl methacrylate, etc.), dammar resin, epoxy resin, divinylbenzene, other plastic resins (e.g., Superplastic, Lowicryl (registered trademark), Epon, Araldite, LR White, and Durcupan), and combination copolymers thereof. Preferred embedding media in this specification are paraffin and paraffin wax.

[0093] For example, when embedding at a temperature below 0°C, specific embedding media include, for example, Optimum Cutting Temperature (OCT) compound (e.g., Tissue-Tek (registered trademark) OCT compound or Tissue-plus (registered trademark) OCT compound), PELCO (registered trademark) cryoembedding compound, PolarStat (trademark), PolarStat Plus (trademark) embedding medium, and Tissue Freezing Medium (TFM (trademark)). These embedding media may contain a water-soluble glycol and a resin. Specifically, for example, they may contain 5 to 15% polyvinyl alcohol and 1 to 10% polyethylene glycol.

[0094] In order to achieve the desired hardness, the embedding medium can be appropriately diluted with an organic solvent or an aqueous solution.

[0095] <Fixed tissue>

[0096] The fixed tissue used in this step is a biological sample for which cell membrane proteins are to be detected by the method of this embodiment.

[0097] The individual, organ, or tissue from which the fixed tissue is derived is not particularly limited. For example, the individual may be a healthy individual, an individual with a potential for developing a disease, or an individual with a disease.

[0098] In this specification, a "healthy individual" refers to an individual in a healthy state. In this specification, a "healthy state" refers to a state in which at least the individual is free of the disease to be examined, and preferably a healthy state free of all diseases and disorders.

[0099] The fixed tissue may include diseased tissue, may have the possibility of including diseased tissue, or may include only normal tissue.

[0100] In the method of this embodiment, the fixed tissue may be derived from a single individual or from multiple individuals simultaneously. The fixed tissue derived from each individual may be derived from a single organ or from multiple organs.

[0101] The type of disease is not particularly limited. Examples include cancer and inflammation. Examples of tissues from which the fixed tissue is derived include cancer tissue and inflammatory tissue. Cell membrane proteins detected by the method of this embodiment may be, for example, cell membrane proteins expressed on the surface of diseased cells. Examples of such cell membrane proteins include cell membrane proteins expressed on the surface of inflammatory cells and cell membrane proteins expressed on the surface of cancer cells.

[0102] The cancer that is the subject of the present invention is not particularly limited. For example, it can be a cancer that expresses CAPRIN-1 protein on the cell membrane surface. Preferred cancers include basal cell carcinoma, Paget's disease, skin cancer, breast cancer, kidney cancer, pancreatic cancer, colorectal cancer, lung cancer, brain tumor, stomach cancer, uterine cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, leukemia, lymphoma, liver cancer, gallbladder cancer, sarcoma, mast cell tumor, adrenocortical carcinoma, Ewing's tumor, Hodgkin's lymphoma, mesothelioma, multiple myeloma, testicular cancer, thyroid cancer, head and neck cancer, Bowen's disease, melanoma, squamous cell carcinoma, extramammary Paget's disease, mycosis fungoides, Sézary syndrome, skin T / NK cell lymphoma, T cell leukemia / lymphoma with skin lesions only, cutaneous B cell lymphoma (indolent group), cutaneous T cell lymphoma breast cancer, combined breast cancer, mixed malignant tumors of the breast, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, neuroepithelial tumors such as gliomas, glioblastoma, neuroblastoma, ependymoma, neuroblastoma, fetal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic myeloid leukemia, schwannoma, ependymoma ... Lymphocytic leukemia, gastrointestinal lymphoma, digestive system lymphoma, small to medium cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, transverse colon cancer, sigmoid colon cancer, rectal cancer, ovarian epithelial cancer, germ cell tumor, stromal cell tumor, pancreatic ductal adenocarcinoma, invasive pancreatic ductal adenocarcinoma, pancreatic adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, giant cell tumor, intraductal papillary mucinous neoplasm, mucinous cystadenocarcinoma, pancreatoblastoma, pancreatic head cell tumor, Frants tumor, serous cystadenocarcinoma, solid papillary carcinoma, gastrinoma The present invention also includes, but is not limited to, glucagonoma, insulinoma, multiple endocrine neoplasia 1 (Wermer's syndrome), non-functioning pancreatic islet cell tumor, somatostatinoma, VIP-producing tumor, cervical cancer, uterine corpus cancer, fibrosarcoma, bone / joint sarcoma, Ewing's sarcoma, Wilms' tumor, hepatoblastoma, soft tissue sarcoma, acute leukemia, chronic leukemia, spinal cord tumor, soft tissue malignant tumor, teratoma group tumor, hypopharyngeal cancer, oropharyngeal cancer, tongue cancer, nasopharyngeal cancer, oral cancer, lip cancer, paranasal sinus cancer, laryngeal cancer, etc. Among head and neck cancers, the present invention also includes, but is not limited to, palpable cancers with the above-mentioned cancers as primary, cancers present under the skin, cancers present in the skin, superficial cancers, cancers present in the dermis, or cancers present in non-parenchymal organs.

[0103] The type of cell membrane protein detected by the method of this embodiment is not particularly limited. It can be any of transmembrane proteins, cell membrane peripheral proteins, and lipid-modified proteins, and can be proteins that are localized on the cell membrane under specific conditions. For example, it can be a cell membrane protein that does not have a transmembrane domain, or a cell membrane protein that is fully or partially exposed to the extracellular space.

[0104] Examples of cell membrane proteins in this specification include disease markers such as cancer markers. Specific examples of cancer markers include proteins such as CAPRIN-1, HER2, CD20, carcinoembryonic antigen (CEA), CA-125, CA19-9, CD117, ALK, BCR-ABL1, BRAF, CFTR, EGFR, IL2RA, RAS, and Claudin8.

[0105] There are no particular limitations on the method for fixing tissues, and methods known in the art can be used. For example, the method described in Hopwood D. "Fixatives and fixation: a review." Histochem J. 1969; 1(4): 323-60, the method described in "Immunohistochemical Staining Methods," 6th edition (2013) (DAKO North America, Inc.), etc. can be used. The reagent used for fixation is not particularly limited. For example, it can be any of a cross-linking agent, a coagulation precipitant, or a combination thereof. Specific examples of fixative reagents include solutions containing glutaraldehyde, formaldehyde, paraformaldehyde, paraformaldehyde-picric acid, periodate-lysine-paraformaldehyde, and zinc ion-formaldehyde as cross-linking agents; methanol, ethanol, acetone, acetic acid-zinc chloride, and methanol-acetone mixtures as coagulation precipitants; or mixtures thereof (e.g., formalin solution). The fixative reagent can be diluted in a buffer solution or the like as needed.

[0106] The conditions for fixation are not particularly limited. Examples of the fixation temperature include below 0°C, 4°C to 45°C, 4°C to 37°C, 4°C to 30°C, and 4°C to 25°C. In addition, the fixation time is not particularly limited. Examples include 30 seconds to 48 hours, 10 minutes to 36 hours, 20 minutes to 24 hours, and 30 minutes to 20 hours. The number of fixations is not particularly limited. For example, it can be performed more than once, more than twice, or more than three times. When multiple fixations are performed, the methods, reagents, and conditions used for each fixation may be the same or different from each other.

[0107] After fixation, dehydration treatment and replacement treatment may be performed as needed. The respective conditions are not particularly limited, and any method known in the art may be used. For example, the method described in the washing step of this process may be used.

[0108] The fixed tissue used in the method of this embodiment can be a tissue piece or a slice thereof obtained by embedding the fixed tissue in the embedding medium. Preferably, the fixed tissue is a slice.

[0109] The method for preparing the slices is not particularly limited. The slices can be prepared by methods known in the art, for example, by manually performing sectioning or by using a thin-cutting device (microtome, vibrating microtome or cryostat, etc.).

[0110] The thickness of the slice is not particularly limited. Specific examples of thickness include 0.5 μm or more, 1 μm or more, 2 μm or more, 3 μm or more, or 4 μm or more. Furthermore, for example, it may be 300 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 80 μm or less, 60 μm or less, 50 μm or less, 30 μm or less, 20 μm or less, 15 μm or less, 10 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, or 5 μm or less. For example, the thickness of the slice may be within the range of 1 μm to 300 μm, 1 μm to 100 μm, 1 μm to 50 μm, 1 μm to 20 μm, 1 μm to 10 μm, 2 μm to 100 μm, 2 μm to 50 μm, 2 μm to 20 μm, or 2 μm to 10 μm.

[0111] The slices may be placed on a support such as a glass slide, or may not be placed on a support, for example, in a state of being suspended in a solution. The slices are preferably placed on a support such as a glass slide. The material of the support is not particularly limited, and may include, for example, a polymer material, a glass material, a plastic material or a metal material. Examples of glass materials include, for example, soda-lime glass, borosilicate glass, crown glass and combinations thereof. The shape of the support is not particularly limited. For example, films, microtiter plates, beads, filters, test strips, glass slides, cover slips and test tubes can be used as supports. The support can be surface-processed as needed. The slices can be attached to the support. The method of attachment is not particularly limited, and examples include methods using matrices such as silane, gelatin, and poly-L-lysine.

[0112] The fixed tissue contains a solid embedding medium. The term "solid embedding medium" means that the embedding medium is not melted. That is, in the fixed tissue used in the method of this embodiment, all or part of the embedding medium is not melted.

[0113] The ratio of the solid embedding medium in the fixed tissue to the total embedding medium is not particularly limited. For example, it may be 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100%.

[0114] It is preferred that the fixed tissue used in this step is not baked and not heated above the melting point of the embedding medium under atmosphere.

[0115] The so-called "melting point" refers to the temperature at which a solid substance melts into a liquid state when heated.

[0116] The heating temperature here varies depending on the type of embedding medium used. For example, in the case of an embedding medium for frozen sections, since the melting point of a typical OCT compound varies depending on the product, it corresponds to a temperature exceeding this temperature when a typical OCT compound is used. Specifically, for example, it is preferable not to expose the sample to an atmosphere above -20°C, above -15°C, above -10°C, above -5°C, or above 0°C for a period of time exceeding a certain temperature. For other embedding media, for example, since the melting point of a typical paraffin wax varies depending on the product, it corresponds to a temperature exceeding this temperature when a typical paraffin wax is used. It is preferable not to expose the sample to an atmosphere above 40°C, above 45°C, above 50°C, above 55°C, above 60°C, or above 62°C for a period of time exceeding a certain temperature.

[0117] The exposure time is not particularly limited as long as it is sufficient for the embedding medium in the fixed tissue to begin melting. For example, it is preferred not to expose the tissue to an atmosphere at a temperature above the melting point of the embedding medium for a period of 1 minute or longer, 2 minutes or longer, 3 minutes or longer, 5 minutes or longer, 6 minutes or longer, 7 minutes or longer, 8 minutes or longer, 9 minutes or longer, 10 minutes or longer, 11 minutes or longer, 12 minutes or longer, 14 minutes or longer, or 15 minutes or longer. For example, it is preferred not to expose the tissue to an atmosphere at a temperature of 50°C or higher for 5 minutes or longer, 55°C or higher for 10 minutes or longer, or 60°C or higher for 15 minutes or longer.

[0118] <Removal solution>

[0119] In this specification, the term "removal solution" refers to a solution that can dissolve the embedding medium. The type of removal solution is not particularly limited. When the embedding medium is a lipophilic substance, for example, a solution containing a surfactant and / or an organic solvent can be used. Preferably, a solution containing an organic solvent is used. On the other hand, when the embedding medium is a hydrophilic substance, for example, water and / or an aqueous solution (containing an aqueous solution of sugar such as sucrose, a buffer solution, etc.) can be used.

[0120] The organic solvent used in this step is not particularly limited. Examples include linear, branched, and cyclic alkanes with 4 to 16 carbon atoms (e.g., heptane, hexadecane); linear, branched, and cyclic dialkyl ethers with 4 to 16 carbon atoms (e.g., diethyl ether, dioctyl ether); aromatic hydrocarbons such as toluene and xylene; alcohols such as methanol, ethanol, and isopropanol; alkyl halides such as chloroform; monoterpenes such as limonene; vegetable oils and fats such as citrus oil and coconut oil; and mixtures thereof. Alternatively, substitutes for these (e.g., xylene substitutes) or removal solutions included with automatic staining systems (e.g., Artisan Cleaning Solution (Agilent)) can be used. Specifically, xylene substitutes include Tissue Clear xylene substitutes, Shandon xylene substitutes, aliphatic hydrocarbons such as heptane and hexadecane, derivatives of aliphatic hydrocarbons such as dioctyl ether, and mixtures thereof.

[0121] The concentration of the organic solvent in the removal solution is not particularly limited. For example, the concentration, in weight percent, may be 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100%. For example, xylene and an aqueous ethanol solution having a concentration of 1% or higher can be used as the removal solution.

[0122] The removal solution used in this step is preferably xylene or a xylene substitute (for example, a xylene substitute containing an aliphatic hydrocarbon).

[0123] <Contact steps>

[0124] In this step, the fixed tissue is brought into contact with the removal solution, whereby all or part of the embedding medium in the fixed tissue is dissolved in the removal solution.

[0125] The contact method is not particularly limited as long as it is a method of contacting the embedding medium in the fixed tissue with the removal solution. For example, the removal solution can be sprayed, spread, or applied to the fixed tissue, or the fixed tissue can be immersed in the removal solution. The contact in this step is preferably by immersion. The contact site in the fixed tissue with the removal solution can be any of the entire or a portion of the fixed tissue. In the case where a portion of the fixed tissue is in contact with the removal solution, care should be taken to ensure that at least the majority of the embedding medium in the fixed tissue is in contact with the removal solution. For example, this step can be carried out in a manner such that 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the embedding medium is in contact with the removal solution.

[0126] The contact conditions in this step are not particularly limited as long as the embedding medium can be dissolved in the removal solution.

[0127] For example, the contact time may be longer at a relatively low temperature that is higher than the freezing point of the removal solution and lower than the melting point of the embedding medium.

[0128] The so-called "freezing point" refers to the temperature at which a liquid substance solidifies into a solid state upon cooling.

[0129] The time in this case is not particularly limited. For example, the contact time can be 3 minutes or more, 5 minutes or more, 6 minutes or more, 8 minutes or more, 10 minutes or more, 11 minutes or more, 12 minutes or more, 13 minutes or more, 14 minutes or more, 15 minutes or more, 17 minutes or more, 19 minutes or more, or 20 minutes or more. The upper limit of the time is not particularly limited. For example, the contact time can be 48 hours or less, 36 hours or less, 30 hours or less, 24 hours or less, 20 hours or less, 18 hours or less, 16 hours or less, 15 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 3 hours or less, 2 hours or less, 90 minutes or less, 60 minutes or less, 30 minutes or less, 25 minutes or less, or 20 minutes or less.

[0130] The lower limit of the temperature in this case is not particularly limited, as long as it is above the freezing point of the removal solution. This temperature varies depending on the type of removal solution used. For example, when xylene is used as the removal solution, a temperature above -25°C, which is the freezing point of xylene, is sufficient; when ethanol is used, a temperature above -114.5°C, which is the freezing point of ethanol, is sufficient. Specific temperatures include, for example, above -100°C, above -50°C, above -24°C, above -10°C, above 0°C, above 1°C, above 5°C, above 10°C, above 15°C, above 20°C, and above 25°C. Furthermore, the upper limit of the temperature in this case is not particularly limited, as long as it is below the melting point of the embedding medium. This temperature varies depending on the type of embedding medium used. For example, when paraffin wax is used as the embedding medium, a temperature between 45°C and 75°C, which is below the melting point of the paraffin wax, is sufficient. Specific temperatures include, for example, less than 75°C, less than 70°C, less than 65°C, less than 60°C, less than 55°C, less than 50°C, less than 45°C, less than 40°C, less than 37°C, less than 35°C, less than 30°C, or less than 27°C. For example, the reaction can be carried out at room temperature (1 to 30°C) or normal temperature (15 to 25°C). Specifically, for example, the reaction can be brought into contact with aromatic hydrocarbons such as xylene at room temperature for 20 minutes or longer.

[0131] The temperature may be maintained at a certain level during the contact period, but may be appropriately changed during the contact period or may change naturally. For example, it is preferable to maintain a temperature lower than the melting point of the embedding medium during the contact period. Specifically, when paraffin is used as the embedding medium, the fixed tissue is preferably maintained at a temperature lower than the melting point of the paraffin wax (e.g., lower than 45°C).

[0132] The contact may be performed in multiple steps. In this case, the removal solution and contact conditions used in each contact may be the same or different. For example, the removal solution may be refreshed each time.

[0133] Alternatively, for example, the removal may be performed at a higher temperature, which is equal to or higher than the melting point of the embedding medium and lower than the boiling point of the removal solution, for a shorter time.

[0134] The so-called "boiling point" refers to the temperature at which the vapor pressure of a liquid is equal to the pressure of the system. In this specification, the "boiling point" specifically refers to the temperature at which the vapor pressure of a liquid is equal to one atmosphere of pressure.

[0135] The contact time in this case is not particularly limited and can be, for example, 3 seconds or longer, 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 20 seconds or longer, 25 seconds or longer, or 30 seconds or longer. The upper limit of the contact time is not particularly limited as long as it does not place excessive stress on the fixed tissue. For example, the contact time can be 1 minute or shorter, 55 seconds or shorter, 50 seconds or shorter, 45 seconds or shorter, 40 seconds or shorter, 35 seconds or shorter, or 30 seconds or shorter.

[0136] The lower limit of the temperature in this case is not particularly limited as long as it is above the melting point of the embedding medium. This temperature varies depending on the type of embedding medium used. For example, when paraffin wax is used as the embedding medium, a temperature equal to or exceeding the melting point of paraffin wax of 45°C to 75°C may be sufficient. Specific temperatures include, for example, temperatures above 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 72°C, or 75°C. Furthermore, the upper limit of the temperature in this case is not particularly limited as long as it is below the boiling point of the removal solution. For example, when xylene is used as the removal solution, a temperature of 138°C to 144°C, below the boiling point of xylene, may be sufficient. When ethanol is used, a temperature below the boiling point of 78.37°C may be sufficient. Specific examples include 140° C. or lower, 130° C. or lower, 120° C. or lower, 110° C. or lower, 100° C. or lower, 90° C. or lower, 85° C. or lower, 80° C. or lower, 78° C. or lower, or 75° C. or lower.

[0137] The proportion of embedding medium removed in this step is not particularly limited as long as it does not inhibit the antigen-antibody reaction using the primary antibody, etc. For example, this step may remove 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the embedding medium.

[0138] Can make the removal solution suitable flow during this step.Flow in this case can be carried out artificially by operation such as stirring, can be carried out naturally by convection etc. In addition, can change the configuration of fixed tissue etc. and make fixed tissue suitable move.

[0139] <Separation Step>

[0140] If necessary, after contact with the removal solution, a separation step of separating the dissolved embedding medium from the fixed tissue may be performed.

[0141] The method of separation is not particularly limited. The dissolved embedding medium is a liquid and the fixed tissue is a solid, so the solid-liquid separation method known in the art can be used. For example, separation can be performed by centrifugation using a dehydrator or the like.

[0142] In cases where a large excess of the removing solution is used in the contacting step, when the removing solution is flowed, or when the removing solution is renewed, the separation step may not be necessary.

[0143] Washing steps

[0144] If necessary, a washing step can be performed to wash the fixed tissue with a removal solution or the like. Washing is performed by bringing the washing solution into contact with the fixed tissue. The contact can be performed using the methods and conditions described above for the contact step.

[0145] Washing solution is just not particularly limited as long as it can remove the embedding medium that has dissolved on the surface of fixed tissue, the removal solution that has dissolved the embedding medium and / or the removal solution in the fixed tissue.For example, a solvent different from the solvent used in the contact step can be used as washing solution among the solvent exemplified as the removal solution.Preferably, when an organic solvent or surfactant is used as the removal solution, a water-soluble solvent is used as washing solution.As specific washing solution, for example, water, any buffer solution (phosphate buffer solution etc.), alcohol (methanol and ethanol etc.) or their mixture can be used.Can preferably use alcohol and / or buffer solution, more specifically ethanol and / or phosphate buffer solution.

[0146] Washing can be performed multiple times. In this case, the washing solution and washing conditions used for each wash can be the same each time, or they can be different for each wash. For example, when xylene is used as the removal solution, washing can be performed with an alcohol such as ethanol, and further washing can be performed with a buffer such as phosphate buffer. In this way, xylene and ethanol can be used independently in this step. For example, the fixed tissue can be contacted with xylene and then with ethanol. The concentration of the ethanol aqueous solution in this case is not particularly limited. For example, in terms of weight %, one or more concentrations of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% can be used. For example, contact with three ethanol solutions of decreasing concentration can be performed. Specifically, for example, washing can be performed by contacting with 100%, 90%, and 80% ethanol solutions in sequence.

[0147] The washing solution can be appropriately flowed during this step. The flow in this case can be artificially performed by operations such as stirring, or can be naturally performed by convection, etc. In addition, the configuration of the fixed tissue can be changed to make the fixed tissue appropriately move.

[0148] Washing may also be performed in any other steps.

[0149] 1-3-2. Antigen Activation Step (S0102)

[0150] The "antigen activation step (S0102)" is an optional step for activating cell membrane proteins in the fixed tissue. This step can be performed after the removal step (S0101). This step can be performed simultaneously with the removal step (S0101).

[0151] Antigen activation is performed by heat treatment, enzyme treatment, or a combination thereof. The specific method of antigen activation is not particularly limited, and any method known in the art can be used. For example, in this step, the methods described by Shi et al. (J Histochemistry & Cytochemistry, 2011, 59: 13-32), D'Amico et al. (J Immunological Methods, 2009, 341: 1-18), and McNicoll and Richmond (Histopathology, 1998, 32: 97-103) can be used. Whether this step is necessary and the treatment performed when implementing this step can be appropriately selected according to the type of antigen, the nature of the fixed tissue, etc.

[0152] This step includes, for example, an enzyme treatment step and / or a heat treatment step, and preferably, this step includes a heat treatment step.

[0153] <Enzyme treatment steps>

[0154] As the enzyme treatment, for example, proteases such as trypsin, DNase, proteinase K, pepsin, pronase and ficin can be used for treatment. As long as the enzyme can show activity, the treatment conditions are not particularly limited. For example, the treatment conditions can be determined based on the most suitable temperature and most suitable pH of the enzyme.

[0155] <Heat treatment step>

[0156] The heat treatment step can be performed by applying a heated antigen activating solution to the fixed tissue. The type of antigen activating solution is not particularly limited, and examples thereof include citric acid buffer, a solution containing a divalent ion chelating agent such as EDTA, tris(hydroxymethyl)methylamine (TRIS) buffer, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES) buffer, 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES) buffer, 2-(N-morpholino)ethanesulfonic acid (TAPS) buffer, N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer, N-tris(hydroxymethyl)methylglycine (Tricine) buffer, glycine-HCl buffer, periodic acid solution, urea solution, lead thiocyanate solution, and the like. The buffer may contain an acidic compound such as potassium dihydrogen phosphate, boric acid, diethylbarbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsonic acid, 2-(N-morpholino)ethanesulfonic acid, or a combination thereof.

[0157] The pH is not particularly limited, and may be, for example, in the range of 6 to 11, 7 to 10, 7.5 to 9.5, or 8 to 9.

[0158] The temperature of the heat treatment is not particularly limited and can be, for example, a temperature range of 90°C to 130°C. Specifically, for example, it can be in the range of 90°C to 130°C, 92°C to 128°C, 95°C to 125°C, or 99°C to 121°C. The heating time is not particularly limited. For example, the treatment time can be 10 minutes or more, 20 minutes or more, 25 minutes or more, 30 minutes or more, 35 minutes or more, or 40 minutes or more. In addition, for example, the treatment time can be 3 hours or less, 2 hours or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, or 45 minutes or less.

[0159] During this process, the conditions can be constant, intermittently, or continuously varied. If the conditions vary, the variation can be artificial or natural. Furthermore, the solution can be caused to flow appropriately. This flow can be artificially achieved through stirring or other manipulations, or naturally through convection or other means. Furthermore, the fixed tissue can be moved appropriately by, for example, changing its placement.

[0160] Furthermore, this step may be performed in multiple steps. In this case, each antigen activation may be performed under different conditions or under the same conditions.

[0161] 1-3-3. Cooling process

[0162] The "cooling step" is an optional step for cooling the fixed tissue. This step can be performed after the above-mentioned antigen activation step.

[0163] For example, this step can be performed when antigen activation is performed by heat treatment or when the temperature is increased for enzyme treatment.

[0164] The cooling method is not particularly limited as long as the temperature of the fixed tissue is lowered to a certain level. For example, a method of allowing the fixed tissue to stand in a low-temperature atmosphere or in a solution, a method of using a cooler such as a refrigerator or freezer, or a combination thereof can be used.

[0165] The temperature after cooling is not particularly limited. For example, it may be a freezing temperature (0°C or lower), a refrigerated temperature (0°C to 5°C), room temperature (1°C to 30°C), or normal temperature (15°C to 25°C). Cooling to room temperature or normal temperature is preferred.

[0166] There are no particular limitations on the cooling rate and cooling time. The cooling time in this step refers to the time required for the fixed tissue to cool to the target temperature. Therefore, if equilibrium is reached after the temperature is lowered and maintained, the time until equilibrium is reached is defined as the duration of this step. As needed, the fixed tissue can be stored for any length of time, such as by allowing it to rest after each step, using this step as an example.

[0167] Generally speaking, the greater the temperature difference between the temperature at the start of cooling and the temperature of the environment in which the fixed tissue is placed, the faster the cooling rate and the shorter the cooling time. The cooling time can be, for example, 10 minutes to 24 hours, 20 minutes to 16 hours, 30 minutes to 12 hours, 35 minutes to 6 hours, 40 minutes to 3 hours, 40 minutes to 2 hours, or 40 minutes to 1 hour.

[0168] In this operation, solution and atmosphere can be suitably flowed. In addition, the flow in this case can be carried out artificially by operations such as stirring, or can be carried out naturally by convection etc. In addition, the configuration of fixed tissue can be changed etc. and fixed tissue can be suitably moved.

[0169] 1-3-4. Nonspecific reaction inhibition step

[0170] The "non-specific reaction inhibition step" is an optional step in which the fixed tissue is brought into contact with a reaction inhibitor to inhibit non-specific reactions in the antibody reaction and / or color development reaction. This step can be performed after the embedding medium removal step. If the antigen activation step is performed, it can be performed simultaneously with or after it. If the cooling step is performed, it can be performed simultaneously with or after it.

[0171] This step can be roughly divided into a blocking step to inhibit nonspecific binding of antibodies and an endogenous reaction inhibition step to inhibit nonspecific color development. Only one of these steps can be performed, or both steps can be performed. Each step can be performed independently multiple times, or one or more other steps can be performed between each step.

[0172] <Blocking step>

[0173] During the blocking step, the fixed tissue is contacted with a blocking solution to inhibit nonspecific binding of the antibodies used.

[0174] The timing of performing this step is not particularly limited, as long as it is after the embedding medium removal step. In the case of an antigen activation step, it can be performed simultaneously with or after it. For example, it can be performed before, simultaneously with, or after the labeling step described below. For example, it can be performed before, simultaneously with, or after the primary antibody reaction step and the secondary antibody reaction step. This step can be performed multiple times, in which case, this step can be performed consecutively or include one or more steps in between.

[0175] As a method for sealing, any method known in the art can be used. Usually, this step is carried out by incubating the fixed tissue for a certain time under a state in which the fixed tissue has been in contact with a sealing solution. For example, when the fixed tissue is configured on a slide, a sealing solution can be placed on the fixed tissue and incubated for a certain time. In addition, for example, when the fixed tissue is suspended in a solution, the solution can be replaced with a sealing solution and incubated for a certain time.

[0176] The blocking solution generally contains protein. The type of protein contained in the blocking solution is not particularly limited. Examples thereof include animal serum protein, immunoglobulin protein, skim milk, non-fat milk, casein, and mixtures thereof.

[0177] For example, the animal from which the serum protein originates is not particularly limited. Examples thereof include serum proteins from goats, horses, humans, mice, rabbits, rats, or pigs. Specifically, for example, bovine serum albumin, fetal bovine serum, horse serum, goat serum, etc. can be used.

[0178] The solvent of sealing solution is not particularly limited, usually buffer.The kind of specific buffer is not particularly limited.For example, phosphate buffer (PBS etc.) can be used, and in specific embodiments, buffer can use tris (hydroxymethyl) methylamine (TRIS) buffer, 2- (N- morpholino) ethanesulfonic acid (TAPS) buffer, N, N- bis (2- hydroxyethyl) glycine (Bicine) buffer, N- tris (hydroxymethyl) methylglycine (Tricine) buffer, 4-2- hydroxyethyl -1- piperazineethanesulfonic acid (HEPES) buffer, 2- { [tris (hydroxymethyl) methyl] amino} ethanesulfonic acid (TES) buffer, acetate buffer, carbonate buffer, citrate buffer and their combination.

[0179] The solvent may contain appropriate additives, for example, surfactants such as Tween 20 and Triton X-100. For example, a buffer solution to which these additives have been added in advance, such as PBS-T (PBS containing 0.05% Tween 20), may be used.

[0180] The pH of the solvent is not particularly limited, but specific examples thereof include 6 to 8, 7 to 8, 7.2 to 7.8, 7.3 to 7.6, and 7.4 to 7.5.

[0181] The protein concentration is not particularly limited, and may be, for example, 0.1% to 30%, 1% to 30%, 5% to 30%, or 10% to 20% in terms of weight volume %.

[0182] <Intrinsic reaction inhibition step>

[0183] In the intrinsic reaction inhibition step, fixed tissue is exposed to a reaction inhibitor of the color development reaction to inhibit nonspecific reactions.

[0184] The timing of performing this step is not particularly limited as long as it is after the embedding medium removal step and before the color development step described below. For example, when performing the antigen activation step, it can be performed simultaneously with or after it, and before the color development step described below. For example, when performing the blocking step, it can be performed before, simultaneously with, or after it, and it can also be performed before, simultaneously with, or after the labeling step described below. For example, it can be performed before, simultaneously with, or after the primary antibody reaction step and the secondary antibody reaction step. This step can be performed multiple times, in which case, this step can be performed consecutively, or it can include one or more steps in between.

[0185] The method for inhibiting endogenous reactions is not particularly limited as long as it can inhibit the endogenous reaction of the fixed tissue that may participate in the color development reaction and hinder the detection of the label bound to the primary antibody described below. For example, it can be appropriately determined according to the type of label used and the color development reaction used.

[0186] Specifically, for example, when a fluorescent substance is used as a label, a quenching agent may be applied to the fixed tissue in order to suppress the autofluorescence of the fixed tissue.

[0187] For example, when peroxidase is used as a label, H2O2 (e.g., 3% H2O2), a peroxidase inhibitor, or a peroxidase inhibitory reagent (Peroxidase Block (DAKO)) can be used to inhibit the peroxidase activity of the fixed tissue.

[0188] For example, when biotin is used as a label, in order to reduce the amount of biotin possessed by the fixed tissue, for example, excess avidin and biotin may be applied to the fixed tissue.

[0189] For example, when phosphatase is used as a label, a phosphatase inhibitor (eg, levamisole) may be applied to the fixed tissue in order to inhibit the activity of the phosphatase in the fixed tissue.

[0190] Alternatively, this step can be performed using a reagent having two or more activities among these.

[0191] The time of this operation is not particularly limited. For example, it can be more than 15 minutes, more than 30 minutes, more than 40 minutes, or more than 60 minutes. In addition, for example, it can be less than 24 hours, less than 16 hours, less than 12 hours, less than 6 hours, less than 3 hours, or less than 2 hours.

[0192] The temperature of this step is not particularly limited and can be carried out, for example, at freezing temperature (0°C or lower), refrigeration temperature (0°C to 5°C), room temperature (1°C to 30°C), or normal temperature (15°C to 25°C).

[0193] During this process, the conditions may be constant, intermittently or continuously. When the conditions change, the change may be artificial or natural.

[0194] 1-3-5. Labeling step (S0103)

[0195] The "labeling step (S0103)" is a required step in which the cell membrane proteins of the fixed tissue are bound to the labeled antibody. This step can be performed after the embedding medium removal step (S0101). If the antigen activation step (S0102) is performed, this step can be performed simultaneously with or after the antigen activation step (S0102). If the nonspecific reaction suppression step is performed, it can be performed simultaneously with or after the blocking step.

[0196] In this step, the method for reacting the labeled antibody with the cell membrane protein in the fixed tissue is not particularly limited as long as the labeled antibody is directly or indirectly bound to the cell membrane protein in the fixed tissue.

[0197] For example, it can be performed by using a labeled antibody immunoreactive with a cell membrane protein, or by using a labeled antibody immunoreactive with a complex of a binding molecule (e.g., an antibody immunoreactive with a cell membrane protein) that binds to a cell membrane protein and the cell membrane protein.

[0198] When using a labeled antibody immunoreactive with a cell membrane protein, a step of binding the labeled antibody to the cell membrane protein is performed for labeling. This step can be performed after the embedding medium removal step. For example, when performing an antigen activation step, this step can be performed simultaneously with or after the antigen activation step. When performing a nonspecific reaction suppression step, this step can be performed simultaneously with or after the blocking step.

[0199] The details of the antibody, antibody solution, and antibody application method in this step are as described later in the "Primary Antibody Reaction Step." The details of the labeling are as described later in the "Secondary Antibody Reaction Step."

[0200] When using a binding molecule that binds to a cell membrane protein, typically, for labeling, a first binding step is performed in which the binding molecule (for example, an antibody immunoreactive with the cell membrane protein) is bound to the membrane protein, and a second binding step is performed in which a labeled antibody immunoreactive to the complex of the binding molecule and the cell membrane protein is bound to the above-mentioned complex.

[0201] The first binding step can be performed after the embedding medium removal step described above. For example, when an antigen activation step is performed, the first binding step can be performed simultaneously with or after the antigen activation step. When a nonspecific reaction inhibition step is performed, it can be performed simultaneously with or after the blocking step. The second binding step can generally be performed after the first binding step. This step can optionally be performed simultaneously with the second binding step.

[0202] The binding step can be carried out more than 3 times. In this case, the binding step after the 2nd time is carried out using a binding molecule that can bind to the complex formed in the binding step before it. In this case, the binding molecule used in the binding step except the last binding step (corresponding to the 2nd binding step) can be labeled or non-labeled binding molecule.

[0203] The type of binding molecule is not particularly limited, but is preferably a binding molecule that specifically binds to a cell membrane protein. For example, antibodies, aptamers, cyclic peptides, receptors or ligands of cell membrane proteins, or combinations thereof, can be used. The binding mode of the binding molecule to the cell membrane protein is not particularly limited. For example, the binding molecule binds to the transmembrane domain of the cell membrane protein, the portion of the cell membrane protein other than the transmembrane domain, such as a portion not embedded in the cell membrane, the intracellular domain or the extracellular domain of the cell membrane protein. The specific details about the binding properties of the binding molecule, the reaction solution, and the application method are in accordance with the content described later in relation to the "primary antibody reaction step."

[0204] The binding scheme of the labeled antibody immunoreactive with the complex of the binding molecule and the cell membrane protein to the complex is not particularly limited. For example, the labeled antibody may bind only after the complex is formed, or may bind to any of the components of the complex (cell membrane protein or binding molecule). The specific details of the labeled antibody are as described below in the "Secondary Antibody Reaction Step."

[0205] When a primary antibody immunoreactive with a cell membrane protein is used as a binding molecule and a labeled secondary antibody immunoreactive with the primary antibody is used as a labeled antibody immunoreactive with the complex, the first binding step is referred to as the primary antibody reaction step and the second binding step is referred to as the secondary antibody reaction step.

[0206] Hereinafter, the details of this step will be described by taking the case where a primary antibody reaction step and a secondary antibody reaction step are performed as an example, but as described above, this step is not limited to performing these steps.

[0207] Primary Antibody Reaction Steps

[0208] The "primary antibody reaction step" is a step in which the cell membrane proteins of the fixed tissue are reacted with a primary antibody that is immunoreactive with the cell membrane proteins. This step can be performed after the embedding medium removal step. For example, if an antigen activation step is performed, this step can be performed simultaneously with or after the antigen activation step. In particular, if a nonspecific reaction suppression step is performed, this step can be performed simultaneously with or after the blocking step.

[0209] The primary antibody used in this step is not particularly limited as long as it is an antibody that is immunoreactive with the cell membrane protein to be detected. Examples thereof include antibodies that can bind to the cell membrane protein to be detected or antibodies that can specifically bind to the cell membrane protein to be detected.

[0210] Specific antibodies can be appropriately determined based on the type of cell membrane protein to be detected. For example, it can be an antibody that can bind to the transmembrane domain of a cell membrane protein, it can also be an antibody that can bind to a portion of a cell membrane protein other than the transmembrane domain, such as a portion that is not embedded in the cell membrane, or it can be an antibody that can bind to the intracellular domain or extracellular domain of a cell membrane protein.

[0211] For example, when the cell membrane protein is CAPRIN-1 protein, an anti-CAPRIN-1 antibody or an antigen-binding fragment thereof can be used. Specific anti-CAPRIN-1 antibodies are not particularly limited. Examples thereof include WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2011 / 096535, WO2013 / 018886, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, and WO2013 / 018893. 3 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, WO2013 / 147169, WO2013 / 147176, WO2015 / 020212, WO2018 / 079740, and WO2019 / 189780.

[0212] Sometimes the primary antibody used in this step has a high binding affinity to the cell membrane protein, making detection easier. For example, the binding constant (affinity constant) K can be used. a (k on / k off ) is preferably at least 10 7 M -1 , at least 10 8 M -1 , at least 5×10 8 M -1 , at least 10 9 M -1 , at least 5×10 9 M -1 , at least 10 10 M -1 , at least 5×10 10 M -1 , at least 10 11 M -1 , at least 5×10 11 M -1 , at least 10 12 M -1 or at least 10 13 M -1 of antibodies.

[0213] The type of antibody is not particularly limited. For example, it can be any type and isotype among IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG3 and IgM. Antigen-binding fragments can be used as antibodies. Examples of antigen-binding fragments include Fab, Fv, F(ab')2 and Fab'. Furthermore, as long as the immunoreactivity with cell membrane proteins is substantially maintained, aggregates, macromolecules and conjugates of immunoglobulins or their antigen-binding fragments can also be used under appropriate circumstances.

[0214] The antibody used in this step may be either a monoclonal antibody or a polyclonal antibody.

[0215] The source of the antibody is not particularly limited. Examples thereof include human antibodies, non-human animal antibodies, recombinant antibodies, humanized antibodies, and chimeric antibodies. In the case where all or a portion of the antibody is derived from a non-human animal antibody, the non-human animal is not particularly limited. For example, antibodies derived from mice, hamsters, rats, guinea pigs, rabbits, ferrets, goats, monkeys, etc. can be used.

[0216] The method for producing and purifying the antibody used in this step is not particularly limited, and any method known in the art can be used. For example, the production and purification methods described in the literature exemplified for anti-CAPRIN-1 antibodies can be used.

[0217] As a method for the primary antibody reaction, any method known in the art can be used. Generally, this step can be performed by incubating the fixed tissue in contact with the primary antibody solution for a predetermined period of time.

[0218] The composition of the antibody solution is not particularly limited as long as it contains antibodies and the antibodies are not denatured.

[0219] The solvent of the antibody solution is not particularly limited, and for example, any buffer solution and commercially available antibody diluent can be used. The specific type of buffer solution is not particularly limited. For example, the buffer solution exemplified in the blocking step can be used, and additives can be included as needed. Among the additives, any substance known in the art can be used, and examples include animal serum and serum proteins (bovine serum albumin, fetal calf serum, goat serum, etc.), surfactants (Tween20, Triton X-100, etc.). A variety of additives can be included, and for example, fetal calf serum, goat serum and Tween20 can be included. The concentration of the additive is not particularly limited. For example, the concentration can be 0.1% to 30%, 1% to 30%, 5% to 30%, or 10% to 20% in terms of weight volume %.

[0220] The pH of the solvent is not particularly limited, but specific examples thereof include 6 to 8, 7 to 8, 7.2 to 7.8, 7.3 to 7.6, and 7.4 to 7.5.

[0221] The solvent may have the same composition as that of the blocking solution or may have a different composition.

[0222] The concentration of the antibody solution is not particularly limited as long as it can detect cell membrane proteins. For example, it can be appropriately set according to the amount of the antigen in the fixed tissue, the type of label connected to the second antibody, etc. Specifically, for example, it can be more than 0.1 μg / mL, more than 0.2 μg / mL, more than 0.3 μg / mL, more than 0.4 μg / mL, more than 0.5 μg / mL, more than 0.6 μg / mL, more than 0.7 μg / mL or more than 0.8 μg / mL. In addition, for example, it can be less than 10 μg / mL, less than 8 μg / mL, less than 7 μg / mL, less than 5 μg / mL, less than 4 μg / mL, less than 3 μg / mL, less than 2.5 μg / mL or a concentration of 2 μg / mL.

[0223] Typically, the incubation is carried out for 12 to 24 hours at a refrigerated temperature or for 15 to 40 minutes at room temperature. However, any conditions known in the art may be used as the incubation conditions, and the incubation is not particularly limited.

[0224] The incubation temperature may be, for example, freezing temperature (0°C or lower), refrigeration temperature (0°C to 5°C), room temperature (1°C to 30°C), or normal temperature (15°C to 25°C).

[0225] Examples of the incubation time include 15 minutes or longer, 30 minutes or longer, 40 minutes or longer, and 60 minutes or longer. Furthermore, for example, the incubation time may be 24 hours or shorter, 16 hours or shorter, 12 hours or shorter, 6 hours or shorter, 3 hours or shorter, or 2 hours or shorter.

[0226] The humidity conditions during incubation are not particularly limited as long as the antibody solution and the fixed tissue can remain in contact with each other during incubation. Preferably, incubation is performed in an environment with a humidity of 100% (eg, in a wet chamber).

[0227] During this step, the conditions can be constant, or can be varied intermittently or continuously. Where the conditions vary, the variation can be artificial or natural.

[0228] Secondary Antibody Reaction Steps

[0229] The "secondary antibody reaction step" is a step in which a labeled secondary antibody immunoreactive with the primary antibody is reacted with the fixed tissue. This step can usually be performed after the primary antibody reaction step. This step can optionally be performed simultaneously with the primary antibody reaction step.

[0230] The same primary antibody reaction procedure can be performed except that a labeled secondary antibody is used instead of the primary antibody.

[0231] The solvent of the antibody solution may have the same composition as that used in the primary antibody reaction step, or may have a different composition.

[0232] As long as it has immunoreactivity with the primary antibody, the type of the secondary antibody is not particularly limited. For example, when the primary antibody has a stable region, an antibody that can bind to the stable region of the primary antibody can be used as the secondary antibody. The animal from which the secondary antibody is derived is not particularly limited, but preferably the secondary antibody is derived from an animal different from the animal from which the primary antibody is derived.

[0233] The type of label is not particularly limited, and examples thereof include luminescent substances, fluorescent substances, enzymes, radioactive substances, biotin, avidin, quantum dots, enzyme substrates, enzyme cofactors, enzyme inhibitors, pigments, and metal ions.

[0234] Examples of the luminescent substance include acridinium ester, 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), luminol and its modified forms, 4-aminophthalhydrazide, various coelenterazines, and luciferin.

[0235] Examples of fluorescent substances include fluorescent dyes such as FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, TAMRA, and Alexa-Fluor dye; and fluorescent proteins such as GFP, EGFP, BFP, and YFP.

[0236] Examples of the enzyme include proteases, peroxidases, phosphatases (e.g., alkaline phosphatase), sulfatases, peptidases, glycosidases, hydrolases, oxidoreductases, lyases, transferases, isomerases, ligases, and synthetases. Peroxidases such as horseradish peroxidase and alkaline phosphatases such as calf intestinal alkaline phosphatase are preferably used.

[0237] Examples of radioactive substances include: 14 C. 123 I. 124 I. 131 I. 125 I, Tc99m, 32 P. 35 S or 3 H et al.

[0238] The label can be directly bound to the antibody, can be bound via a connector, or can form a complex with the antibody via a polymer carrier. The type of the specific connector or polymer carrier is not particularly limited. For example, a peptide connector or the like can be used as a connector well known in the art. For example, as the second antibody in this specification, an enzyme as a label and an antibody can be used as a complex formed via a polymer carrier. In addition, for example, a peroxidase as a label and an antibody can be used as a complex formed via a polymer carrier (for example, Peroxidase Labelled Polymer Conjugated (DAKO Society System) etc.).

[0239] The application method is not particularly limited and can be carried out, for example, in accordance with the application method of the blocking solution in the blocking step.

[0240] 1-3-6. Color development process

[0241] The "color development step" is an optional step in which the fixed tissue is exposed to a reagent to detect the label. This step can be performed simultaneously with or after the labeling step. For example, it can be performed simultaneously with or after the secondary antibody reaction step. If an endogenous reaction inhibition step is performed, it can be performed after that step.

[0242] The color development reaction refers to a reaction that allows detection of the label.

[0243] The reactant is not particularly limited as long as it is a reagent that causes a color development reaction of the label used. For example, a reagent containing an antibody against the label, a reagent containing a substrate when the label is an enzyme, a reagent containing avidin when the label is biotin, and a reagent containing biotin in the case of avidin can be used.

[0244] Substrate can be suitably determined based on the enzyme used as mark.For example, as the substrate using peroxidase as mark, for example, 3,3'-diaminobenzidine (DAB), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic Acid)) (ABTS), 3-amino-9-ethylcarbazole (3-amino-9-ethylcarbazole) (AEC), 3,3', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine) (TMB), Bajoran Purple (Bajoran Purple) and Wei Na Green (VinaGreen) and other color developing reagents can be enumerated.When using peroxidase as mark, preferred color developing reagent is DAB. In addition, for example, when peroxidase is used as a label, examples of substrates include colorimetric reagents such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), Nitro Blue Tetrazolium Chloride (NBT), naphthol AS phosphate, fuchsin, Ferangi Blue, Vulcan Fast Red, and Warp Red.

[0245] If necessary, additional substances or reagents that promote color development can be used. For example, when using DAB, Ni 2+ 、Co 2+ In addition, for example, when naphthol AS phosphate is used, a diazonium salt for forming an azo dye by the enzyme activity of naphthol can be used together.

[0246] The reactants may be used in combination of multiple types. For example, when biotin is used as a label, avidin and biotinylated peroxidase may be used, and a peroxidase substrate may be additionally used.

[0247] The reaction conditions of this step are not particularly limited and can be appropriately determined, for example, based on the concentration of the antibody used and the expected amount of antigen in the fixed tissue.

[0248] The reaction time is not particularly limited. For example, the reaction can be carried out for 5 seconds or longer, 10 seconds or longer, 15 seconds or longer, 16 seconds or longer, 19 seconds or longer, or 20 seconds or longer. Furthermore, the reaction can be carried out for, for example, 60 minutes or shorter, 50 minutes or shorter, 30 minutes or shorter, 25 minutes or shorter, 20 minutes or shorter, 10 minutes or shorter, 9 minutes or shorter, 8 minutes or shorter, or 7 minutes or shorter.

[0249] The temperature is not particularly limited. For example, it may be a freezing temperature (0°C or lower), a refrigerated temperature (0°C to 5°C), a room temperature (1°C to 30°C), or a normal temperature (15°C to 25°C). Preferably, the treatment is carried out at room temperature or normal temperature.

[0250] If necessary, a dehydration treatment may be performed after the color development reaction. The method of the dehydration treatment is not particularly limited. For example, the sample may be slowly dehydrated using ethanol and then further dehydrated using an organic solvent such as xylene. Specifically, for example, the sample may be immersed in aqueous ethanol solutions having concentrations of 70%, 80%, 90%, 95%, and 100% in sequence. Furthermore, the sample may be further dehydrated using xylene.

[0251] 1-3-7. Marking Detection Step (S0104)

[0252] The "label detection step (S0104)" is a required step and is a step for detecting the label bound to the cell membrane protein. This step can be performed after the labeling step (S0103). In the case of a color development step, this step can be performed simultaneously with or after the color development step. This step can be optionally performed simultaneously with the labeling step (S0103), for example, with the secondary antibody reaction step.

[0253] Before detection, other arbitrary staining can be performed in conjunction. For example, staining of the cell nucleus can be performed. The types of other staining are not particularly limited, and examples thereof include DAPI staining, hematoxylin-eosin (HE) staining, Gomori hexamine silver staining (GMS), periodic acid-Schiff (PAS) dye, trichrome blue staining, Mach-A trichrome staining, Prussian blue staining, Giemsa staining, Gram staining, mucin carmine staining, Verhoeff-van Gieson staining, elastic fiber staining, Carbofuchsin staining, Golgi and combinations thereof.

[0254] Before detection, a sealing operation can be performed. The sealing method and the sealing medium used are not particularly limited.

[0255] The method used for detection is not particularly limited. Detection method can be suitably selected according to the property of used labeling and reagent and fixed tissue, and the equipment needed for the detection method can be used. For example, it is possible to detect by visual inspection, using a microscope (for example, an optical microscope such as a stereomicroscope, a confocal microscope, a fluorescence microscope), using a detector (for example, a fluorescence activated cell sorter (FACS), a luminescence photometer, an absorptiometry photometer, etc.), or by a combination thereof. In the case where radioactive material is used as a label, for example, it is possible to detect by autoradiography, a scintillation counter, positron emission tomography (PET) or a combination thereof.

[0256] When the label is visualized by color development using a color developing reagent (for example, a color developing reagent reactive with peroxidase), it is preferably detected in a bright field using an optical microscope.

[0257] Other types of staining can be performed as needed. For example, more than one type of organelle can be stained to determine the location of the marker.

[0258] 1-4. Effect

[0259] According to the detection method of this embodiment, a target cell membrane protein (eg, CAPRIN-1 protein) present on the cell membrane can be detected with high sensitivity and specificity.

[0260] The method of this embodiment can be performed manually or automatically using an automatic staining device or the like.

[0261] 2. Methods for determining treatment

[0262] 2-1. Overview

[0263] The second embodiment of the present invention is a method for determining a treatment. This method includes, as essential steps, a disease marker detection step and a treatment determination step. This method allows determination of a treatment for the individual from whom the fixed tissue was derived.

[0264] 2-2. Methods

[0265] 2-2-1. Disease marker detection process

[0266] The "disease marker detection step" is a step of detecting a disease marker in the embedded fixed tissue.

[0267] This step can be performed according to the method for detecting cell membrane proteins described in Embodiment 1. The difference between the method described in Embodiment 1 and this step is that the cell membrane proteins detected in this step are disease markers.

[0268] Since the disease markers have been described in detail in the embedding medium removal step of the first embodiment, their description is omitted here.

[0269] The individual from whom the fixed tissue of this protocol is derived is not particularly limited and may be, for example, an individual having a disease associated with the disease marker being detected, an individual at risk of having the disease, or a healthy individual.

[0270] 2-2-2. Treatment method determination process

[0271] The "treatment determination step" is a step of determining a treatment for the individual based on the disease marker detected in the disease marker detection step. This step can be performed simultaneously with or after the disease marker detection step.

[0272] The method for determining a treatment regimen is not particularly limited, as long as it is based on the correlation between disease markers and treatment regimens. For example, the presence or absence of a disease marker in a fixed tissue can be determined, and the treatment regimen can be determined based on the correlation between the disease marker and treatment regimen.

[0273] <Determination of whether disease markers are detected>

[0274] The method for determining whether a disease marker is detected is not particularly limited. For example, a disease marker may be determined to be detected when a marker is detected, or a disease marker may be determined to be detected when the detected marker satisfies certain conditions.

[0275] The conditions here are not particularly limited. For example, the amount of labeling in fixed tissue can be quantified and the condition can be that the amount is at least a certain level.

[0276] The method of quantification is not particularly limited. For example, a score value reflecting the staining state can be calculated and quantified. The setting of the score value is, for example, classified into two or more stages, preferably four stages. The following shows an example of setting each score when the score value reflecting the staining state of the cell membrane protein expressed on the cell membrane (e.g., cell surface) is classified into four stages.

[0277] Score 0: The positive cell rate (the proportion of cells where the marker is detected on the cell membrane) is less than 10%.

[0278] Score 1: Although the positive cell rate is 10% or more, the labeling is limited to a part of the cell membrane of the diseased cells (e.g., cancer cells), and the staining intensity is weak.

[0279] Score 2: The positive cell rate is 10% or more, and the marker is localized on the cell membrane of diseased cells (e.g., cancer cells) and shows moderate staining intensity.

[0280] Score 3: Positive cell rate is 10% or more, and the marker is localized on the cell membrane of diseased cells (e.g., cancer cells) and exhibits strong staining intensity.

[0281] The setting of such a score value is stipulated in the American Society of Clinical Oncology in the United States about cancer markers, and is approved by the Japanese Society of Pathology in Japan. In addition, it is also applied to the "HercepTest" that quantifies the amount of Her2, one of cancer antigens, in a patient's sample. Regarding the quantification of Her2, it is stipulated in the ASCO / CAP Her2 inspection guideline, and in Japan, the Her2 inspection guideline including the setting of this score has been formulated by the Trastuzumab Pathology Department. For example, when detecting a protein whose amount on the cell membrane rises based on a disease as a disease marker, a score value can be set according to this example. In addition, although not particularly limited, for example, when the score is more than 1, when it is more than 2, or when it is more than 3, it can be determined that a disease marker has been detected. Preferably, when the score is more than 2 (scores 2 and 3), it is determined that a disease marker has been detected.

[0282] The proportion of diseased cells with detected markers reported for each score value can be estimated using the following formula by increasing the sensitivity of the optical microscope by 4x, 10x, or 20x, counting a minimum of 500 cells within the field of view, and measuring the number of cells showing stained images on the cell membrane for each score value.

[0283] Number of positive cells / total number of cells (around 500) × 100 (%)

[0284] In addition, for example, by comparing the amount of the marker detected in the fixed tissue as the detection object with the amount of the marker in the control tissue (e.g., fixed tissue without the disease or fixed tissue with the disease), it can be determined that the disease marker has been detected. For example, for a disease marker whose expression increases based on the disease, when the amount of the marker is more or statistically significantly more than that in the fixed tissue without the disease, it can be determined that the disease marker has been detected. In this case, on the contrary, for example, when the amount of the marker is less or statistically significantly less than that in the fixed tissue with the disease, it can be determined that the disease marker has not been detected.

[0285] As used herein, "statistically significant" means that there is a significant difference when the difference in the quantities of two substances is statistically processed. Specifically, for example, a significance level (significance level) of less than 5%, 1%, or 0.1% can be given. The test method is not particularly limited as long as it is a known method that can determine whether there is significance. For example, the Student's t-test and the multiple comparison test can be used.

[0286] Furthermore, for example, the determination can be based on a cutoff value for determining that a disease marker has been detected. For example, for a disease marker whose expression increases depending on the disease, it can be determined that the disease marker has been detected when an amount of the marker exceeding the cutoff value is detected.

[0287] <Determining the treatment method>

[0288] The method for determining a treatment is not particularly limited. For example, the treatment can be determined by comparing information on detected disease markers with information on whether or not a correspondence between various disease markers and treatments has been detected. In this case, information on multiple disease markers can be utilized, for example, by combining information on disease markers detected without using the disease marker detection process of this protocol (e.g., disease markers other than cell membrane proteins, disease markers obtained from sources other than fixed tissue, etc.) to determine the treatment.

[0289] As the information on the correspondence between whether or not a disease marker is detected and a therapeutic method, any information known in the relevant technical field can be used.

[0290] In this step, the determination of the detection of a disease marker and the determination of a therapeutic method may be performed by electronic computing equipment such as a computer, or both or either one of them.

[0291] 2-3. Effect

[0292] According to the method of this embodiment, a treatment method for an individual from which a fixed tissue is derived can be determined with high accuracy based on whether a disease marker is detected in the fixed tissue.

[0293] 3. Cell membrane protein detection device

[0294] 3-1. Overview

[0295] The third embodiment of the present invention is a device (0200) for detecting cell membrane proteins. The device of this embodiment includes an embedding medium removal unit (0210), a labeling reaction unit (0230), and a label detection unit (0240) as essential components, and includes an antigen activation reaction unit (0220), a cooling unit, a nonspecific reaction suppression unit, a color development reaction unit, and a control unit (0250) as optional components. The method of this embodiment enables highly specific detection of cell membrane proteins.

[0296] 3-2. Structure

[0297] A functional block diagram of a configuration example of the manufacturing apparatus of the present invention is shown in FIG. Figure 16 The embedding medium, fixed tissue, disease, cell membrane protein, etc. are as described in the first embodiment. The following describes the various components constituting the detection device of this embodiment.

[0298] Furthermore, the cell membrane protein in the device of this embodiment may be, for example, a cell membrane protein expressed on the surface of cancer cells, as described in the first embodiment, and may preferably be a disease marker such as a cancer marker, specifically CAPRIN-1 protein.

[0299] 3-2-1. Embedding medium removal unit (0210)

[0300] The "embedding medium removal unit (0210)" is an essential component of the apparatus of this embodiment, and is configured to remove the embedding medium by bringing fixed tissue containing a solid embedding medium into contact with a removal solution. The embedding medium removal unit is used to carry out the embedding medium removal step (S0101) of the cell membrane protein detection method described in the first embodiment. For example, the apparatus includes a storage unit (0211) for the removal solution, a contact unit (0212) for the fixed tissue and the removal solution, and a holding unit (0213) for the fixed tissue.

[0301] Storage Unit (0211)

[0302] The storage unit (0211) is a container configured to store the removal solution. The specific configuration of the storage unit for the removal solution is not particularly limited.

[0303] The overall shape of the storage unit is not particularly limited as long as it can accommodate the solution therein. For example, it can be any of a polygonal prism (including regular polygonal prisms and shapes approximately corresponding to polygonal prisms), an elliptical cylinder (including cylindrical shapes and shapes approximately corresponding to elliptical cylinders), and a sphere (including shapes approximately corresponding to a sphere).

[0304] The volume of the container is not particularly limited and can be appropriately determined based on the amount of solution used in a single application using the apparatus of this embodiment. For example, when the apparatus of this embodiment is used on a laboratory scale, a volume of approximately 10 mL to 200 mL is sufficient. However, when used on a factory scale and configured to withstand repeated applications over a certain period of time, the volume may be several liters or even tens of liters.

[0305] The material constituting the container is not particularly limited. However, considering that the container contains a solution, it is preferably a material having sufficient strength to contain the solution and sufficient rigidity to maintain its shape while containing the solution, and preferably a material that does not allow the solution to penetrate to the outside and does not decompose or denature the inner wall due to the solution. Examples thereof include, but are not limited to, metals, synthetic resins (plastics), glass, and porcelain.

[0306] Although it depends on the configuration of the contact unit, when the fixed tissue is brought into contact with the solution in the storage unit, the shape and size are not particularly limited as long as they can accommodate at least the portion of the fixed tissue that is subjected to the reaction.

[0307] Depending on the situation, in addition to the opening for passing the fixed tissue, the container may also be equipped with openings for passing liquid (discharge ports and / or water inlets, etc.), flow paths, water flow valves, flow mechanisms such as stirring blades, and filtering mechanisms for removing solid matter from the fixed tissue.

[0308] The configuration of the storage unit used in the embedding medium removal section may be the same as that of the storage unit used in one or more other sections, or may be different from any storage unit used in the other sections.

[0309] Contact unit (0212)

[0310] The contact unit (0212) is configured to bring the fixed tissue held in the holding unit (0213) into contact with the removal solution stored in the storage unit (0211). The specific configuration of the contact unit is not particularly limited. For example, it may be a unit that sprays, spreads, or applies the removal solution to the fixed tissue, or it may be a unit that immerses the fixed tissue in the removal solution.

[0311] When the holding unit is located away from the storage unit, any mechanism for reducing the distance therebetween may be provided, such as a mechanism for moving the holding unit and / or the storage unit, a flow path capable of transporting the removal solution from the storage unit to the vicinity of the holding unit, or a combination thereof.

[0312] The structure of the contact unit used in the embedding medium removal section may be the same as that of the contact unit used in one or more other sections, or may be different from any contact unit used in other sections. In addition, it may be configured so as to be commonly used in two or more sections.

[0313] <Holding unit (0213)>

[0314] The holding unit (0213) is configured to hold a fixed tissue containing a solid embedding medium. The specific configuration of the holding unit for the fixed tissue is not particularly limited. For example, a holding unit commonly used in immunochemical staining of fixed tissue can be used. For example, the holding unit can be configured to hold, clamp, or support the fixed tissue, or can be configured to accommodate the fixed tissue therein.

[0315] The structures capable of being grasped include any of structures that are integrated with each other and any structures that can be installed and removed, such as holding units using magnetic forces such as permanent magnets or temporary magnets, hanging units such as hooks or ropes, restraining units such as chucks, or combinations thereof, without any particular limitation.

[0316] The clampable structure includes any of a structure that clamps the entire fixed tissue (such as a structure in which the fixed tissue is arranged in a space between partitions) and a structure that clamps a part of the fixed tissue (such as a structure in which the fixed tissue is arranged upright between low partitions), without particular limitation.

[0317] The structure that can be placed includes any structure that can place fixed tissue, and is not particularly limited. The surface shape of the placement surface is not particularly limited, and can be a structure that can limit the position of the fixed tissue, or a structure that does not limit the position of the fixed tissue. In addition, it can be a shape with more than one hole on its surface (a shape composed of linear structures that cross or do not cross each other, etc.), or it can be a shape without holes. In the case of having holes, as long as the fixed tissue does not fall off, the size, number and shape of the holes are not particularly limited.

[0318] The structure capable of accommodating a fixed tissue internally includes any structure capable of accommodating a fixed tissue internally and having an internal space connected to an external space, without particular limitation. The structure in which the internal space is connected to the external space includes any of a structure that always maintains communication with the external space and a structure that is connected to the external space only under specific conditions. As a structure that always maintains communication with the external space, for example, a structure that surrounds the fixed tissue with a material having holes such as a mesh, cloth, filter, or a combination thereof, or a structure that accommodates the fixed tissue inside the material. As a structure that is connected to the external space only under specific conditions, for example, a structure (for example, a container, etc.) that accommodates the fixed tissue inside a space composed of a material that does not have holes that can penetrate a drug can be mentioned. In this case, an opening that can be opened and closed according to conditions can be added.

[0319] The material of the holding unit is not particularly limited. For example, fiber materials, organic resins, glass-based materials, metal materials, or combinations thereof can be used. In addition, the holding unit can be composed of a rigid body or at least a portion thereof can be composed of a soft body.

[0320] When the holding unit has a portion that comes into contact with the drug, the portion is preferably made of a material that is not decomposed or denatured by the drug.

[0321] exist Figure 16 In the embodiment described above, the fixed tissue is held by a single holding unit until it reaches the marker detection unit (0240). However, regardless of the differences in the configuration of the holding unit, each unit may be provided with a holding unit independently of the other units, and the fixed tissue may be transferred between the holding units of different units. Furthermore, the configuration of the holding unit used in the embedding medium removal unit may be the same as that used in one or more other units, or may be different from any holding unit used in the other units.

[0322] Temperature Control Unit

[0323] The temperature control unit is configured to control the temperature of the fixed tissue. In particular, the temperature control unit is configured to maintain the fixed tissue at a temperature lower than the melting point of the embedding medium. The specific configuration of the temperature control unit is not particularly limited.

[0324] For example, it can be constructed in a manner that can directly adjust the temperature of the fixed tissue itself, or it can be constructed in a manner that can adjust the temperature of the solution surrounding the fixed tissue. The temperature control unit can be constructed in a manner that can keep heat, or it can be constructed in a manner that can change the temperature. As a structure that can keep heat, for example, a heat insulating member, a gas layer, a vacuum layer, or a structure using a combination thereof can be cited. In the case of a structure that can change the temperature, it can be constructed in a manner that can perform both heating and cooling in one body, or it can be constructed in a manner that can only perform either one. Specifically, for example, a thermostat, a heater and a cooler for liquid or solid, an exothermic agent and an endothermic agent using a chemical reaction, a storage tank for a high-temperature or low-temperature solution, etc. can be cited. The temperature control unit can be installed in any one or more of the holding unit (0213), the contact unit (0212), and the storage unit (0211), or it can be provided with a temperature control unit separately from them.

[0325] The fixed tissue is preferably maintained at a temperature below the melting point of the embedding medium by a temperature control unit. The temperature to be controlled can be appropriately set according to the type of embedding medium and is not particularly limited. The temperature is as described in the first embodiment.

[0326] Washing unit

[0327] The washing unit is configured to wash the surface of the fixed tissue. The specific configuration of the washing unit is not particularly limited. For example, it can be configured to store or supply the washing solution described in the first embodiment. For example, the configuration capable of storing the washing solution is similar to the above-described storage unit (0211), except that the stored liquid is a washing solution instead of a removal solution.

[0328] The structure of the washing unit used in the embedding medium removal section may be the same as that used in one or more other sections, or may be different from any washing unit used in other sections. In addition, it may be configured so as to be commonly used in two or more sections.

[0329] 3-2-2. Antigen activation reaction part (0220)

[0330] The antigen activation reaction section (0220) is an optional component of the apparatus of this embodiment, and is configured to activate cell membrane proteins in the fixed tissue that has passed through the embedding medium removal section (0210). The antigen activation reaction section (0220) is used to carry out the antigen activation step (S0102) of the cell membrane protein detection method described in the first embodiment. The antigen activation reaction section (0220) typically includes an antigen activation unit (0221), a contact unit (0222), a fixed tissue holding unit, and a washing unit.

[0331] The contact unit (0222), the holding unit and the washing unit are described in accordance with the description of each unit in the embedding medium removal unit (0210).

[0332] <Antigen Activation Unit (0221)>

[0333] The antigen activation unit (0221) is configured to activate cell membrane proteins of fixed tissues. The specific configuration of the antigen activation unit (0221) can be appropriately selected depending on the type of antigen activation treatment to be performed and is not particularly limited.

[0334] For example, when the antigen activation treatment is based on an enzyme treatment step, the enzyme solution storage unit described in the first embodiment can be used, and when the antigen activation treatment is based on a heat treatment step, the antigen activation solution storage unit described in the first embodiment can be used. The storage units for various liquids are similar to the storage unit for the removal solution (0211), except when the stored solution is an enzyme solution or an antigen activation solution.

[0335] The antigen activation unit is preferably configured so that the temperature of the liquid can be adjusted. The specific configuration is not particularly limited. The temperature to be adjusted is the temperature during the antigen activation step described in the first embodiment, and is similar to the temperature control unit in the embedding medium removal unit (0210). Even when only one step is performed at a time, a storage unit for solutions corresponding to both steps may be provided. Furthermore, storage units for multiple solutions may be provided for each of the enzyme solution and the antigen activation solution.

[0336] 3-2-3. Cooling section

[0337] The cooling unit is an optional component of the apparatus of this embodiment, and is configured to cool the fixed tissue that has passed through the antigen activation reaction unit (0220). The cooling unit is used to implement the cooling step in the cell membrane protein detection method described in the first embodiment. The cooling unit generally includes a cooling unit and a holding unit for the fixed tissue, and may further include a contact unit and a washing unit depending on the situation.

[0338] The holding unit, contact unit and washing unit are described in accordance with the description of each unit in the embedding medium removal unit (0210).

[0339] Cooling unit

[0340] The cooling unit is configured to lower the temperature of the fixed tissue in contact. The specific configuration is not particularly limited as long as it can lower the temperature of the fixed tissue. For example, it can be configured to release heat from the fixed tissue or to absorb heat from the fixed tissue. The specific configuration is not particularly limited, but is basically the same as the temperature control unit in the embedding medium removal section (0210), except that the temperature to be controlled is the temperature in the cooling process of the first embodiment, for the purpose of cooling. Alternatively, only the cooling function of the unit that can also heat the tissue may be used.

[0341] In particular, for cooling herein, the fixed tissue may be contacted with any solid, solution, gas or combination thereof. Thus, the cooling unit may be configured to contact the fixed tissue with a solid, solution, gas or the like having a temperature lower than that of the fixed group.

[0342] 3-2-4. Nonspecific reaction inhibition section

[0343] The nonspecific reaction inhibition section is an arbitrary component of the device of this embodiment, and is configured in a manner that enables the fixed tissue that has passed through the embedding medium removal section (0210) to contact the reaction inhibitor. Depending on the circumstances, it can be configured in a manner that can process the fixed tissue that has passed through the antigen activation reaction section (0220) and / or the cooling section. The nonspecific reaction inhibition section is used to implement the nonspecific reaction inhibition step in the cell membrane protein detection method described in the first embodiment. The nonspecific reaction inhibition section usually includes a reaction inhibitor storage unit, a contact unit, and a holding unit for the fixed tissue, and, depending on the circumstances, further includes a washing unit.

[0344] The contact unit, holding unit and washing unit are described in the same manner as the respective units in the embedding medium removal unit (0210).

[0345] The specific structure of the reaction inhibitor storage unit can be appropriately selected according to the type of nonspecific reaction inhibition treatment to be implemented, and is not particularly limited. For example, when the nonspecific reaction inhibition treatment is based on a blocking step, a storage unit for the blocking solution described in the first embodiment can be used. When the nonspecific reaction inhibition treatment is based on an endogenous reaction inhibition step, a storage unit for the reaction inhibitor of the color development reaction described in the first embodiment can be used. Regarding the storage units for various liquids, except when the stored solution is a reaction inhibitor, a storage unit for a removal solution can be used. Even if only one step is performed in a single implementation, a storage unit for solutions corresponding to two steps can be provided. In addition, a storage unit for multiple reaction inhibitors can be provided.

[0346] 3-2-5. Labeling reaction section (0230)

[0347] The labeling reaction section (0230) is an essential component of the device of this scheme, and is constructed in a manner that allows the above-mentioned cell membrane protein of the fixed tissue that has passed through the embedding medium removal section (0210) to react with the labeled antibody that has immunoreactivity with the above-mentioned cell membrane protein. Depending on the situation, it can be constructed in a manner that can process the fixed tissue that has passed through the antigen activation reaction section (0220), the cooling section and / or the non-specific reaction inhibition section. The labeling reaction section (0230) is used to implement the labeling step (S0103) in the detection method of the cell membrane protein described in the first scheme. The labeling reaction section (0230) usually includes a labeled antibody storage unit (0233), its contact unit (0234) and a holding unit for the fixed tissue, and depending on the situation, further includes a binding molecule storage unit (0231), its contact unit (0232), and a washing unit.

[0348] The contact units (0232, 0234), the holding unit and the washing unit are described in accordance with the description of each unit in the embedding medium removing section (0210).

[0349] When multiple binding steps are performed, a binding molecule storage unit (0231) for storing binding molecules used in each binding step may be provided. Specifically, for example, when a primary antibody reaction step and a secondary antibody reaction step are performed, a storage unit for the primary antibody solution described in the first embodiment may be provided as the binding molecule storage unit (0231), and a storage unit for the secondary antibody solution described in the first embodiment may be provided as the labeled antibody storage unit (0233).

[0350] The storage unit for each solution is similar to the storage unit for the removal solution (0211), except that the stored solution is the antibody solution or the binding molecule solution described in the first embodiment. Even when only a binding reaction using a labeled antibody is performed in one experiment, a storage unit for the binding molecule solution may be provided. Furthermore, storage units for multiple antibodies or binding molecules may be provided.

[0351] 3-2-6. Color reaction section

[0352] The color development reaction section is an optional component of the device of this embodiment, and is configured to allow the fixed tissue that has passed through the labeling reaction section (0230) to be exposed to a reagent for detection. The color development step in the cell membrane protein detection method described in the first embodiment is carried out through the color development reaction section. The color development reaction section generally includes a reagent storage section, a contact unit, and a holding unit for the fixed tissue, and may further include a washing unit as needed.

[0353] The contact unit, holding unit, and washing unit are described in the embedding medium removal unit (0210). The reagent storage unit is described in the removal solution storage unit (0211), except that the stored solution is the reagent described in the first embodiment.

[0354] 3-2-7. Mark Detection Unit (0240)

[0355] The label detection unit (0240) is an essential component of the device of this embodiment and is configured to detect the labeled antibody bound to the immobilized tissue after passing through the labeling reaction unit. The label detection step (S0104) of the cell membrane protein detection method described in the first embodiment is carried out by the label detection unit (0240). The label detection unit generally includes a detection unit (0241), an analysis unit (0242), and a fixed tissue holding unit.

[0356] Regarding the holding unit, follow the description of the holding unit (0213) in the embedding medium removing section (0210).

[0357] <Detection unit (0241)>

[0358] The detection unit (0241) is configured to detect a label bound to a cell membrane protein in a fixed tissue. The configuration of the detection unit (0241) can be appropriately selected based on the label and reagent used, and the properties of the fixed tissue, and is not particularly limited.

[0359] Specifically, the apparatus may include, for example, a microscope (e.g., an optical microscope such as a stereo microscope, a confocal microscope, or a fluorescence microscope), a detector (e.g., a fluorescence activated cell sorter (FACS), a luminescence photometer, an absorptiometry photometer, etc.), an autoradiography apparatus, a scintillation counter, a positron emission tomography (PET), or a combination thereof. Various detection units may be provided as needed.

[0360] <Analysis Unit (0242)>

[0361] The analyzing unit (0242) is configured to analyze the signal data of the marker detected by the detecting unit (0241). The configuration of the analyzing unit (0242) can be appropriately selected according to the detecting unit (0241) used and is not particularly limited.

[0362] For example, an analysis unit commonly used in analyzing signal data using each detection unit may be provided, or an analysis mechanism built into the detection unit used may be used as the analysis unit of this embodiment. Typically, the analysis unit includes a computer. When the analysis unit includes a computer, the specific configuration of the hardware and software components is not particularly limited; for example, the configuration described below regarding the control unit may be employed.

[0363] The specific content of the analysis processing performed here is not particularly limited. For example, it may include converting the signal into image data or removing noise from the signal data. If necessary, the system may be configured to further perform a determination process for whether a disease marker as described in the second embodiment has been detected.

[0364] The analyzing unit may further include an output mechanism for displaying the analysis result to a user.

[0365] 3-2-8. Control Unit (0250)

[0366] The control unit (0250) is an optional component of the apparatus of this embodiment, and is configured to adjust the communication between each unit and the fixed tissue and the treatment conditions in each unit. For example, the control unit (0250) allows the fixed tissue to communicate with a specified unit at a specified time, and to be treated under specified treatment conditions (such as the type of reaction solution and temperature conditions) for a specified period of time.

[0367] The control unit may be configured so that the apparatus of this embodiment can execute the series of methods described in the first embodiment, or may be configured so that a portion of the steps of the method described in the first embodiment can be executed. Typically, the control unit includes a computer.

[0368] The control unit is composed of a hardware unit, or a hardware unit and a software unit. The hardware unit is composed of a CPU (0251), a volatile memory (0252), a non-volatile memory (0253), an interface (0254), a system bus (0255) connecting these, and peripheral devices. The peripheral devices are not limited, and examples thereof include clocks and water level sensors. The software unit is composed of programs that can be executed on the hardware memory.

[0369] In the control unit, for example, various programs (e.g., a communication control program, a processing condition control program) stored in the non-volatile memory (0253) of the hardware unit are expanded on the volatile memory (0252) and executed sequentially, thereby realizing the functions of each unit by processing, storing, and outputting the data on the memory and the data input through the interface (0254). For example, by activating the contact unit when a programmed predetermined time has elapsed, the fixed tissue can be brought into contact with the target drug, etc., and after a certain period of time, the contact unit is activated again to stop the contact. In addition, for example, by activating the temperature control unit and the cooling unit when a programmed predetermined time has elapsed, the fixed tissue can be adjusted to a predetermined temperature, and after a certain period of time, the contact unit is activated again to stop the contact. Furthermore, in the reaction in each part, when information that the conditions deviate from the target is input from various sensors such as temperature sensors and pH sensors via the interface (0254), the reaction condition control program developed on the volatile memory (0252) can be executed to add reagents and / or operate the temperature control unit in a manner so as to adjust the conditions to within the target range.

[0370] If necessary, a display unit for presenting information to the user may be provided, and the content of the information is controlled by the display control unit. The hardware of the display unit is not particularly limited, and examples thereof include a display.

[0371] In addition, as needed, an input device for the user to input information may be provided. Examples of the input device include a keyboard, a mouse, a touch panel, and a stylus. The information input from the input device is controlled by the input / output control unit. In addition to the input of data and commands from the input device, the input / output control unit also controls the output of various data output from the processing device. The information required to be input by the user is not particularly limited. For example, in addition to the time of each process and the setting of reaction conditions, the user may be required to specify the process to be implemented and the benchmark for updating the solution in the storage unit (for example, turbidity, time, etc.).

[0372] Example

[0373] Hereinafter, the present invention will be described in more detail based on examples, but the scope of the present invention is not limited to these examples.

[0374] Example 1: Detection of CAPRIN-1 protein using immunohistochemical staining

[0375] (Paraffin removal process)

[0376] From various paraffin-embedded human cancer tissue sections (breast cancer tissue, lung cancer tissue, pancreatic cancer tissue, kidney cancer tissue, colorectal cancer tissue, gastric cancer tissue, ovarian cancer tissue and prostate cancer tissue; manufactured by USBIOMAX; using sections of different samples from 1,361 people) with a thickness of 4 μm, paraffin was removed using xylene for 20 minutes. Xylene was replaced every 5 minutes, and replaced 3 times. Next, 100%, 90%, and 80% ethanol aqueous solutions were used instead of xylene, and washing was performed for 5 minutes each using the same steps. These steps were performed at room temperature. Then, PBS-T (phosphate buffer (PBS) containing 0.05% Tween20, pH 7.4) was used to wash 3 times for 5 minutes each.

[0377] (Antigen Activation Process)

[0378] Various human cancer tissue sections that had undergone the paraffin removal process were placed in a staining bottle filled with 10 mM citrate buffer (pH 6.0) containing 0.05% Tween 20, and heated at 9°C for 40 minutes. The sections were then allowed to stand at room temperature for 40 minutes or more to cool.

[0379] (Nonspecific inhibition reaction step)

[0380] (Peroxidase endogenous reaction inhibition step)

[0381] Excess water around the sections of various human cancer tissues that had undergone the antigen activation step was removed, and an appropriate amount of Peroxidase Block (manufactured by DAKO) was added dropwise and allowed to stand at room temperature for 5 minutes.

[0382] (Protein blocking)

[0383] After washing three times with PBS-T for 5 minutes each time, a PBS-T solution containing 10% FBS was added as a blocking solution, and the cells were incubated in a humidified chamber at room temperature for 1 hour.

[0384] (Labeling process: primary antibody reaction step)

[0385] A primary antibody solution containing the mouse anti-CAPRIN-1 monoclonal antibody prepared in WO2013 / 018891 was added to sections of various human cancer tissues that had undergone a nonspecific inhibition reaction step and incubated overnight at 4°C in a humidified chamber. The primary antibody solution was prepared by adding the mouse anti-CAPRIN-1 antibody to a PBS-T solution containing 5% FBS and 20% normal goat serum at a final concentration of 2 μg / mL.

[0386] (Labeling process: secondary antibody reaction step)

[0387] After the primary antibody reaction step, various human cancer tissue sections were washed three times with PBS-T for 10 minutes each time, and then an appropriate amount of Peroxidase Labeled Polymer Conjugated (manufactured by BIOCARE) was added dropwise to the sections and incubated at room temperature in a humidified chamber for 30 minutes.

[0388] (Color development process)

[0389] After undergoing the secondary antibody reaction step, sections of various human cancer tissues were washed three times with PBS-T for 10 minutes each time. DAB colorimetric solution (manufactured by BIOCARE) was then added to the sections and allowed to stand at room temperature for approximately 20 seconds to develop color. The colorimetric solution was then discarded, and the sections were washed three times with PBS-T for 10 minutes each time, followed by rinsing with distilled water. The sections were then immersed in aqueous ethanol solutions at concentrations of 70%, 80%, 90%, 95%, and 100% for 1 minute each, followed by dehydration by incubation in xylene overnight.

[0390] (Seal)

[0391] Various human cancer tissue sections that had undergone the color development step were mounted using cover glasses and Glycergel Mounting Medium (manufactured by BIOCARE) as a mounting medium.

[0392] (observe)

[0393] Using a 4x objective lens of an optical microscope, CAPRIN-1 protein staining images of cancer cells in mounted sections of cancer tissue were observed.

[0394] The staining images obtained for ovarian cancer tissue are shown in Figure 1 In the figure, A shows the staining image of the cell nucleus and CAPRIN-1 protein, and B shows the staining image of CAPRIN-1 protein with the cell nucleus signal excluded. As indicated by the arrow in the figure, the signal of CAPRIN-1 protein localized on the cell membrane is observed at the cell edge ( Figure 1 B).

[0395] Therefore, it was found that this staining method can detect the signal on the cell membrane of the cell membrane protein with high precision.

[0396] Furthermore, it is understood that in this embodiment, as the antibody used in the primary antibody reaction step, even if applied to WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2 The mouse anti-CAPRIN-1 monoclonal antibodies or rabbit anti-CAPRIN-1 monoclonal antibodies produced in WO2013 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, and WO2015 / 020212 can also detect cell membrane signals of cell membrane proteins with high precision as described above.

[0397] Comparative Example 1: CAPRIN-1 protein detection using immunohistochemical staining

[0398] Paraffin-embedded sections of various human cancer tissues (manufactured by BIOMAX) derived from the same samples as in Example 1 were subjected to immunohistochemical staining according to the procedures described in Example 1, except that they were heat-treated (baked) at 62°C for 15 minutes prior to paraffin removal in the paraffin removal step and were heat-treated at 121°C in the antigen activation step.

[0399] The staining images obtained for ovarian cancer tissue are shown in Figure 2As shown by the arrow in the figure, the CAPRIN-1 protein is localized on the cell membrane, but the signal of the CAPRIN-1 protein is observed not only on the cell membrane but also in the cytoplasm, and the signal is observed in a manner that the entire cell is covered ( Figure 2 B).

[0400] Therefore, it was found that, unlike the staining method of Example 1, the detection accuracy of the signal on the cell membrane of the cell membrane protein was significantly reduced by performing baking.

[0401] Example 2: Determination of CAPRIN-1-positive cancer cells

[0402] First, using a 4x objective lens on an optical microscope, images of CAPRIN-1 protein staining and the signal intensity of positive staining were observed in cancer cells within the mounted sections of cancer tissue in Example 1 and Comparative Example 1. Next, the objective lens was switched to 10x or 20x, and the ratio of cancer cells with specific staining observed on the cell membrane relative to all cells in the cancer tissue (CAPRIN-1 positive cell ratio) was calculated in all sections derived from each sample.

[0403] Based on the CAPRIN-1 positive cell rate and the local presence of CAPRIN-1 signals, each cancer tissue sample was classified into the following scores of 0 to 3. Among these scores, scores 2 and 3 were determined to be CAPRIN-1 positive cancer tissue samples.

[0404] Score 0 (no CAPRIN-1 protein overexpression): The CAPRIN-1 positive cell rate (the proportion of cells that specifically express signals on the cell membrane) is less than 10%.

[0405] Score 1 (no overexpression of CAPRIN-1 protein): The CAPRIN-1 positive cell rate is 10% or higher, but the signal is limited to a part of the cancer cell membrane and the staining intensity is weak.

[0406] Score 2 (CAPRIN-1 protein overexpression): The CAPRIN-1 positive cell rate is 10% or more, and the signal is localized on the membrane of cancer cells, with a moderate staining intensity.

[0407] Score 3 (CAPRIN-1 protein overexpression): The CAPRIN-1 positive cell rate is 10% or more, and the signal is localized on the membrane of cancer cells, with strong staining intensity.

[0408] Tissue samples with scores of 2 and 3 were determined to be CAPRIN-1-positive cancer tissue samples.

[0409] The illustrative staining images for each score are shown in Figures 3 to 10 For example, in gastric cancer tissue, Figure 2 The sample with fraction 0 as shown ( Figure 3 ) and the sample with fraction 1 ( Figure 4 ) were judged as CAPRIN-1 negative. The signal on the cell membrane of these samples was weak and the boundaries between cells were unclear. On the other hand, in gastric cancer tissues, Figure 5 and 6 The sample of fraction 2 as shown ( Figure 5 ) and samples of fraction 3 ( Figure 6 ) were identified as CAPRIN-1-positive samples. In these samples, strong signals were observed on the cell membrane, and the boundaries between cells were clear.

[0410] The same determination was made in other cancer tissues. For example, in renal cancer tissue, the signal on the cell membrane was weak. Figure 7 and 8 The sample with fraction 0 as shown ( Figure 7 ) and the sample with fraction 1 ( Figure 8 ) was judged as CAPRIN-1 negative. On the other hand, strong signal on the cell membrane was observed. Figure 9 and 10 The sample of fraction 2 as shown ( Figure 9 ) and samples of fraction 3 ( Figure 10 ) samples were judged to be CAPRIN-1 positive.

[0411] The results of the determination showed that the proportion of CAPRIN-1-positive cancer tissue samples among the samples stained in Example 1, that is, the ratio of CAPRIN-1-positive cancer tissue samples to the number of each cancer tissue sample, was 75% for breast cancer tissue, 65% for lung cancer tissue, 70% for pancreatic cancer tissue, 70% for kidney cancer tissue, 60% for colorectal cancer tissue, 60% for gastric cancer tissue, 65% for ovarian cancer tissue, and 90% for prostate cancer tissue.

[0412] On the other hand, when determination was performed based on the samples stained in Comparative Example 1, the proportion of CAPRIN-1-positive cancer tissue samples was 52% for breast cancer tissue, 54% for lung cancer, 58% for pancreatic cancer, 55% for kidney cancer, 49% for colorectal cancer, 40% for stomach cancer, 45% for ovarian cancer, and 54% for prostate cancer, which were results that were significantly different from the determination results based on staining in Example 1.

[0413] Therefore, it can be seen that the reduction in dyeing accuracy due to baking in the paraffin removal step also has a significant influence on the determination results.

[0414] In addition, in this comparative example, as the antibody used in the primary antibody reaction step, even if the antibodies described in WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2013 / 018894, WO2013 / 018892, WO2013 / 01889 1. The same decrease in staining accuracy as described above was also observed with the mouse anti-CAPRIN-1 monoclonal antibodies or rabbit anti-CAPRIN-1 monoclonal antibodies prepared in WO2013 / 018889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640, and WO2015 / 020212.

[0415] Example 3: Detection of CAPRIN-1 protein using immunohistochemical staining

[0416] Various paraffin-embedded human cancer tissue sections (manufactured by BIOMAX) derived from the same samples as in Example 1 were subjected to immunohistochemical staining as described in Example 1, except that the antibody concentration of the primary antibody solution used in the primary antibody reaction step was set to 0.8 μg / mL and the development time in the color development step was set to approximately 7 minutes. CAPRIN-1-positive cancer tissue samples were identified in the same manner as in Example 2.

[0417] The staining images obtained for ovarian cancer tissue are shown in Figure 12 It can be seen that even if the antibody concentration and the color development time are changed, the staining image ( Figure 11 )Dyeing with the same precision.

[0418] The results of the determination showed that the proportion of CAPRIN-1-positive cancer tissues was 80% in breast cancer tissue, 70% in lung cancer, 80% in pancreatic cancer, 80% in kidney cancer, 70% in colorectal cancer, 55% in gastric cancer, 55% in ovarian cancer, and 80% in prostate cancer, which was roughly consistent with the determination results based on staining in Example 1.

[0419] In addition, in this embodiment, as the antibody used in the primary antibody reaction step, even if applied to WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2013 / 01 The mouse anti-CAPRIN-1 monoclonal antibodies or rabbit anti-CAPRIN-1 monoclonal antibodies prepared in WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640 and WO2015 / 020212 were also used to obtain the same proportion of CAPRIN-1-positive cancer tissue samples in the number of each cancer tissue sample as above.

[0420] Example 4: Detection of CAPRIN-1 protein using immunohistochemical staining

[0421] Various paraffin-embedded human cancer tissues (manufactured by BIOMAX) derived from the same samples as in Example 1 were subjected to heat treatment at 121°C in the antigen activation step. Immunohistochemical staining was performed as described in Example 1, and CAPRIN-1-positive cancer tissue samples were determined in the same manner as in Example 2.

[0422] The staining images obtained for ovarian cancer tissue are shown in Figure 13 It can be seen that even if the heating temperature in the antigen activation step is changed, the staining image obtained in Example 1 ( Figure 11 )Dyeing with the same precision.

[0423] The results of the determination showed that the proportion of CAPRIN-1-positive cancer tissue samples was 77% in breast cancer tissue, 67% in lung cancer, 75% in pancreatic cancer, 75% in kidney cancer, 65% in colorectal cancer, 60% in gastric cancer, 60% in ovarian cancer, and 85% in prostate cancer, which was roughly consistent with the determination results based on staining in Example 1.

[0424] In addition, in this embodiment, as the antibody used in the primary antibody reaction step, even if applied to WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2013 / 01 The mouse anti-CAPRIN-1 monoclonal antibodies or rabbit anti-CAPRIN-1 monoclonal antibodies prepared in WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640 and WO2015 / 020212 were also used to obtain the same proportion of CAPRIN-1-positive cancer tissue samples in the number of each cancer tissue sample as above.

[0425] Example 5: Detection of CAPRIN-1 protein by immunohistochemical staining

[0426] Various paraffin-embedded human cancer tissue sections (manufactured by BIOMAX) derived from the same samples as in Example 1 were subjected to immunohistochemical staining as described in Example 1, except that the antigen activation step was heat treated at 121°C, the antibody concentration of the primary antibody solution used in the primary antibody reaction step was set to 0.8 μg / mL, and the color development time in the color development step was set to approximately 7 minutes. CAPRIN-1-positive cancer tissue samples were identified in the same manner as in Example 2.

[0427] The staining images obtained for ovarian cancer tissue are shown in Figure 14 It can be seen that even if the heating temperature, antibody concentration and color development time in the antigen activation process are changed, the staining image obtained in Example 1 can be obtained ( Figure 11 )Dyeing with the same precision.

[0428] The results of the determination showed that the proportion of CAPRIN-1-positive cancer tissue samples was 79% in breast cancer tissue, 68% in lung cancer, 77% in pancreatic cancer, 76% in kidney cancer, 62% in colorectal cancer, 62% in gastric cancer, 61% in ovarian cancer, and 86% in prostate cancer, which was roughly consistent with the determination results based on staining in Example 1.

[0429] In addition, in this embodiment, as the antibody used in the primary antibody reaction step, even if applied to WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2013 / 01 The mouse anti-CAPRIN-1 monoclonal antibodies or rabbit anti-CAPRIN-1 monoclonal antibodies prepared in WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640 and WO2015 / 020212 were also used to obtain the same proportion of CAPRIN-1-positive cancer tissue samples in the number of each cancer tissue sample as above.

[0430] Example 6: Detection of CAPRIN-1 protein using immunohistochemical staining

[0431] Various paraffin-embedded human cancer tissue sections (BIOMAX) derived from the same samples as in Example 1 were subjected to the paraffin removal step as described below. Immunohistochemical staining was performed as described in Example 1, and CAPRIN-1-positive cancer tissue samples were identified in the same manner as in Example 2.

[0432] (Paraffin removal process)

[0433] Paraffin-embedded human cancer tissues (BIOMAX) were paraffin-deparaffinized using Artisan Cleaning Solution (Agilent) over 15 minutes. The Artisan Cleaning Solution was replaced every 5 minutes twice. This process was performed at room temperature. The tissues were then washed three times with PBS-T, each for 5 minutes.

[0434] The results of the determination showed that the proportion of CAPRIN-1-positive cancer tissue samples was 78% in breast cancer tissue, 66% in lung cancer, 76% in pancreatic cancer, 79% in kidney cancer, 63% in colorectal cancer, 64% in gastric cancer, 62% in ovarian cancer, and 88% in prostate cancer, which was roughly consistent with the determination results based on staining in Example 1.

[0435] In addition, in this embodiment, as the antibody used in the primary antibody reaction step, even if applied to WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2013 / 01 The mouse anti-CAPRIN-1 monoclonal antibodies or rabbit anti-CAPRIN-1 monoclonal antibodies prepared in WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640 and WO2015 / 020212 were also used to obtain the same proportion of CAPRIN-1-positive cancer tissue samples in the number of each cancer tissue sample as above.

[0436] Example 7: Detection of CAPRIN-1 protein using immunohistochemical staining

[0437] Various paraffin-embedded human cancer tissue sections (manufactured by BIOMAX) derived from the same samples as in Example 1 were subjected to immunohistochemical staining in the same manner as in Example 6, except that the antibody concentration of the primary antibody solution used in the primary antibody reaction step was set to 0.8 μg / mL and the development time in the color development step was set to approximately 7 minutes. CAPRIN-1-positive cancer tissue samples were identified in the same manner as in Example 2.

[0438] The results of the determination showed that the proportion of CAPRIN-1-positive cancer tissue samples was 77% in breast cancer tissue, 67% in lung cancer, 72% in pancreatic cancer, 72% in kidney cancer, 68% in colorectal cancer, 58% in gastric cancer, 64% in ovarian cancer, and 82% in prostate cancer, which was roughly consistent with the determination results based on staining in Example 1.

[0439] In addition, in this embodiment, as the antibody used in the primary antibody reaction step, even if applied to WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2013 / 01 The mouse anti-CAPRIN-1 monoclonal antibodies or rabbit anti-CAPRIN-1 monoclonal antibodies prepared in WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640 and WO2015 / 020212 were also used to obtain the same proportion of CAPRIN-1-positive cancer tissue samples in the number of each cancer tissue sample as above.

[0440] Example 8: Detection of CAPRIN-1 protein using immunohistochemical staining

[0441] Various paraffin-embedded human cancer tissue sections (manufactured by BIOMAX) derived from the same samples as in Example 1 were subjected to heat treatment at 121°C in the antigen activation step. Immunohistochemical staining was performed in the same manner as in Example 6, and CAPRIN-1-positive cancer tissue samples were identified in the same manner as in Example 2.

[0442] The results of the determination showed that the proportion of CAPRIN-1-positive cancer tissue samples was 80% in breast cancer tissue, 70% in lung cancer, 70% in pancreatic cancer, 78% in kidney cancer, 69% in colorectal cancer, 55% in gastric cancer, 60% in ovarian cancer, and 90% in prostate cancer, which was roughly consistent with the determination results based on staining in Example 1.

[0443] In addition, in this embodiment, as the antibody used in the primary antibody reaction step, even if applied to WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2013 / 01 8889, WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640 and WO2015 / 020212, and the same proportion of CAPRIN-1-positive cancer tissue samples in the number of each cancer tissue sample was obtained as described above.

[0444] Example 9: Detection of CAPRIN-1 protein using immunohistochemical staining

[0445] Various paraffin-embedded human cancer tissue sections (manufactured by BIOMAX) derived from the same samples as in Example 1 were subjected to immunohistochemical staining in the same manner as in Example 6, except that the antigen activation step was heat treated at 121°C, the antibody concentration of the primary antibody solution used in the primary antibody reaction step was set to 0.8 μg / mL, and the color development time in the color development step was set to approximately 7 minutes. CAPRIN-1-positive cancer tissue samples were identified in the same manner as in Example 2.

[0446] The results of the determination showed that the proportion of CAPRIN-1-positive cancer tissue samples was 75% in breast cancer tissue, 65% in lung cancer, 80% in pancreatic cancer, 80% in kidney cancer, 62% in colorectal cancer, 55% in gastric cancer, 65% in ovarian cancer, and 80% in prostate cancer, which was roughly consistent with the determination results based on staining in Example 1.

[0447] In addition, in this embodiment, as the antibody used in the primary antibody reaction step, even if applied to WO2010 / 016526, WO2011 / 096517, WO2011 / 096528, WO2011 / 096519, WO2011 / 096533, WO2011 / 096534, WO2013 / 018894, WO2013 / 018892, WO2013 / 018891, WO2013 / 01 The mouse anti-CAPRIN-1 monoclonal antibodies or rabbit anti-CAPRIN-1 monoclonal antibodies prepared in WO2013 / 018883, WO2013 / 125636, WO2013 / 125654, WO2013 / 125630, WO2013 / 125640 and WO2015 / 020212 were also used to obtain the same proportion of CAPRIN-1-positive cancer tissue samples in the number of each cancer tissue sample as above.

[0448] From the above results, it was found that the staining method of the present invention can detect cell membrane proteins with high accuracy even when the type of the removal solution and other conditions are changed to some extent.

[0449] All publications, patents, and patent applications cited in this specification are hereby incorporated by reference into this specification as they are.

[0450] Explanation of symbols

[0451] S0101 Embedding medium removal process

[0452] S0102 Antigen Activation Process

[0453] S0103 Tokenization process

[0454] S0104 Marking Detection Process

[0455] 0210 Embedding medium removal unit

[0456] 0211 Removal solution storage unit

[0457] 0212, 0222, 0232, 0234 contact units

[0458] 0213 Holding Unit

[0459] 0220 Antigen Activation Response Unit

[0460] 0221 Antigen Activation Unit

[0461] 0230 Labeling reaction unit

[0462] 0231 Binding Molecular Storage Unit

[0463] 0233 Labeled Antibody Storage Unit

[0464] 0240 Marking Detection Department

[0465] 0241 Detection Unit

[0466] 0242 Analysis Unit

[0467] 0250 Control Department

[0468] 0251CPU

[0469] 0252 Volatile Memory

[0470] 0253 Non-volatile Memory

[0471] 0254 Interface

[0472] 0255 bus.

Claims

1. A method for detecting cell membrane proteins, comprising removing an embedding medium from an embedded fixed tissue to detect cell membrane proteins, the method comprising: an embedding medium removal step of bringing the fixed tissue containing the embedding medium in a solid state into contact with a removal solution to remove the embedding medium; A labeling step of binding the cell membrane protein of the fixed tissue after the embedding medium removal step with a labeled antibody; and After the labeling step, a label detection step is performed to detect the labeled antibody bound to the cell membrane protein.

2. The method according to claim 1, wherein the embedding medium removal step is a step of removing the embedding medium by bringing the fixed tissue into contact with the removal solution at a temperature lower than the melting point of the embedding medium. The method according to claim 1 , wherein the embedding medium is paraffin. 4 . The method according to claim 3 , wherein the embedding medium removal step is a step of removing the embedding medium by bringing the fixed tissue into contact with the removal solution while maintaining the fixed tissue at a temperature lower than 45° C. 5 . The method according to claim 1 , wherein the removal solution is a solution containing a surfactant and / or an organic solvent. 6 . The method according to claim 1 , wherein the contacting in the embedding medium removal step is immersion. 7 . The method according to claim 1 , further comprising, after the embedding medium removal step, an antigen activation step of activating the cell membrane protein in the fixed tissue.

8. The method according to claim 7, wherein the antigen activation step comprises a heating step at 90-130°C. 9 . The method according to claim 7 or 8 , further comprising a cooling step of cooling the fixed tissue after the antigen activation step.

10. The method according to any one of claims 1 to 9, wherein the labeling step comprises a primary antibody reaction step and a secondary antibody reaction step. In the primary antibody reaction step, the cell membrane protein of the fixed tissue after the embedding medium removal step is reacted with a primary antibody, wherein the primary antibody has immunoreactivity with the cell membrane protein. In the secondary antibody reaction step, a labeled secondary antibody is reacted with the fixed tissue after the primary antibody reaction step, and the labeled secondary antibody has immunoreactivity with the primary antibody. The method according to claim 10 , wherein the labeled secondary antibody is a complex of an antibody immunoreactive with the primary antibody and peroxidase bound to a polymer carrier. 12 . The method according to claim 11 , wherein the label detection step is a step of detecting the cell membrane protein that has developed color using a color developing reagent reactive with the peroxidase. 13 . The method according to claim 11 , wherein the color developing reagent is 3,3′-diaminobenzidine (DAB). 14 . The method according to claim 1 , wherein the cell membrane protein is a cell membrane protein expressed on the surface of cancer cells. 15 . The method according to claim 1 , wherein the cell membrane protein is a disease marker.

16. The method of claim 15, wherein the disease is cancer. The method according to claim 15 or 16, wherein the disease marker is CAPRIN-1 protein.

18. A method of determining a treatment for an individual from whom fixed tissue was derived, the method comprising: a step of detecting a disease marker in the embedded fixed tissue using the method according to any one of claims 15 to 17; and A treatment determination step of determining a treatment for the individual based on the disease marker detected in the step.

19. A device for detecting cell membrane proteins in embedded fixed tissue, comprising: an embedding medium removing section for removing the embedding medium by bringing the fixed tissue containing the embedding medium in a solid state into contact with a removal solution; a labeling reaction section for binding the cell membrane protein of the fixed tissue that has passed through the embedding medium removal section to a labeled antibody; and A label detection section detects the labeled antibody bound to the cell membrane protein in the fixed tissue that has passed through the labeling reaction section. 20 . The apparatus according to claim 19 , wherein the embedding medium removing section comprises a temperature regulating unit for making the temperature of the fixed tissue lower than the melting point of the embedding medium. 21 . The device according to claim 19 , further comprising an antigen activation reaction section for activating the cell membrane protein in the fixed tissue that has passed through the embedding medium removal section.

22. The device according to any one of claims 19 to 21, wherein the cell membrane protein is a cell membrane protein expressed on the surface of cancer cells.

23. The device according to any one of claims 19 to 22, wherein the cell membrane protein is a disease marker.

24. The device of claim 23, wherein the disease is cancer.

25. The device according to claim 23 or 24, wherein the disease marker is CAPRIN-1 protein.

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