WO2024204325A1 - 細胞膜タンパク質を検出する方法 - Google Patents

細胞膜タンパク質を検出する方法 Download PDF

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Publication number
WO2024204325A1
WO2024204325A1 PCT/JP2024/012221 JP2024012221W WO2024204325A1 WO 2024204325 A1 WO2024204325 A1 WO 2024204325A1 JP 2024012221 W JP2024012221 W JP 2024012221W WO 2024204325 A1 WO2024204325 A1 WO 2024204325A1
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cell membrane
fixed tissue
embedding medium
cancer
membrane protein
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English (en)
French (fr)
Japanese (ja)
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隆喜 井戸
文義 岡野
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Toray Industries Inc
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Toray Industries Inc
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Priority to CN202480012001.XA priority Critical patent/CN120677381A/zh
Priority to EP24780431.3A priority patent/EP4692791A1/en
Priority to JP2024526940A priority patent/JPWO2024204325A1/ja
Priority to KR1020257034502A priority patent/KR20250166224A/ko
Priority to AU2024242849A priority patent/AU2024242849A1/en
Publication of WO2024204325A1 publication Critical patent/WO2024204325A1/ja
Priority to MX2025010250A priority patent/MX2025010250A/es
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a method for detecting cell membrane proteins in fixed, embedded tissue and a method for determining a treatment for an individual from which the fixed tissue is derived, using the same.
  • Specific cell membrane proteins on cancer cells can be used as disease markers to detect cancer due to their cancer specificity, and various antibody drugs that target these proteins as antigens are used in cancer treatment as cancer treatment drugs with few side effects.
  • the cytopasmic-activation and proliferation-associated protein 1 (CAPRIN-1) protein is expressed on the cell membrane surface of many cancer cells, and is known to be a promising cancer disease marker (Patent Document 1).
  • antibodies against the CAPRIN-1 protein are known to be promising for use as a medicine for the treatment and/or prevention of cancer (Patent Document 2).
  • the objective of the present invention is to provide a highly accurate method for staining cell membrane proteins.
  • a method for detecting a cell membrane protein in an embedded fixed tissue comprising: an embedding medium removal step of contacting the fixed tissue containing a solid-state embedding medium with a removal solution to remove the embedding medium; an antigen activation step of activating the cell membrane protein in the fixed tissue after the removal step; a primary antibody reaction step of reacting the cell membrane protein of the fixed tissue with a primary antibody that is immunologically reactive with the cell membrane protein; a secondary antibody reaction step of reacting a labeled secondary antibody that is immunologically reactive with the primary antibody with the fixed tissue after the primary antibody reaction step; and a labeling detection step of detecting the labeled secondary antibody that has bound to the cell membrane protein via the primary antibody after the secondary antibody reaction step.
  • a method for detecting a cell membrane protein by removing an embedding medium from an embedded fixed tissue comprising: an embedding medium removal step of contacting the fixed tissue containing a solid-state embedding medium 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 a labeling detection step of detecting the labeled antibody bound to the cell membrane protein after the labeling step.
  • the method according to [1-2] further comprising an antigen retrieval step of activating the cell membrane protein in the fixed tissue after the embedding medium removal step.
  • the labeling step includes a primary antibody reaction step in which the cell membrane protein of the fixed tissue after the embedding medium removal step is reacted with a primary antibody that is immunologically reactive with the cell membrane protein, and a secondary antibody reaction step in which a labeled secondary antibody that is immunologically reactive with the primary antibody is reacted with the fixed tissue after the primary antibody reaction step.
  • the embedding medium removal step is a step of removing the embedding medium by contacting the fixed tissue with the removal solution without baking the fixed tissue.
  • the embedding medium removing step is a step of removing the embedding medium by contacting the fixed tissue with the removal solution at a temperature lower than the melting point of the embedding medium.
  • the removal solution is a solution containing a surfactant and/or an organic solvent.
  • the contact in the embedding medium removal step is immersion.
  • the antigen retrieval step includes a heat treatment step at 90 to 130°C.
  • [6] The method according to any one of [1] to [5], further comprising a cooling step of cooling the fixed tissue after the antigen retrieval step.
  • the labeled secondary antibody is a complex in which an antibody immunologically reactive with the primary antibody and peroxidase are bound to a polymer carrier.
  • 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.
  • the color-developing reagent is 3,3'-diaminobenzidine (DAB).
  • [10-1] The method according to any one of [1] to [9], wherein the embedding medium is paraffin.
  • the embedding medium removing 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 below 45°C.
  • the cell membrane protein is a cell membrane protein expressed on the surface of a cancer cell.
  • the cell membrane protein is a disease marker.
  • the disease is cancer.
  • a method for determining a treatment method for an individual from which a fixed tissue is derived comprising: a step of detecting a disease marker in the embedded fixed tissue using the method according to any one of [11] to [13]; and a treatment method determination step of determining a treatment method for the individual based on the disease marker detected by the above step.
  • An apparatus for detecting cell membrane proteins in embedded fixed tissue comprising: an embedding medium removal section that removes the embedding medium by contacting the fixed tissue containing a solid-state embedding medium with a removal solution; a labeling reaction section that binds the cell membrane protein of the fixed tissue that has passed through the embedding medium removal section with a labeled antibody; and a label detection section that detects the labeled antibody that has bound to the cell membrane protein in the fixed tissue that has passed through the labeling reaction section.
  • the embedding medium removal unit is provided with a temperature control means for keeping the fixed tissue below the melting point of the embedding medium.
  • the detection method of the present invention makes it possible to detect cell membrane proteins with high accuracy in embedded, fixed tissue.
  • the method for determining a treatment method of the present invention makes it possible to determine an appropriate treatment method based on expression information of cell membrane proteins detected with high accuracy.
  • 1 shows exemplary stained images of ovarian cancer tissues obtained by the staining method of Example 1.
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei is excluded.
  • the arrows indicate cells whose cell membranes are appropriately stained, and the scale bar indicates 50 ⁇ m.
  • 1 shows exemplary stained images of ovarian cancer tissues by the staining method of Comparative Example 1.
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image excluding the signal of cell nuclei.
  • the arrowhead indicates a cell inappropriately stained up to the cytoplasm, and the scale bar indicates 50 ⁇ m.
  • the figures show stained images of gastric cancer tissues judged to have a score of 0 (CAPRIN-1 negative).
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei has been excluded.
  • the scale bar indicates 50 ⁇ m.
  • the figures show stained images of gastric cancer tissues judged to be score 1 (CAPRIN-1 negative).
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei has been excluded.
  • the scale bar indicates 50 ⁇ m.
  • the figures show stained images of gastric cancer tissues judged to be score 2 (CAPRIN-1 positive).
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei has been excluded.
  • the scale bar indicates 50 ⁇ m.
  • the figures show stained images of gastric cancer tissues judged to be score 3 (CAPRIN-1 positive).
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei has been excluded.
  • the scale bar indicates 50 ⁇ m.
  • the figures show stained images of kidney 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 excluding the signal of cell nuclei.
  • the scale bar indicates 50 ⁇ m.
  • the figures show stained images of kidney cancer tissues judged to be score 1 (CAPRIN-1 negative).
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image excluding the signal of cell nuclei.
  • the scale bar indicates 50 ⁇ m.
  • the figures show stained images of kidney cancer tissues judged to be score 2 (CAPRIN-1 positive).
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei has been excluded.
  • the scale bar indicates 50 ⁇ m.
  • the figures show stained images of kidney cancer tissues judged to be score 3 (CAPRIN-1 positive).
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image excluding the signal of cell nuclei.
  • the scale bar indicates 50 ⁇ m. 1 shows exemplary stained images of ovarian cancer tissues obtained by the staining method of Example 1.
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei has been excluded.
  • the scale bar indicates 50 ⁇ m. 1 shows exemplary stained images of ovarian cancer tissues obtained by the staining method of Example 3.
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei has been excluded.
  • the scale bar indicates 50 ⁇ m.
  • FIG. 1 shows exemplary stained images of ovarian cancer tissues obtained by the staining method of Example 4.
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei has been excluded.
  • the scale bar indicates 50 ⁇ m.
  • 1 shows exemplary stained images of ovarian cancer tissues obtained by the staining method of Example 5.
  • A shows a stained image of cell nuclei and CAPRIN-1 protein.
  • B shows a stained image in which the signal of cell nuclei has been excluded.
  • the scale bar indicates 50 ⁇ m.
  • 1 is a flow chart illustrating an exemplary embodiment of a method for detecting a cell membrane protein.
  • FIG. 11 is an exemplary functional block diagram of a cell membrane protein detection device according to the third embodiment.
  • FIG. 2 is a diagram illustrating an example of the hardware configuration of a control unit of the cell membrane protein detection device.
  • the first aspect of the present invention is a method for detecting cell membrane proteins.
  • the method of this aspect includes 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 non-specific reaction inhibition step, and a color development step as optional steps.
  • cell membrane proteins can be detected with high specificity.
  • Embedding medium refers to the reagent used for embedding.
  • Embedding refers to the process of permeating a biological sample with a reagent and hardening the biological sample.
  • Fixed tissue refers to a biological sample that contains fixed tissue.
  • Fixation refers to a process performed to protect a biological sample from deterioration due to autolysis or putrefaction by denaturing and/or precipitating proteins in the biological sample. Fixation in this specification includes any fixation process that uses a crosslinking agent, a coagulation precipitant, or a combination thereof.
  • biological sample refers to a sample composed of cells of an organism.
  • biological sample includes both samples isolated from an individual organism and samples containing cells prepared outside of a vitro system.
  • the individual organisms referred to herein are not particularly limited as long as they are eukaryotic organisms, but include, for example, mammalian organisms, including primates, pet animals, livestock, and sports animals. Preferred organisms include humans, horses, pigs, cows, sheep, goats, dogs, and cats.
  • Cell membrane protein refers to a protein present on the cell membrane.
  • “cell membrane protein” includes transmembrane proteins, cell membrane surface proteins, and lipid-modified proteins.
  • Cell membrane surface proteins and lipid-modified proteins are proteins that do not have a transmembrane domain.
  • a "transmembrane domain” is a protein domain that has affinity for the lipid bilayer that constitutes the cell membrane and penetrates the lipid bilayer.
  • the protein domain that is exposed on the outside of the cell is called the extracellular domain
  • the protein domain that is exposed on the inside of the cell is called the intracellular domain.
  • some lipid-modified proteins and cell membrane surface proteins are not entirely embedded in the cell membrane, and the entire protein can be called an extracellular domain or an intracellular domain.
  • Solid state refers to a state in which there is no fluidity.
  • the solid state preferably does not include a semi-solid state such as a gel.
  • immunologically reactive refers to the property of an antibody binding to an antigen or a partial polypeptide thereof in vivo. For example, it refers to having the activity of binding to an antigen based on an antigen-antibody reaction.
  • binding to an antigen includes both specific and non-specific binding.
  • an “antibody” refers to an immunoglobulin or antigen-binding fragment thereof that specifically binds to another molecule.
  • antibody includes both monoclonal and polyclonal antibodies and antigen-binding fragments thereof.
  • Label refers to a substance whose presence is detectable. As used herein, labeling a substance refers to covalently linking the substance to a label.
  • Baking refers to the process of heating in an atmosphere.
  • “baking” specifically refers to the process of heating fixed tissue in an atmosphere to a temperature above the melting point of the embedding medium. This process is usually performed to partially melt the embedding medium and to attach the tissue to a glass slide or the like.
  • disease marker refers to a biological molecule (e.g., a nucleic acid molecule or a protein molecule) that serves as an indicator for determining whether or not a person has a disease or is at risk of developing the disease.
  • the term particularly refers to a protein that serves as an indicator for determining whether or not a person has a disease or is at risk of developing the disease.
  • cancer means a malignant neoplasm and is used interchangeably with “tumor.”
  • cancer includes primary cancer, metastatic cancer, metastatic cancer, and recurrent cancer.
  • CAPRIIN-1 cytopasmic-activation and proliferation-associated protein 1 protein
  • CAPRIN-1 protein refers to a cell membrane protein that is expressed mainly on the cell membrane of cancer cells.
  • the CAPRIN-1 protein also includes proteins that have biological functions equivalent to those of human CAPRIN-1 protein, such as homologs (i.e., homologs or orthologs), mutants such as genetic polymorphisms, and derivatives.
  • Treatment refers to a procedure to completely or partially cure or alleviate a disease or condition or its associated symptoms, or to prevent or delay its progression in an individual who already has the condition.
  • prevention refers to a procedure to prevent or delay the onset of a disease or condition in an individual who may be susceptible to the disease or condition.
  • Therapeutic method or “prophylactic method” refers to a method used for therapeutic or prophylactic treatment.
  • the method of this embodiment includes an embedding medium removing step, a labeling step, and a label detection step as essential steps, and an antigen retrieval step, a cooling step, a non-specific reaction inhibition step, and a color development step as optional steps. Each step is described in detail below.
  • Embedding medium removal process (S0101)
  • the "embedding medium removing step (S0101)" is an essential step, in which the fixed tissue containing the embedding medium in a solid state is brought into contact with a removal solution to remove the embedding medium.
  • the embedding medium is not particularly limited as long as it is a medium capable of embedding and hardening a tissue, and can be appropriately selected according to, for example, the type of fixed tissue to be fixed, the temperature conditions for embedding, the detection method used in the label detection step, and the like.
  • specific embedding media include, for example, wax, paraffin (e.g., Paraplast, Broloid, and Tissuecan), paraffin wax, acrylic resins, methacrylic acid resins (e.g., ethylene glycol dimethacrylate, glycol methacrylate, butyl methacrylate, hydroxypropyl methacrylate, methyl methacrylate, etc.), dammar resins, epoxy resins, divinylbenzene, other plastic resins (e.g., Sparplastics, Lowicryl®, Epon, Araldite, LR White, and Durcupan, etc.), and combination copolymers thereof.
  • Preferred embedding media herein are paraffin and paraffin wax.
  • specific embedding media include, for example, Optimum Cutting Temperature (OCT) compound (e.g., Tissue-Tek® O.C.T. compound or Tissue-plus® O.C.T. compound), PELCO® freezing embedding compound, PolarStat®, PolarStat Plus® embedding medium, and Tissue Freezing Medium (TFM®).
  • OCT Optimum Cutting Temperature
  • PELCO® freezing embedding compound e.g., Tissue-Tek® O.C.T. compound or Tissue-plus® O.C.T. compound
  • PELCO® freezing embedding compound
  • PolarStat® PolarStat Plus® embedding medium
  • Tissue Freezing Medium Tissue Freezing Medium
  • the embedding medium may be diluted with an organic solvent or aqueous solution to achieve the desired hardness.
  • the fixed tissue used in this step is the biological sample for which cell membrane proteins are to be detected by the method of this embodiment.
  • the individual, organ, and tissue from which the fixed tissue is derived are not particularly limited.
  • the individual may be a healthy individual, an individual possibly suffering from a disease, or an individual suffering from a disease.
  • the term “healthy individual” refers to an individual in a healthy state.
  • the term “healthy state” refers to a state in which the individual is at least not afflicted by the disease being tested, and preferably a healthy state free of any disease or disorder.
  • the fixed tissue may include diseased tissue, may potentially include diseased tissue, or may include only normal tissue.
  • the fixed tissue may be derived from a single individual, or may be derived from multiple individuals simultaneously.
  • the fixed tissues derived from each individual may be derived from a single organ or multiple organs.
  • the type of disease is not particularly limited. Examples include cancer and inflammation, and examples of tissues from which fixed tissues are derived include cancer tissue and inflamed tissue.
  • the cell membrane protein detected by the method of this embodiment may be, for example, a cell membrane protein expressed on the surface of diseased cells, and examples of such cell membrane proteins include a cell membrane protein expressed on the surface of inflammatory cells and a cell membrane protein expressed on the surface of cancer cells.
  • the cancers targeted by the present invention are not particularly limited.
  • the cancer may be one in which CAPRIN-1 protein is expressed on the cell membrane surface.
  • Preferred cancers include basal cell carcinoma, Paget's disease, skin cancer, breast cancer, kidney cancer, pancreatic cancer, colon 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, Sezary syndrome, skin cancer, and leukemia.
  • T/NK cell lymphoma T cell leukemia/lymphoma with lesions only in the skin
  • cutaneous B cell lymphoma indolent group
  • cutaneous T cell lymphoma breast adenocarcinoma
  • hybrid breast adenocarcinoma malignant mixed tumor of the breast
  • intraductal papillary adenocarcinoma lung adenocarcinoma
  • squamous cell carcinoma small cell carcinoma, large cell carcinoma
  • neuroepithelial tissue tumors such as glioma, glioblastoma, neuroblastoma, ependymoma, neuronal cell tumor, embryonal neuroectodermal tumor, neurilemmoma, neurofibroma, meningioma, chronic lymphocytic leukemia , gastrointestinal lymphoma, digestive lymphoma, small to medium cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, transverse
  • the type of cell membrane protein detected by the method of this embodiment is not particularly limited. It may be any of transmembrane proteins, cell membrane surface proteins, and lipid-modified proteins, and may be a protein that is localized on the cell membrane under specific conditions. For example, it may be a cell membrane protein that does not have a transmembrane domain, or a cell membrane protein that is exposed in whole or in part to the outside of the cell.
  • Cell membrane proteins in this specification can include, for example, disease markers such as cancer markers.
  • cancer markers include, for example, 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.
  • the method of fixing the fixed tissue is not particularly limited, and any method 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 or the method described in "Immunohistochemical Staining Methods" 6th Edition (2013) (DAKO North America, Inc.) can be used.
  • the reagent used for fixation is not particularly limited. For example, it may be any of a crosslinking agent, a coagulation precipitant, or a combination thereof.
  • fixatives include crosslinking agents such as glutaraldehyde, formaldehyde, paraformaldehyde, paraformaldehyde-picric acid, periodic acid-lysine-paraformaldehyde, and zinc ion-formaldehyde solutions; coagulation precipitants such as methanol, ethanol, acetone, acetate-zinc chloride, and methanol-acetone mixtures; and mixtures thereof (e.g., formalin solutions). Fixatives can be used by diluting them with a buffer solution or the like, as necessary.
  • crosslinking agents such as glutaraldehyde, formaldehyde, paraformaldehyde, paraformaldehyde-picric acid, periodic acid-lysine-paraformaldehyde, and zinc ion-formaldehyde solutions
  • coagulation precipitants such as methanol, ethanol, acetone, acetate-zinc chloride, and methanol-acetone mixture
  • fixation conditions are not particularly limited.
  • fixation temperatures include 0°C or lower, 4°C to 45°C, 4°C to 37°C, 4°C to 30°C, and 4°C to 25°C.
  • fixation time examples include 30 seconds to 48 hours, 10 minutes to 36 hours, 20 minutes to 24 hours, and 30 minutes to 20 hours.
  • fixation can be performed once or more, twice or more, or three or more times. When fixation is performed multiple times, the fixation method, reagents, and conditions used for each fixation may be the same or different.
  • fixation, dehydration and replacement treatments can be performed as necessary. There are no particular limitations on the conditions for each, and any method known in the art can be used. For example, the method described in the washing step of this process can be used.
  • the fixed tissue used in the method of this embodiment may be either a tissue fragment in which the fixed tissue is embedded in the embedding medium described above, or a section thereof.
  • the fixed tissue is a section.
  • Sections can be prepared using methods known in the art, for example, by manual tomography or by using a thin-sectioning device (such as a microtome, vibratome, or cryostat).
  • a thin-sectioning device such as a microtome, vibratome, or cryostat.
  • the thickness of the slice is not particularly limited. Specific thicknesses include, for example, 0.5 ⁇ m or more, 1 ⁇ m or more, 2 ⁇ m or more, 3 ⁇ m or more, or 4 ⁇ m or more. Also, for example, the thickness can be 300 ⁇ m or less, 200 ⁇ m or less, 150 ⁇ m or less, 100 ⁇ m or less, 80 ⁇ m or less, 60 ⁇ m or less, 50 ⁇ m or less, 30 ⁇ m or less, 20 ⁇ m or less, 15 ⁇ m or less, 10 ⁇ m or less, 8 ⁇ m or less, 7 ⁇ m or less, 6 ⁇ m or less, or 5 ⁇ m or less.
  • the thickness of the slice may be within the range of 1 ⁇ m to 300 ⁇ m, 1 ⁇ m to 100 ⁇ m, 1 ⁇ m to 50 ⁇ m, 1 ⁇ m to 20 ⁇ m, 1 ⁇ m to 10 ⁇ m, 2 ⁇ m to 100 ⁇ m, 2 ⁇ m to 50 ⁇ m, 2 ⁇ m to 20 ⁇ m, 2 ⁇ m to 10 ⁇ m, etc.
  • the section may be placed on a support such as a glass slide, or may not be placed on a support and may be, for example, suspended in a solution.
  • the section is placed on a support such as a glass slide.
  • the material of the support is not particularly limited, but may include, for example, a polymeric material, a glass material, a plastic material, or a metal material. Examples of glass materials include soda-lime glass, borosilicate glass, crown glass, and combinations thereof.
  • the shape of the support is not particularly limited. For example, membranes, microtiter plates, beads, filters, test strips, slides, cover glasses, and test tubes can be used as supports.
  • the support may be surface-treated as necessary.
  • the section can be attached to the support.
  • the method of attachment is not particularly limited, but examples include methods using matrices such as silane, gelatin, and poly-L-lysine.
  • the fixed tissue includes an embedding medium in a solid state.
  • the embedding medium being in a solid state means that the embedding medium is not melted. In other words, in the fixed tissue used in the method of this embodiment, the embedding medium is not melted in whole or in part.
  • the ratio of the solid-state embedding medium to the total embedding medium in the fixed tissue 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%.
  • the fixed tissue used in this process has not been baked and has not been heated above the melting point of the embedding medium in the atmosphere.
  • Melting point refers to the transition temperature at which a substance in a solid state melts into a liquid state when heated.
  • the heating temperature here varies depending on the type of embedding medium used.
  • the melting point of a typical O.C.T. compound is -15°C to -5°C, although this varies depending on the product, so when using such a medium, temperatures exceeding this temperature apply.
  • Specific temperatures for example, it is preferable that the medium is not exposed to an atmosphere of -20°C or higher, -15°C or higher, -10°C or higher, -5°C or higher, or 0°C or higher for a certain period of time.
  • the melting point of a typical paraffin wax is 45°C to 75°C, although this varies depending on the product, so when using such a medium, temperatures exceeding this temperature apply. It is preferable that the medium is not exposed to an atmosphere of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, or 62°C or higher for a certain period of time.
  • the exposure time here is not particularly limited as long as it is a time sufficient for the embedding medium in the fixed tissue to begin to melt.
  • the tissue is not exposed to an atmosphere with a temperature above the melting point of the embedding medium for 1 minute or more, 2 minutes or more, 3 minutes or more, 5 minutes or more, 6 minutes or more, 7 minutes or more, 8 minutes or more, 9 minutes or more, 10 minutes or more, 11 minutes or more, 12 minutes or more, 14 minutes or more, or 15 minutes or more.
  • the tissue is not exposed to an atmosphere with a temperature of 50°C or more for 5 minutes or more, an atmosphere with a temperature of 55°C or more for 10 minutes or more, or an atmosphere with a temperature of 60°C or more for 15 minutes or more.
  • the term "removal solution” refers to a solution capable of dissolving the embedding medium.
  • the type of removal solution is not particularly limited.
  • the embedding medium is a lipophilic substance, for example, a solution containing a surfactant and/or an organic solvent can be used.
  • a solution containing an organic solvent is used.
  • the embedding medium is a hydrophilic substance, for example, water and/or an aqueous solution (such as an aqueous solution containing a sugar such as sucrose, a buffer solution, etc.) can be used.
  • the organic solvent used in this step is not particularly limited.
  • linear, branched, and cyclic alkanes having 4 to 16 carbon atoms (heptane, hexadecane, etc.); linear, branched, and cyclic dialkyl ethers having 4 to 16 carbon atoms (diethyl ether, dioctyl ether, etc.); aromatic hydrocarbons such as toluene and xylene; alcohols such as methanol, ethanol, and isopropyl alcohol; alkyl halides such as chloroform; monoterpenes such as limonene; vegetable oils such as citrus oil and coconut oil; or mixtures of these.
  • substitutes for these e.g., xylene substitutes, etc.
  • removal solutions provided with automatic staining devices e.g., Artisan Cleaning Solution (Agilent)
  • xylene substitutes include Tissue Clear xylene substitute, Shandon xylene substitute, aliphatic hydrocarbons such as heptane and hexadecane, derivatives of aliphatic hydrocarbons such as dioctyl ether, and mixtures thereof.
  • the concentration of the organic solvent contained in the removal solution is not particularly limited.
  • the concentration by weight percent can be 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%.
  • xylene and one or more concentrations of aqueous ethanol solutions can be used as the removal solution.
  • the removal solution used in this process is preferably xylene or a xylene substitute (e.g., one that contains an aliphatic hydrocarbon).
  • Step> the fixed tissue is brought into contact with a removal solution, so that all or part of the embedding medium in the fixed tissue is dissolved in the removal solution.
  • the contact method is not particularly limited as long as it is a method that allows contact between the removal solution and the embedding medium in the fixed tissue.
  • the removal solution can be sprayed, sprinkled, or applied to the fixed tissue, or the fixed tissue can be immersed in the removal solution.
  • the contact in this step is preferably immersion.
  • the contact location of the fixed tissue with the removal solution may be any of the whole or part of the fixed tissue, but if the removal solution comes into contact with a part of the fixed tissue, care should be taken to ensure that the removal solution comes into contact with at least most of the embedding medium in the fixed tissue.
  • this step can be carried out so that the removal solution comes into contact with 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the embedding medium.
  • the contact conditions in this step are not particularly limited as long as the embedding medium is soluble in the removal solution.
  • contact can be performed for a relatively long period of time 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.
  • Freezing point refers to the transition temperature at which a liquid substance solidifies into a solid state upon cooling.
  • the time in this case is not particularly limited, but can be, for example, 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 contact can be for 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.
  • the lower limit of the temperature is not particularly limited as long as it is higher than 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, the temperature may be higher than the freezing point of xylene, which is -25°C, and when ethanol is used, the temperature may be higher than the freezing point of ethanol, which is -114.5°C.
  • Specific temperatures include, for example, temperatures of -100°C or higher, -50°C or higher, -24°C or higher, -10°C or higher, 0°C or higher, 1°C or higher, 5°C or higher, 10°C or higher, 15°C or higher, 20°C or higher, and 25°C or higher.
  • the upper limit of the temperature is not particularly limited as long as it is lower than the melting point of the embedding medium.
  • This temperature varies depending on the type of embedding medium used.
  • the temperature may be lower than the melting point of paraffin wax, which is 45°C to 75°C.
  • Specific temperatures include, for example, temperatures below 75°C, below 70°C, below 65°C, below 60°C, below 55°C, below 50°C, below 45°C, below 40°C, below 37°C, below 35°C, below 30°C, or below 27°C.
  • it can be performed at room temperature (1°C to 30°C) or normal temperature (15°C to 25°C).
  • contact with an aromatic hydrocarbon such as xylene can be performed at normal temperature for 20 minutes or more.
  • the temperature may be maintained at a constant level during contact, or may be changed appropriately during contact or may vary naturally.
  • the contact may be performed multiple times.
  • the removal solution and the contact conditions used for each contact may be the same each time, or may be different for each contact.
  • the removal solution may be renewed for each contact.
  • it can be performed at a relatively high temperature, for example, above the melting point of the embedding medium and below the boiling point of the removal solution, for a relatively short time.
  • Biting point refers to the temperature at which the vapor pressure of a liquid is equal to the pressure of the system. In this specification, “boiling point” specifically refers to the temperature at which the vapor pressure of a liquid is equal to one atmosphere.
  • the time in this case is not particularly limited, but can be, for example, 3 seconds or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 20 seconds or more, 25 seconds or more, or 30 seconds or more.
  • the upper limit of the time is not particularly limited as long as it is within a range that does not cause excessive stress on the fixed tissue. For example, contact can be made for 1 minute or less, 55 seconds or less, 50 seconds or less, 45 seconds or less, 40 seconds or less, 35 seconds or less, or 30 seconds or less.
  • the lower limit of the temperature is not particularly limited as long as it is equal to or higher than the melting point of the embedding medium.
  • This temperature varies depending on the type of embedding medium used. For example, when paraffin is used as the embedding medium, the temperature may be equal to or higher than the melting point of paraffin wax, which is 45°C to 75°C. Specific temperatures include, for example, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 72°C or higher, or 75°C or higher.
  • the upper limit of the temperature is not particularly limited as long as it is lower than the boiling point of the removal solution.
  • the temperature when xylene is used as the removal solution, the temperature may be lower than the boiling point of xylene, which is 138°C to 144°C, and when ethanol is used, the temperature may be lower than the boiling point of ethanol, which is 78.37°C.
  • Specific temperatures include, for example, 140°C or less, 130°C or less, 120°C or less, 110°C or less, 100°C or less, 90°C or less, 85°C or less, 80°C or less, 78°C or less, or 75°C or less.
  • the percentage of the embedding medium removed by this process is not particularly limited as long as it does not inhibit the antigen-antibody reaction using a primary antibody or the like.
  • this process removes 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the embedding medium.
  • the removal solution can be made to flow as needed.
  • the flow can be artificial, such as by stirring, or can occur naturally, such as by convection.
  • the fixed tissue can be moved as needed, such as by changing the position of the fixed tissue.
  • contact with the removal solution can be followed by a separation step to separate the dissolved embedding medium and the fixed tissue.
  • the method of separation is not particularly limited. Since the dissolved embedding medium is liquid and the fixed tissue is solid, any method for separating solids and liquids known in the art can be used. For example, separation can be performed by centrifugation using a dehydrator or the like.
  • the separation step may not be necessary if a large excess of removal solution is used in the contact step, if the removal solution is made to flow, or if the removal solution is renewed.
  • a washing step can be performed to wash away any remaining removal solution from the fixed tissue. Washing is performed by contacting the fixed tissue with a washing solution. The contact can be performed by the method and under the conditions described above for the contact step.
  • the cleaning solution is not particularly limited as long as it can remove the dissolved embedding medium remaining on the surface of the fixed tissue, the removal solution in which the embedding medium has dissolved, and/or the removal solution in the fixed tissue.
  • a solvent different from the solvent used in the contact step may be used as the cleaning solution.
  • a water-soluble solvent is used as the cleaning solution.
  • Specific examples of the cleaning solution that can be used include water, any buffer solution (such as phosphate buffer), alcohol (such as methanol and ethanol), or a mixture of these.
  • alcohol and/or a buffer solution more specifically, ethanol and/or phosphate buffer, can be used.
  • Washing can be performed multiple times.
  • the washing solution and washing conditions used for each wash may be the same each time, or may be different for each wash.
  • xylene is used as the removal solution
  • washing with an alcohol such as ethanol can be performed, and then washing with a buffer solution such as a phosphate buffer can be performed.
  • a buffer solution such as a phosphate buffer
  • xylene and ethanol can be used independently, and for example, the fixed tissue can be contacted with xylene and then with ethanol.
  • the concentration of the ethanol solution is not particularly limited.
  • concentrations can be used in weight percent: 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%.
  • concentrations can be used in weight percent: 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%.
  • three different concentrations of ethanol solutions can be contacted in order of decreasing concentration. Specifically, for example, washing can be performed by contacting the tissue with 100%, 90% and 80% ethanol solutions in sequence.
  • the washing solution can be made to flow as appropriate.
  • the flow can be artificially performed by stirring or the like, or can be natural by convection or the like.
  • the fixed tissue can be moved as appropriate, for example by changing the position of the fixed tissue. Washing can also be performed in any other step.
  • Antigen retrieval step (S0102) is an optional step for activating cell membrane proteins in the fixed tissue. This step can be carried out after the removal step (S0101). Optionally, this step can be carried out simultaneously with the removal step (S0101).
  • Antigen activation is performed by heat treatment, enzyme treatment, or a combination thereof.
  • any method known in the art can be used.
  • the methods described in Shi et al. J Histochemistry & Cytochemistry, 2011, 59:13-32
  • D'Amico et al. J Immunological Methods, 2009, 341:1-18
  • McNicoll and Richmond Histopathology, 1998, 32:97-103 can be used.
  • the necessity of this step and the treatment to be performed when this step is performed can be appropriately selected depending on the type of antigen and the properties of the fixed tissue.
  • This process includes, for example, an enzyme treatment step and/or a heat treatment step, and preferably includes a heat treatment step.
  • the enzyme treatment can be carried out using proteases such as trypsin, DNase, proteinase K, pepsin, pronase, and ficin.
  • proteases such as trypsin, DNase, proteinase K, pepsin, pronase, and ficin.
  • the treatment conditions are not particularly limited as long as the enzyme can exhibit activity.
  • the treatment conditions can be determined based on the optimum temperature and optimum pH of the enzyme.
  • the heat treatment step can be performed by applying a heated antigen retrieval solution to the fixed tissue.
  • the type of antigen retrieval solution is not particularly limited, but examples thereof include citrate 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 include, for example, an acidic compound such as potassium dihydrogen phosphate, boric acid, diethylbarbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, 2-(N-morpholino)ethanesulfonic acid, or combinations thereof.
  • an acidic compound such as potassium dihydrogen phosphate, boric acid, diethylbarbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, 2-(N-morpholino)ethanesulfonic acid, or combinations thereof.
  • the pH is not particularly limited, but can be in the range of, for example, 6 to 11, 7 to 10, 7.5 to 9.5, or 8 to 9.
  • the temperature of the heat treatment is not particularly limited, but can be, for example, in the range of 90°C to 130°C. Specifically, it can be, for example, 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.
  • the treatment can be performed for 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.
  • the treatment time can be, for example, 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.
  • the conditions may be constant or may change intermittently or continuously. If the conditions change, the change may be artificial or natural.
  • the solution may be made to flow as appropriate. In this case, the flow may be artificial, such as by stirring, or natural, such as by convection.
  • the fixed tissue may be moved as appropriate, such as by changing the position of the fixed tissue.
  • each antigen activation can be carried out under different conditions or the same conditions.
  • the "cooling step” is an optional step in which the fixed tissue is cooled. This step can be performed after the antigen retrieval step.
  • this step can be performed when antigen activation is performed by heat treatment or when the temperature is increased for enzyme treatment.
  • the cooling method is not particularly limited as long as it can lower the temperature of the fixed tissue to a certain temperature.
  • a method of leaving the tissue in a low-temperature atmosphere or in a solution, a method using a cooling device such as a refrigerator or freezer, or a combination of these methods can be used.
  • the temperature after cooling is not particularly limited. For example, it can be freezing temperature (0°C or below), refrigerated temperature (0°C to 5°C), room temperature (1°C to 30°C), or normal temperature (15°C to 25°C). It is preferably cooled to room temperature or normal temperature.
  • the cooling rate and cooling time are not particularly limited.
  • the cooling time in this step refers to the time required for the fixed tissue to drop to the desired temperature. Therefore, if the temperature reaches equilibrium after the drop and is maintained at that temperature, the time until equilibrium is reached is the time for this step. If necessary, the fixed tissue can be stored for any length of time by leaving it to stand after this step or any other step.
  • 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.
  • the solution and atmosphere can be made to flow as appropriate.
  • the flow in this case can be artificial, such as by stirring, or can be natural, such as by convection.
  • the fixed tissue can be moved as appropriate, such as by changing the position of the fixed tissue.
  • Non-specific reaction suppression step is an optional step in which the fixed tissue is contacted with a reaction suppressor to suppress non-specific reactions in the antibody reaction and/or color reaction. This step can be performed after the embedding medium removal step. If the antigen retrieval step is performed, this step can be performed simultaneously with or after the antigen retrieval step. If the cooling step is performed, this step can be performed simultaneously with or after the cooling step.
  • This process is broadly divided into a blocking step that suppresses non-specific binding of antibodies, and an endogenous reaction suppression step that suppresses non-specific color reactions. Either one of these steps may be performed, or both steps may be performed. Each step may be performed independently multiple times, and one or more other steps may be performed between each step.
  • ⁇ Blocking step> the fixed tissue is contacted with a blocking solution to prevent non-specific binding of the antibody used.
  • the timing of this step is not particularly limited, so long as it is performed after the embedding medium removal step. If an antigen activation step is performed, it can be performed simultaneously with or after that. For example, it may be performed before, simultaneously with or after the labeling step described below. For example, it may be performed before, simultaneously with or after the primary antibody reaction step and the secondary antibody reaction step. This step may be performed multiple times, in which case the steps may be performed consecutively with one another, and one or more steps may be included in between.
  • any method known in the art can be used for blocking.
  • this step is performed by incubating the fixed tissue in contact with the blocking solution for a certain period of time.
  • the blocking solution can be placed on the fixed tissue and incubated for a certain period of time.
  • the solution can be replaced with the blocking solution and incubated for a certain period of time.
  • Blocking solutions generally contain proteins. There are no particular limitations on the type of protein contained in the blocking solution. Examples include animal serum proteins, immunoglobulin proteins, skim milk, non-fat milk, casein, and mixtures thereof.
  • the animal from which the serum protein is derived is not particularly limited.
  • serum proteins from goats, horses, humans, mice, rabbits, rats, or pigs include serum proteins from goats, horses, humans, mice, rabbits, rats, or pigs.
  • bovine serum albumin, fetal bovine serum, horse serum, goat serum, etc. can be used.
  • the solvent of the blocking solution is not particularly limited, but is usually a buffer solution.
  • the specific type of buffer solution is not particularly limited.
  • a phosphate buffer solution such as PBS
  • the buffer solution can be a tris(hydroxymethyl)methylamine (TRIS) buffer solution, a 2-(N-morpholino)ethanesulfonic acid (TAPS) buffer solution, an N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer solution, an N-tris(hydroxymethyl)methylglycine (Tricine) buffer solution, a 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES) buffer solution, a 2- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ ethanesulfonic acid (TES) buffer solution, an acetate buffer solution, a carbonate buffer solution, a citrate buffer solution, or a combination thereof.
  • TAS tris(hydroxymethyl)methylamine
  • TAPS 2-(N-morpholino)ethan
  • the solvent may contain additives as appropriate, for example, surfactants such as Tween 20 and Triton X-100.
  • surfactants such as Tween 20 and Triton X-100.
  • a buffer solution to which these additives have already been added such as PBS-T (PBS containing 0.05% Tween 20), can be used.
  • the pH of the solvent is not particularly limited. Specific pH values include, for example, 6 to 8, 7 to 8, 7.2 to 7.8, 7.3 to 7.6, and 7.4 to 7.5.
  • the protein concentration is not particularly limited.
  • the concentration can be 0.1% to 30%, 1% to 30%, 5% to 30%, or 10% to 20% by weight and volume.
  • Endogenous response inhibition step In the endogenous reaction inhibition step, the fixed tissue is contacted with an inhibitor of the color reaction to inhibit non-specific reactions.
  • the timing of this step is not particularly limited as long as it is performed after the embedding medium removal step and before the color development step described below.
  • an antigen activation step it can be performed simultaneously with or after that and before the color development step described below.
  • a blocking step it may be performed before, simultaneously with or after that, and it may be performed before, simultaneously with or after the labeling step described below.
  • it may be performed before, simultaneously with or after the primary antibody reaction step and the secondary antibody reaction step. This step may be performed multiple times, in which case the steps may be performed consecutively with one another and one or more steps may be included in between.
  • the method for suppressing endogenous reactions is not particularly limited as long as it can suppress endogenous reactions of fixed tissues that may be involved in color reactions and inhibit detection of the label bound to the primary antibody described below.
  • it can be appropriately determined depending on the type of label used and the color reaction used.
  • a quenching agent can be applied to the fixed tissue to suppress the autofluorescence of the fixed tissue.
  • H2O2 e.g., 3% H2O2
  • a peroxidase inhibitor or a peroxidase inhibitor reagent can be used to suppress the activity of peroxidase in the fixed tissue.
  • biotin is used as a label
  • an excess of avidin and biotin can be applied to the fixed tissue to reduce the amount of biotin present in the fixed tissue.
  • a phosphatase inhibitor such as levamisole
  • a phosphatase inhibitor can be applied to the fixed tissue to suppress the activity of phosphatase in the fixed tissue.
  • this step can be performed using a reagent that has two or more of these activities.
  • the duration of this process is not particularly limited. For example, it can be 15 minutes or more, 30 minutes or more, 40 minutes or more, or 60 minutes or more. It can also be 24 hours or less, 16 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, or 2 hours or less.
  • the temperature of this process is not particularly limited.
  • it can be carried out under temperature conditions such as freezing temperature (0°C or below), refrigerated temperature (0°C to 5°C), room temperature (1°C to 30°C), or normal temperature (15°C to 25°C).
  • the conditions may be constant or may change intermittently or continuously. If the conditions change, the change may be artificial or natural.
  • Labeling step (S0103) is an essential step, and is a step of binding the cell membrane protein of the fixed tissue with the labeled antibody. This step can be performed after the embedding medium removal step (S0101). Antigen When the activation step (S0102) is performed, this step can be performed simultaneously with or after the antigen activation step (S0102). When the non-specific reaction suppression step is performed, it can be performed simultaneously with the blocking step. Or it can be done later.
  • the method for reacting the labeled antibody with the cell membrane protein of the fixed tissue is not particularly limited, as long as it is a method that can directly or indirectly bind the labeled antibody to the cell membrane protein in the fixed tissue.
  • this may be done by using a labeled antibody that is immunologically reactive with the cell membrane protein, or by using a labeled antibody that is immunologically reactive with a complex of the cell membrane protein and a binding molecule that binds to the cell membrane protein (e.g., an antibody that is immunologically reactive with the cell membrane protein).
  • a step of binding the labeled antibody to the cell membrane protein is carried out to perform labeling.
  • This step can be carried out after the embedding medium removal step.
  • an antigen activation step is carried out, this step can be carried out simultaneously with or after the antigen activation step.
  • a non-specific reaction suppression step is carried out, this step can be carried out particularly simultaneously with or after the blocking step.
  • labeling typically involves a first binding step in which the binding molecule (e.g., an antibody that is immunologically reactive with the cell membrane protein) is bound to the membrane protein, and a second binding step in which a labeled antibody that is immunologically reactive with the complex of the binding molecule and the cell membrane protein is bound to the complex.
  • the binding molecule e.g., an antibody that is immunologically reactive with the cell membrane protein
  • a second binding step in which a labeled antibody that is immunologically reactive with the complex of the binding molecule and the cell membrane protein is bound to the complex.
  • the first binding step can be performed after the embedding medium removal step. For example, if an antigen activation step is performed, the first binding step can be performed simultaneously with or after the antigen activation step. If a non-specific reaction suppression step is performed, the first binding step can be performed simultaneously with or after the blocking step.
  • the second binding step can usually be performed after the first binding step. Optionally, this step can be performed simultaneously with the second binding step.
  • the binding step may be performed three or more times.
  • the second and subsequent binding steps are performed using a binding molecule capable of binding to the complex formed in the previous binding step.
  • the binding molecule used in the binding steps other than the last binding step (corresponding to the second binding step) may be labeled or may be an unlabeled binding molecule.
  • the type of binding molecule is not particularly limited, but is preferably a binding molecule that specifically binds to a cell membrane protein.
  • a binding molecule that specifically binds to a cell membrane protein.
  • the binding mode between the binding molecule and the cell membrane protein is not particularly limited.
  • the binding molecule binds to the transmembrane domain of the cell membrane protein, a portion of the cell membrane protein other than the transmembrane domain, for example, a portion not embedded in the cell membrane, an intracellular domain or an extracellular domain of the cell membrane protein.
  • the specific details of the binding properties of the binding molecule, the reaction solution, and the application method are as described below in relation to the "primary antibody reaction step.”
  • the binding mode between the labeled antibody having immunological reactivity to the complex of the binding molecule and the cell membrane protein and the complex is not particularly limited.
  • the labeled antibody may be capable of binding only after the complex is formed, or may be capable of binding to either one of the components of the complex (the cell membrane protein or the binding molecule). Specific details regarding the labeled antibody are as described below in relation to the "secondary antibody reaction step.”
  • the first binding step is called the primary antibody reaction step
  • the second binding step is called the secondary antibody reaction step.
  • the "primary antibody reaction step” is a step in which a cell membrane protein of the fixed tissue is reacted with a primary antibody that is immunologically reactive with the cell membrane protein. This step can be performed after the embedding medium removal step. For example, when an antigen activation step is performed, this step can be performed simultaneously with or after the antigen activation step. When a non-specific reaction suppression step is performed, this step can be performed particularly simultaneously with or after the blocking step.
  • the primary antibody used in this step is not particularly limited as long as it is an antibody that has immunological reactivity with the cell membrane protein to be detected.
  • it may be an antibody that can bind to or specifically bind to the cell membrane protein of interest.
  • Specific antibodies can be appropriately determined based on the type of cell membrane protein to be detected. For example, they may be antibodies capable of binding to the transmembrane domain of a cell membrane protein, or they may be antibodies capable of binding to a portion of a cell membrane protein other than the transmembrane domain, for example, a portion that is not embedded in the cell membrane, or they may be antibodies capable of binding to the intracellular or extracellular domain of a cell membrane protein.
  • an anti-CAPRIN-1 antibody or an antigen-binding fragment thereof can be used.
  • Specific anti-CAPRIN-1 antibodies are not particularly limited. For example, see WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2011/096535, WO2013/018886, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/0188 89, WO2013/018883, WO2013/125636, WO2013/125654, WO2013/125630, WO2013/125640, WO2013/147169, WO2013/147176, WO2015/020212, WO2018/079740, and WO2019/189780.
  • the primary antibody used in this step may be easier to detect if it has a high binding affinity to the cell membrane protein.
  • an antibody having a binding constant (affinity constant) K a (k on /k off ) of 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 may be used.
  • the type of antibody is not particularly limited.
  • any class and isotype may be used, such as 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'.
  • aggregates, polymers, and conjugates of immunoglobulins or their antigen-binding fragments can also be used in appropriate cases, as long as the immunological reactivity with cell membrane proteins is substantially maintained.
  • the antibody used in this step may be either a monoclonal or polyclonal antibody.
  • the origin of the antibody is not particularly limited. Examples include human antibodies, non-human animal antibodies, recombinant antibodies, humanized antibodies, and chimeric antibodies.
  • 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.
  • the antibody production and purification methods used in this step are not particularly limited, and any method known in the art can be used.
  • the production and purification methods described in the literature exemplified for anti-CAPRIN-1 antibodies may be used.
  • any method known in the art can be used for the primary antibody reaction. Typically, this step is performed by incubating the fixed tissue in a solution of the primary antibody for a certain period of time.
  • composition of the antibody solution is not particularly limited as long as it contains an antibody and the antibody is not denatured.
  • the solvent of the antibody solution is not particularly limited, and may be, for example, any buffer solution or commercially available antibody diluent.
  • the specific type of buffer is not particularly limited.
  • the buffer solution exemplified for the blocking step may be used, and additives may be included as necessary. Any substance known in the art may be used as the additive, and examples of such additives include animal serum and serum proteins (bovine serum albumin, fetal bovine serum, goat serum, etc.), surfactants (Tween 20, Triton X-100, etc.). Multiple additives may be included, and may include, for example, fetal bovine serum, goat serum, and Tween 20.
  • the concentration of the additive is not particularly limited. For example, the concentration may be 0.1% to 30%, 1% to 30%, 5% to 30%, or 10% to 20% by weight and volume.
  • the pH of the solvent is not particularly limited. Specific pH values include, for example, 6 to 8, 7 to 8, 7.2 to 7.8, 7.3 to 7.6, and 7.4 to 7.5.
  • the solvent may have the same composition as the blocking solution, or it may have a different composition.
  • the concentration of the antibody solution is not particularly limited as long as the cell membrane protein can be detected.
  • it can be appropriately set depending on the amount of antigen in the fixed tissue and the type of label linked to the secondary antibody.
  • the concentration can be, for example, 0.1 ⁇ g/mL or more, 0.2 ⁇ g/mL or more, 0.3 ⁇ g/mL or more, 0.4 ⁇ g/mL or more, 0.5 ⁇ g/mL or more, 0.6 ⁇ g/mL or more, 0.7 ⁇ g/mL or more, or 0.8 ⁇ g/mL or more.
  • the concentration can be, for example, 10 ⁇ g/mL or less, 8 ⁇ g/mL or less, 7 ⁇ g/mL or less, 5 ⁇ g/mL or less, 4 ⁇ g/mL or less, 3 ⁇ g/mL or less, 2.5 ⁇ g/mL or less, or 2 ⁇ g/mL.
  • incubation is performed at refrigerated temperature for 12 to 24 hours or at room temperature for 15 to 40 minutes, but any incubation conditions known in the art can be used and are not particularly limited.
  • the incubation temperature can be, for example, freezing temperature (0°C or below), refrigerated temperature (0°C to 5°C), room temperature (1°C to 30°C), or normal temperature (15°C to 25°C).
  • the incubation time can be, for example, 15 minutes or more, 30 minutes or more, 40 minutes or more, or 60 minutes or more. It can also be, for example, 24 hours or less, 16 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, or 2 hours or less.
  • the humidity conditions during incubation are not particularly limited as long as the antibody solution and the fixed tissue can continue to be in contact during incubation.
  • incubation is performed in an environment with 100% humidity (e.g., in a moist chamber).
  • the conditions may be constant, or may change intermittently or continuously. If the conditions change, the change may be artificial or natural.
  • the "secondary antibody reaction step” is a step of reacting a fixed tissue with a labeled secondary antibody that has immunological reactivity with the primary antibody. This step can usually be performed after the primary antibody reaction step. Optionally, this step may be performed simultaneously with the primary antibody reaction step.
  • the solvent for the antibody solution may be of the same composition as that used in the primary antibody reaction step, or may be of a different composition.
  • the type of secondary antibody is not particularly limited as long as it has immunological reactivity with the primary antibody.
  • the primary antibody has a constant region
  • an antibody capable of binding to the constant 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.
  • the type of label is not particularly limited. Examples include luminescent substances, fluorescent substances, enzymes, radioactive substances, biotin, avidin, quantum dots, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, and metal ions.
  • luminescent substances include acridinium esters, 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), luminol and its modified forms, 4-aminophthalhydrazide, various coelenterazines, and luciferin.
  • fluorescent substances include fluorescent dyes such as FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, TAMRA, and Alexa-Fluor dyes; fluorescent proteins such as GFP, EGFP, BFP, and YFP; and so on.
  • fluorescent dyes such as FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, TAMRA, and Alexa-Fluor dyes
  • fluorescent proteins such as GFP, EGFP, BFP, and YFP; and so on.
  • Enzymes include, for example, 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 used.
  • radioactive substances examples include 14 C, 123 I, 124 I, 131 I, 125 I, Tc99m, 32 P, 35 S, or 3 H.
  • the label may be directly bound to the antibody, or may be bound via a linker or the like, or may form a complex with the antibody via a polymer carrier or the like.
  • linker or polymer carrier There are no particular limitations on the specific type of linker or polymer carrier.
  • a linker known in the art such as a peptide linker, may be used.
  • a complex formed between an enzyme as a label and an antibody via a polymer carrier may be used.
  • a complex formed between a peroxidase as a label and an antibody via a polymer carrier for example, Peroxidase Labelled Polymer Conjugated (manufactured by DAKO) may be used.
  • the application method is not particularly limited. For example, it can be performed in accordance with the application method of the blocking solution in the blocking step.
  • Color development step is an optional step in which the fixed tissue is exposed to a reaction agent to make the label detectable. This step can be performed simultaneously with or after the labeling step. For example, this step can be performed simultaneously with or after the secondary antibody reaction step. If an endogenous reaction suppression step is performed, this step can be performed after that.
  • a color reaction is a reaction that makes a label detectable.
  • the reaction agent is not particularly limited as long as it is an agent that induces a color reaction of the label used.
  • an agent containing an antibody against the label, an agent containing a substrate when the label is an enzyme, an agent containing avidin when the label is biotin, an agent containing biotin when the label is avidin, etc. can be used.
  • the substrate can be appropriately determined based on the enzyme used as the label.
  • the substrate when peroxidase is used as the label, can be, for example, 3,3'-diaminobenzidine (DAB), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3-amino-9-ethylcarbazole (AEC), 3,3',5,5'-tetramethylbenzidine (TMB), Bajoran Purple, Vina Green, or other color-developing reagents.
  • DAB 3,3'-diaminobenzidine
  • ABTS 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
  • AEC 3-amino-9-ethylcarbazole
  • TMB 3,3',5,5'-tetramethylbenzidine
  • Bajoran Purple Vina Green
  • Vina Green or other color-developing reagents.
  • examples of substrates include color-developing 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.
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate
  • NBT Nitro Blue Tetrazolium Chloride
  • naphthol AS phosphate naphthol AS phosphate
  • fuchsin fuchsin
  • Ferangi Blue Ferangi Blue
  • Vulcan Fast Red Vulcan Fast Red
  • Warp Red Warp Red
  • a substance or agent that accelerates the color-developing reaction can be additionally used.
  • divalent ions such as Ni2 + or Co2 + can be used, or other commercially available enzyme reaction accelerators can be used.
  • naphthol AS phosphate a diazonium salt can be used in combination to form an azo dye by the enzyme activity of naphthol.
  • Reactants can be used in combination with multiple types. For example, if biotin is used as a label, avidin and biotinylated peroxidase can be used, and a peroxidase substrate can be added.
  • the reaction conditions for this step are not particularly limited. For example, they can be appropriately determined according to the concentration of the antibody used and the expected amount of antigen in the fixed tissue.
  • the reaction time is not particularly limited.
  • the reaction can be carried out for 5 seconds or more, 10 seconds or more, 15 seconds or more, 16 seconds or more, 19 seconds or more, or 20 seconds or more.
  • the reaction can be carried out for, for example, 60 minutes or less, 50 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 10 minutes or less, 9 minutes or less, 8 minutes or less, or 7 minutes or less.
  • the temperature is not particularly limited.
  • it can be freezing temperature (0°C or below), refrigerated temperature (0°C to 5°C), room temperature (1°C to 30°C), or normal temperature (15°C to 25°C).
  • the processing is performed at room temperature or normal temperature.
  • a dehydration treatment can be performed after the color reaction.
  • the method of dehydration treatment is not particularly limited.
  • ethanol can be used for gradual dehydration, and then further dehydration can be performed using an organic solvent such as xylene.
  • the sample can be immersed in 70%, 80%, 90%, 95%, and 100% ethanol aqueous solutions in that order. Further dehydration can be performed using xylene.
  • Label detection step (S0104) is an essential step, which is a step of detecting the label bound to the cell membrane protein. This step can be performed after the labeling step (S0103). When a color development step is performed, this step can be performed simultaneously with or after the color development step. Optionally, this step can be performed simultaneously with the labeling step (S0103), for example, simultaneously with the secondary antibody reaction step.
  • any other staining may be performed in combination.
  • cell nuclei may be stained.
  • the types of other staining are not particularly limited, but examples include DAPI staining, hematoxylin-eosin (HE) staining, Gomori methenamine silver staining (GMS), periodic acid-Schiff (PAS) stain, trichrome blue staining, Masson's trichrome staining, Prussian blue staining, Giemsa staining, Gram staining, mucicarmine staining, Verhoeff-van Gieson staining, Elastica staining, carbol fuchsin staining, Golgi staining, and combinations thereof.
  • Encapsulation can be performed prior to detection. There are no particular limitations on the encapsulation method and encapsulant used.
  • the detection method can be appropriately selected depending on the label and reagent used, and the properties of the fixed tissue, and the equipment required for that detection method can be used.
  • detection can be performed by visual inspection, using a microscope (e.g., optical microscope such as a stereomicroscope, confocal microscope, or fluorescent microscope), using a detector (e.g., fluorescence activated cell sorting (FACS), luminescence photometer, absorption photometer, etc.), or a combination thereof.
  • FACS fluorescence activated cell sorting
  • luminescence photometer luminescence photometer
  • absorption photometer etc.
  • detection can be performed, for example, by autoradiography, a scintillation counter, positron emission tomography (PET), or a combination thereof.
  • a color-developing reagent e.g., a color-developing reagent reactive with peroxidase
  • it is preferably detected in bright field using an optical microscope.
  • one or more organelles can be stained to determine the location of the label.
  • a target cell membrane protein eg, CAPRIN-1 protein
  • a target cell membrane protein present on a cell membrane can be detected with high sensitivity and specificity.
  • This method can be performed manually, but can also be performed automatically using an automatic staining device, etc.
  • a second aspect of the present invention is a method for determining a treatment method.
  • the method of this aspect includes a disease marker detection step and a treatment method determination step as essential steps.
  • a treatment method for an individual from which a fixed tissue is derived can be determined.
  • the "disease marker detecting step” is a step of detecting a disease marker in the embedded fixed tissue.
  • This step can be carried out in accordance with the method for detecting cell membrane proteins described in the first embodiment.
  • the method described in the first embodiment differs from this step in that the cell membrane proteins detected in this step are disease markers.
  • the individual from which the fixed tissue of this embodiment is derived is not particularly limited. For example, it may be an individual who has a disease correlated with the disease marker to be detected, an individual who may have the disease, or a healthy individual.
  • the "treatment method determination step” is a step of determining a treatment method 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.
  • the method of determining the treatment method is not particularly limited as long as it is based on the correspondence between the disease marker and the treatment method. For example, it is possible to determine whether or not a disease marker has been detected in the fixed tissue, and to determine the treatment method from the obtained determination result based on the correspondence between the disease marker and the treatment method.
  • the method of determining whether or not a disease marker has been detected is not particularly limited.
  • the disease marker may be determined to have been detected when a label is detected, or the disease marker may be determined to have been detected when the detected label satisfies a certain condition.
  • the conditions here are not particularly limited.
  • the amount of label in the fixed tissue can be quantified and the condition can be that it is equal to or greater than a certain amount.
  • the method of quantification is not particularly limited.
  • a score value reflecting the staining state can be calculated and quantified.
  • the score values are set, for example, in two or more stages, preferably in four stages.
  • An example of setting each score when classifying the score values reflecting the staining state of cell membrane proteins expressed on the cell membrane (e.g., cell surface) into four stages is shown below.
  • - Score 0 The positive cell rate (the percentage of cells in which the label is detected on the cell membrane) is less than 10%.
  • - Score 1 The positive cell rate is 10% or more, but the label is limited to a part of the cell membrane of the diseased cell (e.g., cancer cell), and shows weak staining intensity.
  • - Score 2 The positive cell rate is 10% or more, the label is localized to the cell membrane of the diseased cell (e.g., cancer cell), and shows moderate staining intensity.
  • - Score 3 The positive cell rate is 10% or more, the label is localized to the cell membrane of the diseased cell (e.g., cancer cell), and shows strong staining intensity.
  • score values are determined by the American Society of Clinical Oncology in the United States for cancer markers, and are recognized by the Japanese Society of Pathology in Japan. They are also applied to the "Hercept Test" which quantifies the amount of Her2, a cancer antigen, present in a patient sample. The quantification of Her2 is determined in the ASCO/CAP Her2 Testing Guideline, and in Japan, the Trastuzumab Pathology Committee has established a Her2 Testing Guide that includes the setting of this score. For example, when detecting a protein whose amount on the cell membrane increases based on a disease as a disease marker, the score value can be set according to this example.
  • a disease marker has been detected when the score is 1 or more, 2 or more, or 3 or more.
  • a disease marker is determined to have been detected when the score is 2 or more (scores 2 and 3).
  • the percentage of diseased cells in which the label is detectable, as indicated by each score value can be estimated by increasing the sensitivity of the optical microscope by 4 times, 10 times, or 20 times, counting at least 500 cells in the field of view, and counting the cells that show a stained image on the cell membrane indicated by each score value, using the following formula: Number of positive cells/total number of cells (around 500) x 100 (%)
  • a disease marker may be determined to have been detected by comparing the amount of label detected in the fixed tissue to be detected with the amount of label in a control tissue (e.g., a fixed tissue not having the disease, or a fixed tissue having the disease). For example, in the case of a disease marker whose expression increases based on a disease, it can be determined that a disease marker has been detected if the amount of label detected is greater than or statistically significantly greater than the amount of label in the fixed tissue not having the disease. Conversely, in this case, it can be determined that a disease marker has not been detected if the amount of label detected is less than or statistically significantly less than the amount of label in the fixed tissue having the disease.
  • a control tissue e.g., a fixed tissue not having the disease, or a fixed tissue having the disease.
  • “statistically significant” means that there is a significant difference when the quantitative difference between two things is statistically processed. Specific examples include cases where the risk rate (significance level) is less than 5%, 1%, or 0.1%. There are no particular limitations on the testing method, so long as it is a publicly known method that can determine the presence or absence of significance. For example, the Student's t-test or multiple comparison test can be used.
  • the determination may be based on a cutoff value for determining that a disease marker has been detected.
  • the disease marker can be determined to have been detected when an amount of marker that exceeds the cutoff value is detected.
  • the treatment method can be determined by collating information on the detected disease marker with information on the correspondence between the presence or absence of detection of various disease markers and the treatment method.
  • information on a plurality of disease markers can be used, and the treatment method can be determined in combination with information on disease markers (e.g., disease markers other than cell membrane proteins, disease markers obtained from tissues other than fixed tissues, etc.) detected without using the disease marker detection process of this embodiment.
  • disease markers e.g., disease markers other than cell membrane proteins, disease markers obtained from tissues other than fixed tissues, etc.
  • Any information known in the technical field can be used as information on the relationship between the presence or absence of detection of a disease marker and a treatment method.
  • either or both of the determination that a disease marker has been detected and the decision on a treatment method can be performed by a computer or other electronic calculator.
  • a treatment method for an individual from which the fixed tissue is derived can be determined with high accuracy based on whether or not a disease marker is detected in the fixed tissue.
  • the third aspect of the present invention is a cell membrane protein detection device (0200).
  • the device of this aspect includes an embedding medium removal unit (0210), a labeling reaction unit (0230), and a label detection unit (0240) as essential components, and an antigen activation reaction unit (0220), a cooling unit, a non-specific reaction inhibition unit, a color reaction unit, and a control unit (0250) as optional components.
  • an embedding medium removal unit (0210) a labeling reaction unit (0230), and a label detection unit (0240) as essential components
  • an antigen activation reaction unit 0220
  • cooling unit a cooling unit
  • non-specific reaction inhibition unit a color reaction unit
  • a control unit (0250) as optional components.
  • cell membrane proteins can be detected with high specificity.
  • FIG. 16 A functional block diagram of a configuration example of the manufacturing device of the present invention is shown in Figure 16.
  • the embedding medium, fixed tissue, disease, cell membrane protein, etc. are as described in the first embodiment.
  • Each part constituting the detection device of this embodiment will be described below.
  • the cell membrane protein in the device of this embodiment may be, for example, a cell membrane protein expressed on the surface of a cancer cell, as described in the first embodiment, and may preferably be a disease marker such as a cancer marker, specifically, CAPRIN-1 protein, etc.
  • the "embedding medium removal unit (0210)" is an essential component of the device of this embodiment, and is configured to allow the embedding medium to be removed by contacting the fixed tissue containing the solid embedding medium with a removal solution.
  • the embedding medium removing unit performs the embedding medium removing step (S0101) in the method for detecting a cell membrane protein according to the first aspect.
  • the storage means (0211) is a container configured to store the removal solution.
  • the specific configuration of the storage means for the removal solution is not particularly limited.
  • the overall shape of the storage means is not particularly limited as long as it can contain the solution inside.
  • it may be any shape such as a polygonal prism (including a regular polygonal prism and an approximately polygonal prism), an elliptical cylinder (including a cylindrical liquid and an approximately elliptical cylinder), or a sphere (including an approximately spherical shape).
  • the volume of the container is not particularly limited, and may be determined appropriately according to the amount of solution used in one run with the device of this embodiment. For example, when the device of this embodiment is used on a laboratory scale, a volume of about 10 mL to 200 mL is sufficient, but when used on a factory scale or configured to withstand repeated runs over a certain period of time, the volume can be several liters or more, or even several tens of liters or more.
  • the material that the container is made of but considering that a solution will be stored inside, it is preferable that the material has the strength to store the solution and the rigidity to maintain its shape when stored inside, that the solution does not penetrate to the outside, and that at least the inner wall is not decomposed or altered by the solution.
  • the material include, but are not limited to, metal, synthetic resin (plastic), glass, and porcelain.
  • the contacting means when the fixed tissue is brought into contact with the solution in the storage means, there are no particular limitations as long as the shape and size are such that at least the part of the fixed tissue to be subjected to the reaction can be stored.
  • the container may be equipped with additional openings for passing liquid (such as an outlet and/or water inlet), flow paths, flow valves, flow mechanisms such as stirring blades, and filtering mechanisms for removing solid matter derived from the fixed tissue, in addition to the opening for passing the fixed tissue.
  • liquid such as an outlet and/or water inlet
  • flow paths such as an outlet and/or water inlet
  • flow valves such as stirring blades
  • filtering mechanisms for removing solid matter derived from the fixed tissue, in addition to the opening for passing the fixed tissue.
  • the configuration of the storage means used in the embedding medium removal section may be the same as the storage means used in one or more other sections, or may be different from any of the storage means used in the other sections.
  • the contact means (0212) is configured to be able to bring the fixed tissue held in the holding means (0213) into contact with the removal solution stored in the storage means (0211).
  • the specific configuration of the contact means is as follows: There are no particular limitations to the method, and the method may be, for example, a method capable of spraying, scattering, or applying the removal solution to the fixed tissue, or a method capable of immersing the fixed tissue in the removal solution.
  • any mechanism for reducing the distance may be provided.
  • Such mechanisms include, for example, a mechanism for moving the holding means and/or the storage means, a flow path capable of transporting the removal solution from the storage means to a position close to the holding means, or a combination thereof.
  • the configuration of the contact means used in the embedding medium removal unit may be the same as the contact means used in one or more other units, or may be different from any of the contact means used in the other units. It may also be configured so that it can be commonly used in two or more units.
  • the holding means (0213) is configured to hold the fixed tissue including the solid-state embedding medium.
  • the specific configuration of the holding means for the fixed tissue is not particularly limited.
  • a holding means that is commonly used in staining can be used.
  • the holding means can be configured to hold, clamp, or place a fixed tissue, or can be configured to accommodate a fixed tissue inside the holding means. You may do so.
  • the graspable configuration includes, but is not limited to, both an integrated configuration and a detachable configuration, such as a magnetic holding means such as a permanent magnet or temporary magnet, a hanging means such as a hook or a string, a fastening means such as a chuck, or a combination of these.
  • a magnetic holding means such as a permanent magnet or temporary magnet
  • a hanging means such as a hook or a string
  • a fastening means such as a chuck, or a combination of these.
  • the clamping configuration includes both a structure that clamps the entire fixed tissue (such as a structure in which the fixed tissue is placed in a space sandwiched between partitions) and a structure that clamps only a portion of the fixed tissue (such as a structure in which the fixed tissue is placed upright between low partitions), and is not particularly limited.
  • the configuration on which the fixed tissue can be placed includes any configuration on which the fixed tissue can be placed, and is not particularly limited.
  • the surface shape of the placement surface is not particularly limited, and may be a configuration that allows the position of the fixed tissue to be restricted, or a configuration that does not restrict the position of the fixed tissue.
  • the surface may have one or more holes (such as a shape composed of linear structures that may or may not intersect with each other), or may have no holes. If holes are present, the size, number, and shape of the holes are not particularly limited as long as the fixed tissue does not fall out.
  • the configuration capable of accommodating fixed tissue therein includes any configuration capable of accommodating fixed tissue therein and in which the internal space communicates with the external space, and is not particularly limited.
  • the configuration in which the internal space communicates with the external space includes both a configuration in which communication with the external space is constantly maintained, and a configuration in which communication with the external space is only maintained under specific conditions. Examples of configurations in which communication with the external space is constantly maintained include a configuration in which the fixed tissue is surrounded by a material having holes, such as a mesh, cloth, filter, or a combination thereof, or a configuration in which the fixed tissue is accommodated within the material.
  • Examples of configurations that communicate with the external space only under specific conditions include a configuration (such as a container) in which the fixed tissue is accommodated within a space made of a material that does not have holes through which a drug can enter. In this case, an opening that can be opened or closed depending on the conditions can be additionally included.
  • the material of the holding means is not particularly limited.
  • a fiber material, an organic resin, a glass-based material, a metal material, or a combination of these can be used.
  • the holding means may be made of a rigid body, or at least a part of the holding means may be made of a soft body.
  • the holding means has a portion that comes into contact with the drug, it is preferable that the portion be made of a material that is not decomposed or denatured by the drug.
  • each unit can be provided with a holding means independent of the other units, and can be configured to allow the fixed tissue to be transferred between the holding means of different units.
  • the configuration of the holding means used in the embedding medium removal unit may be the same as the holding means used in one or more other units, or may be different from any of the holding means used in the other units.
  • the temperature control means is configured to be able to adjust the temperature of the fixed tissue.
  • the temperature control means here is configured to be able to maintain the fixed tissue at a temperature lower than the melting point of the embedding medium.
  • the specific configuration of the temperature control means is not particularly limited.
  • the temperature of the fixed tissue itself may be directly adjustable, or the temperature of the solution surrounding the fixed tissue may be adjustable.
  • the temperature adjustment means may be configured to maintain heat, or to change the temperature.
  • Examples of configurations that can maintain heat include configurations that use insulating materials, gas layers, vacuum layers, or combinations thereof. In the case of a configuration that can change the temperature, it may be configured to enable both heating and cooling, or to enable only one of them. Specific examples include thermostats, heaters and coolers for liquids or solids, heat generating agents and heat absorbing agents that are generated by chemical reactions, and storage tanks for high or low temperature solutions.
  • One or more of the holding means (0213), contact means (0212), and storage means (0211) may be equipped with a temperature adjustment means, or a temperature adjustment means may be provided separately from them.
  • the temperature of the fixed tissue is maintained below the melting point of the embedding medium by the temperature control means.
  • the temperature can be appropriately set depending on the type of embedding medium, and is not particularly limited. The temperature is as described in the first embodiment.
  • the cleaning means is configured to be capable of cleaning the surface of the fixed tissue.
  • the specific configuration of the cleaning means is not particularly limited. For example, it can be configured to be capable of storing or supplying the cleaning solution described in the first aspect.
  • the configuration capable of storing is similar to the above-mentioned storage means (0211) except that the liquid to be stored is not a removal solution but a cleaning solution.
  • the configuration of the cleaning means used in the embedding medium removal unit may be the same as the cleaning means used in one or more other units, or may be different from any of the cleaning means used in the other units. It may also be configured so that it can be used in common by two or more units.
  • the antigen activation reaction unit (0220) is an optional component of the device of this embodiment, and is configured to be capable of activating cell membrane proteins in fixed tissue that have been subjected to the embedding medium removal unit (0210).
  • the antigen activation reaction unit (0220) performs the antigen activation step (S0102) in the method for detecting cell membrane proteins described in the first embodiment.
  • the antigen activation reaction unit (0220) usually includes an antigen activation means (0221), a contact means (0222), a means for holding the fixed tissue, and a washing means.
  • the contacting means (0222), holding means and cleaning means shall conform to the description of each means in the embedding medium removal section (0210).
  • the antigen activating means (0221) is configured to activate cell membrane proteins of fixed tissue.
  • the specific configuration of the antigen activating means (0221) is not particularly limited and can be appropriately selected depending on the type of antigen activation treatment to be performed.
  • the enzyme liquid storage means described in the first embodiment can be used, and if the antigen activation process is based on a heat treatment step, the antigen activation solution storage means described in the first embodiment can be used.
  • the storage means for various liquids are similar to the removal solution storage means (0211) except that the solution stored is an enzyme liquid or antigen activation solution.
  • the antigen activation means is preferably configured so that the temperature of the liquid can be adjusted.
  • the adjusted temperature is the temperature in the antigen activation process described in the first embodiment, it is similar to the temperature control means in the embedding medium removal unit (0210). Even if only one step is performed in a single run, storage means for solutions corresponding to both steps can be provided, and storage means for multiple types of solutions for each of the enzyme liquid and antigen activation solution can be provided.
  • Cooling section is an optional component of the device of this embodiment, and is configured to be capable of cooling the fixed tissue that has passed through the antigen activation reaction section (0220).
  • the cooling section performs the cooling step in the method for detecting cell membrane proteins described in the first embodiment.
  • the cooling section usually includes a cooling means and a means for holding the fixed tissue, and may further include a contacting means and a washing means.
  • the holding means, contacting means and cleaning means shall conform to the description of each means in the embedding medium removal section (0210).
  • the cooling means is configured to be able to lower the temperature of the fixed tissue that is in contact with it.
  • the fixed tissue may be configured to be able to dissipate heat, or may be configured to be able to absorb heat from the fixed tissue.
  • the specific configuration it basically conforms to the temperature adjustment means in the embedding medium removal unit (0210) except that the purpose is cooling and the temperature to be adjusted is the temperature in the cooling step of the first embodiment. Only the cooling function of the means capable of heating may be used.
  • the cooling here can bring the fixed tissue into contact with any solid, solution, gas or combination thereof.
  • the cooling means may be configured to bring the fixed tissue into contact with a solid, solution, gas, etc. that is at a lower temperature than the fixed tissue.
  • Non-specific reaction suppression unit is an optional component of the device of this embodiment, and is configured to be able to contact the fixed tissue that has passed through the embedding medium removal unit (0210) with a reaction suppressor. In some cases, it may be configured to be able to process the fixed tissue that has passed through the antigen activation reaction unit (0220) and/or the cooling unit.
  • the non-specific reaction suppression unit performs the non-specific reaction suppression step in the method for detecting cell membrane proteins described in the first embodiment.
  • the non-specific reaction suppression unit usually includes a reaction suppressor storage means, a contact means, and a fixed tissue holding means, and in some cases, further includes a washing means.
  • the contact means, holding means and cleaning means shall conform to the description of each means in the embedding medium removal section (0210).
  • the specific configuration of the reaction inhibitor storage means can be appropriately selected depending on the type of nonspecific reaction inhibition treatment performed, and is not particularly limited.
  • the storage means for the blocking solution described in the first embodiment can be used, and when the nonspecific reaction inhibition treatment is based on an endogenous reaction inhibition step, the storage means for the reaction inhibitor for the color reaction described in the first embodiment can be used.
  • the storage means for various liquids are similar to the storage means for the removal solution, except that the solution stored is a reaction inhibitor. Even when only one step is performed in a single implementation, storage means for solutions corresponding to both steps can be provided, and storage means for multiple types of reaction inhibitors can be provided.
  • the labeling reaction section (0230) is an essential component of the device of this embodiment, and is configured to be capable of reacting the cell membrane protein of the fixed tissue that has passed through the embedding medium removal section (0210) with a labeled antibody that is immunologically reactive with the cell membrane protein. In some cases, it may be configured to be capable of processing the fixed tissue that has passed through the antigen activation reaction section (0220), the cooling section, and/or the non-specific reaction suppression section.
  • the labeling reaction section (0230) performs the labeling step (S0103) in the method for detecting a cell membrane protein described in the first embodiment.
  • the labeling reaction section (0230) usually includes a labeled antibody storage means (0233), a contact means (0234), and a fixed tissue holding means, and in some cases, further includes a binding molecule storage means (0231), a contact means (0232), and a washing means.
  • the contacting means (0232, 0234), holding means and washing means shall conform to the description of each means in the embedding medium removal section (0210).
  • a binding molecule storage means (0231) for storing the binding molecules used in each binding step can be provided.
  • the binding molecule storage means (0231) can include a storage means for the primary antibody solution described in the first aspect
  • the labeled antibody storage means (0233) can include a storage means for the secondary antibody solution described in the first aspect.
  • the storage means for each solution is similar to the storage means for the removal solution (0211) except that the stored solution is the antibody solution or binding molecule solution described in the first embodiment. Even if only a binding reaction using a labeled antibody is performed in one run, a storage means for the binding molecule solution can be provided, and storage means for multiple types of antibodies or binding molecules can be provided.
  • the color reaction section is an optional component of the device of this embodiment, and is configured to allow exposure of the fixed tissue that has passed through the labeling reaction section (0230) to a reactant so that the label becomes detectable.
  • the color reaction section performs the color development step in the method for detecting cell membrane proteins described in the first embodiment.
  • the color reaction section usually includes a reactant storage section, a contact means, and a means for holding the fixed tissue, and may further include a washing means.
  • the contact means, holding means, and washing means are the same as those described for the embedding medium removal section (0210).
  • the reactant storage means is the same as the removal solution storage means (0211) except that the stored solution is the reactant described in the first embodiment.
  • the label detection unit (0240) is an essential component of the device of this embodiment, and is configured to be capable of detecting the labeled antibody of the labeled antibody-bound immobilized tissue that has passed through the labeling reaction unit.
  • the label detection unit (0240) performs the label detection step (S0104) in the method for detecting cell membrane proteins according to the first embodiment.
  • the label detection unit usually includes a detection means (0241), an analysis means (0242), and a holding means for the fixed tissue.
  • the holding means shall conform to the description of the holding means (0213) in the embedding medium removal section (0210).
  • the detection means (0241) is configured to be capable of detecting a label bound to a cell membrane protein in a fixed tissue.
  • the configuration of the detection means (0241) can be appropriately selected depending on the label and reagent used, the properties of the fixed tissue, and the like, and is not particularly limited.
  • a microscope e.g., optical microscope such as a stereo microscope, a confocal microscope, or a fluorescence microscope
  • a detector e.g., fluorescence activated cell sorting (FACS), a luminometer, an absorptiometer, etc.
  • FACS fluorescence activated cell sorting
  • a luminometer e.g., a luminometer
  • an absorptiometer e.g., a laser scanner
  • autoradiography e.g., a scintillation counter
  • PET positron emission tomography
  • the analysis means (0242) is configured to be capable of analyzing the signal data of the label detected by the detection means (0241).
  • the configuration of the analysis means (0242) varies depending on the detection means (0241) used. It can be selected appropriately and is not particularly limited.
  • an analysis means that is normally used to analyze signal data using each detection means may be provided, or an analysis mechanism built into the detection means used may be used as the analysis means of this embodiment.
  • the analysis means includes a computer.
  • the specific configuration of the hardware means and software means is not particularly limited, but for example, the configuration described below for the control unit can be adopted.
  • the specific content of the analysis process performed here is not particularly limited. For example, a process of converting the signal into image data, a process of removing noise from the signal data, etc. may be performed. If necessary, the device may be further configured to be capable of performing a process of determining whether or not the disease marker described in the second aspect has been detected.
  • the analysis means may further include an output mechanism for displaying the analysis results to a user.
  • Control unit (0250) is an optional component of the device of this embodiment, and is configured to be able to adjust the connection between each unit and the fixed tissue and the processing conditions in each unit.
  • the control unit (0250) allows the fixed tissue to be connected to a predetermined unit at a predetermined timing, and the fixed tissue is processed under predetermined processing conditions (such as the type of reaction solution and temperature conditions) for a predetermined period of time.
  • the control unit may be configured to enable the device of this embodiment to execute the series of methods described in the first embodiment, or may be configured to enable the device to execute some of the steps of the methods described in the first embodiment.
  • the control unit includes a computer.
  • the control unit is composed of hardware means, or hardware means and software means
  • the hardware means is composed of the following components: a CPU (0251), a volatile memory (0252), a non-volatile memory (0253), an interface (0254), a system bus (0255) connecting these, and peripheral devices.
  • Peripheral devices include, but are not limited to, a clock, a water level sensor, and the like.
  • the software means is composed of programs that can be executed on the hardware memory, and the like.
  • various programs e.g., communication control programs and processing condition control programs
  • various programs stored in the non-volatile memory (0253) of the hardware means are expanded on the volatile memory (0252), and the programs are executed sequentially, thereby processing, storing, outputting, etc., data in the memory and data input via the interface (0254).
  • the contact means by operating the contact means when a programmed predetermined time is reached, the fixed tissue can be brought into contact with the target drug, etc., and the contact means can be operated again after a certain time has passed to stop the contact.
  • the temperature control means or cooling means when a programmed predetermined time is reached, the fixed tissue can be adjusted to a predetermined temperature, and the contact means can be operated again after a certain time has passed to stop the contact. Furthermore, if information indicating that the target conditions are deviated from during the reaction in each unit is input via the interface (0254) from various sensors such as a temperature sensor or a pH sensor, the reaction condition control program expanded on the volatile memory (0252) can be executed, and a drug can be added or the temperature control means can be operated to adjust the conditions to within the target range.
  • a display unit that presents information to the user can be provided, the contents of which are controlled by the display control unit.
  • the display control unit There are no particular limitations on the hardware of the display unit, but examples include a display.
  • an input device through which the user inputs information may be provided, and examples of input devices include a keyboard, mouse, touch panel, and touch pen.
  • the information input from the input device is controlled by an input/output control unit.
  • the input/output control unit controls the input of data and commands from the input device as well as the output of various data output from the processing device.
  • the user may be asked to set the time and reaction conditions for each process, as well as to specify the process to be performed and the criteria for updating the solution in the storage means (e.g., turbidity, period, etc.).
  • Example 1 Detection of CAPRIN-1 protein by immunohistochemical staining 1 (Paraffin removal process) Paraffin was removed from 4 ⁇ m-thick paraffin-embedded human cancer tissue sections (breast cancer tissue, lung cancer tissue, pancreatic cancer tissue, kidney cancer tissue, colon cancer tissue, gastric cancer tissue, ovarian cancer tissue, and prostate cancer tissue; US BIOMAX; sections derived from 1,361 different subjects were used) using xylene for 20 minutes. The xylene was replaced three times every 5 minutes. Next, washing was performed using 100%, 90%, and 80% ethanol aqueous solutions instead of xylene, for 5 minutes each, in the same manner. These steps were performed at room temperature. Then, washing was performed three times for 5 minutes each using PBS-T (phosphate buffer solution (PBS) containing 0.05% Tween 20, pH 7.4).
  • PBS-T phosphate buffer solution
  • Non-specific inhibition reaction process (Peroxidase endogenous reaction inhibition step) Excess water was removed from the surroundings of each type of human cancer tissue section that had been subjected to the antigen activation step, and an appropriate amount of Peroxidase Block (manufactured by DAKO) was added dropwise and allowed to stand at room temperature for 5 minutes.
  • a primary antibody solution containing the mouse anti-CAPRIN-1 monoclonal antibody prepared in WO2013/018891 was added to various human cancer tissue sections that had been subjected to the nonspecific inhibition reaction step, and the sections were incubated overnight in a moist chamber at 4° C.
  • the primary antibody solution was prepared by adding the above-mentioned mouse anti-CAPRIN-1 antibody to a PBS-T solution containing 5% FBS and 20% normal goat serum to a final concentration of 2 ⁇ g/mL.
  • FIG. 1 The stained images obtained for ovarian cancer tissue are shown in Figure 1.
  • A shows the stained image of the cell nucleus and CAPRIN-1 protein
  • B shows the stained image of CAPRIN-1 protein excluding the signal of the cell nucleus.
  • the signal of CAPRIN-1 protein localized on the cell membrane was observed to border the cells ( Figure 1B).
  • the antibodies used in the primary antibody reaction step are those disclosed in WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/018889, WO20 It was found that the signals of cell membrane proteins on the cell membrane can be detected with high accuracy, similar to the above, by applying 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.
  • Comparative Example 1 Detection of CAPRIN-1 Protein by Immunohistochemical Staining
  • Various paraffin-embedded human cancer tissue sections manufactured by BIOMAX derived from the same specimens as in Example 1 were subjected to immunohistochemical staining according to the steps described in Example 1, except that a heat treatment (baking) was performed at 62°C for 15 minutes before paraffin removal in the paraffin removal step, and that the heat treatment in the antigen activation step was performed at 121°C.
  • Example 2 Determination of CAPRIN-1 positive cancer cells
  • a 4x objective lens of an optical microscope was used to observe CAPRIN-1 protein stained images and positive stained signal intensities in cancer cells in the cancer tissues of the sections mounted in Example 1 and Comparative Example 1.
  • the objective lens was switched to 10x or 20x, and the ratio of cancer cells showing specific coloring in the cell membrane (CAPRIN-1 positive cell rate) to all cells of the cancer tissues in all sections derived from each specimen was calculated.
  • each cancer tissue sample was classified into the following scores of 0 to 3. Of these scores, scores of 2 and 3 were determined to be CAPRIN-1 positive cancer tissue samples.
  • Score 0 (no CAPRIN-1 protein overexpression): CAPRIN-1 positive cell rate (proportion of cells exhibiting a signal specific to the cell membrane) is less than 10%.
  • Score 1 (no CAPRIN-1 protein overexpression): CAPRIN-1 positive cell rate is 10% or more, but the signal is localized to a part of the cancer cell membrane, and the staining intensity is weak.
  • Score 2 (CAPRIN-1 protein overexpression): CAPRIN-1 positive cell rate is 10% or more, the signal is localized to the cancer cell membrane, and the staining intensity is moderate.
  • Score 3 (CAPRIN-1 protein overexpression): CAPRIN-1 positive cell rate is 10% or more, the signal is localized to the cancer cell membrane, and the staining intensity is strong. Tissue specimens with scores 2 and 3 are determined to be CAPRIN-1 positive cancer tissue specimens.
  • Illustrative staining images for each score are shown in Figures 3 to 10.
  • the specimen with a score of 0 Figure 3
  • the specimen with a score of 1 Figure 4
  • the signal on the cell membrane was weak and the boundaries between cells were not clear.
  • the specimen with a score of 2 Figure 5
  • the specimen with a score of 3 Figure 6
  • a strong signal was observed on the cell membrane and the boundaries between cells were clear.
  • the proportion of CAPRIN-1 positive cancer tissue specimens in the samples stained in Example 1 i.e., the proportion of CAPRIN-1 positive cancer tissue specimens in the number of each type of cancer tissue specimen, was 75% for breast cancer tissue, 65% for lung cancer tissue, 70% for pancreatic cancer tissue, 70% for kidney cancer tissue, 60% for colon cancer tissue, 60% for gastric cancer tissue, 65% for ovarian cancer tissue, and 90% for prostate cancer tissue.
  • the percentage of CAPRIN-1 positive cancer tissue specimens was 52% for breast cancer tissue, 54% for lung cancer, 58% for pancreatic cancer, 55% for kidney cancer, 49% for colon cancer, 40% for gastric cancer, 45% for ovarian cancer, and 54% for prostate cancer, which was significantly different from the results based on the staining in Example 1.
  • the antibodies used in the primary antibody reaction step are those disclosed in WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/
  • a similar decrease in staining accuracy was observed when 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 were used.
  • Example 3 Detection of CAPRIN-1 protein by immunohistochemical staining 2
  • immunohistochemical staining was performed as described in Example 1, except that the antibody concentration of the primary antibody solution used in the primary antibody reaction step was 0.8 ⁇ g/mL and the color development time in the color development step was approximately 7 minutes.
  • CAPRIN-1 positive cancer tissue specimens were determined in the same manner as in Example 2.
  • CAPRIN-1 positive cancer tissues was 80% for breast cancer tissue, 70% for lung cancer, 80% for pancreatic cancer, 80% for kidney cancer, 70% for colon cancer, 55% for gastric cancer, 55% for ovarian cancer, and 80% for prostate cancer, which was generally consistent with the results based on staining in Example 1.
  • the antibodies used in the primary antibody reaction step are those disclosed in WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/018889, WO20
  • 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 used, the same percentage of CAPRIN-1 positive cancer tissue specimens as above was obtained.
  • Example 4 Detection of CAPRIN-1 protein by immunohistochemical staining method 3
  • immunohistochemical staining was performed as described in Example 1, except that the heat treatment in the antigen activation step was performed at 121°C, and CAPRIN-1 positive cancer tissue specimens were evaluated in the same manner as in Example 2.
  • the stained image obtained for the ovarian cancer tissue is shown in Figure 13. It was found that even if the heating temperature in the antigen activation process was changed, staining with the same accuracy as the stained image obtained in Example 1 ( Figure 11) was possible.
  • the percentage of CAPRIN-1 positive cancer tissue samples was 77% for breast cancer tissue, 67% for lung cancer, 75% for pancreatic cancer, 75% for kidney cancer, 65% for colon cancer, 60% for gastric cancer, 60% for ovarian cancer, and 85% for prostate cancer, which was generally consistent with the determination results based on the staining in Example 1.
  • the antibodies used in the primary antibody reaction step are those disclosed in WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/018889, WO20
  • 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 used, the same percentage of CAPRIN-1 positive cancer tissue specimens as above was obtained.
  • Example 5 Detection of CAPRIN-1 protein by immunohistochemical staining 4
  • immunohistochemical staining was performed as described in Example 1, except that the heat treatment in the antigen activation step was performed at 121°C, the antibody concentration of the primary antibody solution used in the primary antibody reaction step was 0.8 ⁇ g/mL, and the color development time in the color development step was approximately 7 minutes.
  • CAPRIN-1 positive cancer tissue specimens were determined in the same manner as in Example 2.
  • the stained image obtained for the ovarian cancer tissue is shown in Figure 14. It was found that even if the heating temperature, antibody concentration, and color development time in the antigen activation process were changed, staining with the same accuracy as the stained image obtained in Example 1 ( Figure 11) was possible.
  • the percentage of CAPRIN-1 positive cancer tissue samples was 79% for breast cancer tissue, 68% for lung cancer, 77% for pancreatic cancer, 76% for kidney cancer, 62% for colon cancer, 62% for gastric cancer, 61% for ovarian cancer, and 86% for prostate cancer, which was generally consistent with the results based on the staining in Example 1.
  • the antibodies used in the primary antibody reaction step are those disclosed in WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/018889, WO20
  • 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 used, the same percentage of CAPRIN-1 positive cancer tissue specimens as above was obtained.
  • Example 6 Detection of CAPRIN-1 protein by immunohistochemical staining 5
  • immunohistochemical staining was carried out in accordance with the description of Example 1, except that the paraffin removal process was carried out as follows, and CAPRIN-1 positive cancer tissue specimens were determined in the same manner as in Example 2.
  • Paraffin removal process Paraffin-embedded human cancer tissues (BIOMAX) were deparaffinized using Artisan Cleaning Solution (Agilent) for 15 minutes. The Artisan Cleaning Solution was replaced twice every 5 minutes. This step was carried out at room temperature. The tissues were then washed three times with PBS-T for 5 minutes each.
  • the percentage of CAPRIN-1 positive cancer tissue samples was 78% for breast cancer tissue, 66% for lung cancer, 76% for pancreatic cancer, 79% for kidney cancer, 63% for colon cancer, 64% for gastric cancer, 62% for ovarian cancer, and 88% for prostate cancer, which was generally consistent with the results based on the staining in Example 1.
  • the antibodies used in the primary antibody reaction step are those disclosed in WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/018889, WO20
  • 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 used, the same percentage of CAPRIN-1 positive cancer tissue specimens as above was obtained.
  • Example 7 Detection of CAPRIN-1 protein by immunohistochemical staining 6
  • immunohistochemical staining was performed 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 0.8 ⁇ g/mL and the color development time in the color development step was approximately 7 minutes, and CAPRIN-1 positive cancer tissue specimens were determined in the same manner as in Example 2.
  • the percentage of CAPRIN-1 positive cancer tissue samples was 77% for breast cancer tissue, 67% for lung cancer, 72% for pancreatic cancer, 72% for kidney cancer, 68% for colon cancer, 58% for gastric cancer, 64% for ovarian cancer, and 82% for prostate cancer, which was generally consistent with the determination results based on the staining in Example 1.
  • the antibodies used in the primary antibody reaction step are those disclosed in WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/018889, WO20
  • 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 used, the same percentage of CAPRIN-1 positive cancer tissue specimens as above was obtained.
  • Example 8 Detection of CAPRIN-1 protein by immunohistochemical staining method 7
  • immunohistochemical staining was performed in the same manner as in Example 6, except that the heat treatment in the antigen activation step was carried out at 121°C, and CAPRIN-1 positive cancer tissue specimens were determined in the same manner as in Example 2.
  • CAPRIN-1 positive cancer tissue samples was 80% for breast cancer tissue, 70% for lung cancer, 70% for pancreatic cancer, 78% for kidney cancer, 69% for colon cancer, 55% for gastric cancer, 60% for ovarian cancer, and 90% for prostate cancer, which was generally consistent with the results based on staining in Example 1.
  • the antibodies used in the primary antibody reaction step are those disclosed in WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/018889, WO20
  • 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 used, the same percentage of CAPRIN-1 positive cancer tissue specimens as above was obtained.
  • Example 9 Detection of CAPRIN-1 protein by immunohistochemical staining 8
  • immunohistochemical staining was performed in the same manner as in Example 6, except that the heating treatment in the antigen activation step was performed at 121°C, the antibody concentration of the primary antibody solution used in the primary antibody reaction step was 0.8 ⁇ g/mL, and the color development time in the color development step was approximately 7 minutes.
  • CAPRIN-1 positive cancer tissue specimens were determined in the same manner as in Example 2.
  • the percentage of CAPRIN-1 positive cancer tissue samples was 75% for breast cancer tissue, 65% for lung cancer, 80% for pancreatic cancer, 80% for kidney cancer, 62% for colon cancer, 55% for gastric cancer, 65% for ovarian cancer, and 80% for prostate cancer, which was generally consistent with the determination results based on the staining in Example 1.
  • the antibodies used in the primary antibody reaction step are those disclosed in WO2010/016526, WO2011/096517, WO2011/096528, WO2011/096519, WO2011/096533, WO2011/096534, WO2013/018894, WO2013/018892, WO2013/018891, WO2013/018889, WO20
  • 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 used, the same percentage of CAPRIN-1 positive cancer tissue specimens as above was obtained.

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